Respiration in Archaea and Bacteria
Advances in Photosynthesis and Respiration VOLUME 16 Series Editor: GOVINDJEE Uni...
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Respiration in Archaea and Bacteria
Advances in Photosynthesis and Respiration VOLUME 16 Series Editor: GOVINDJEE University of Illinois, Urbana, Illinois, U.S.A.
Consulting Editors: Christine FOYER, Harpenden, U.K. Elisabeth GANTT, College Park, Maryland, U.S.A. John H. GOLBECK, University Park, Pennsylvania, U.S.A. Susan S. GOLDEN, College Station, Texas, U.S.A. Wolfgang JUNGE, Osnabrück, Germany Hartmut MICHEL, Frankfurt am Main, Germany Kirmiyuki SATOH, Okayama, Japan James Siedow, Durham, North Carolina, U.S.A.
The scope of our series, beginning with volume 11, reflects the concept that photosynthesis and respiration are intertwined with respect to both the protein complexes involved and to the entire bioenergetic machinery of all life. Advances in Photosynthesis and Respiration is a book series that provides a comprehensive and state-of-the-art account of research in photosynthesis and respiration. Photosynthesis is the process by which higher plants, algae, and certain species of bacteria transform and store solar energy in the form of energy-rich organic molecules. These compounds are in turn used as the energy source for all growth and reproduction in these and almost all other organisms. As such, virtually all life on the planet ultimately depends on photosynthetic energy conversion. Respiration, which occurs in mitochondrial and bacterial membranes, utilizes energy present in organic molecules to fuel a wide range of metabolic reactions critical for cell growth and development. In addition, many photosynthetic organisms engage in energetically wasteful photorespiration that begins in the chloroplast with an oxygenation reaction catalyzed by the same enzyme responsible for capturing carbon dioxide in photosynthesis. This series of books spans topics from physics to agronomy and medicine, from femtosecond processes to season long production, from the photophysics of reaction centers, through the electrochemistry of intermediate electron transfer, to the physiology of whole orgamisms, and from X-ray christallography of proteins to the morphology or organelles and intact organisms. The goal of the series is to offer beginning researchers, advanced undergraduate students, graduate students, and even research specialists, a comprehensive, up-to-date picture of the remarkable advances across the full scope of research on photosynthesis, respiration and related processes. The titles published in this series are listed at the end of this volume and those of forthcoming volumes on the back cover.
Respiration in Archaea and Bacteria Diversity of Prokaryotic Respiratory Systems
Edited by
Davide Zannoni Universita di Bologna, Italy
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 1-4020-2002-3 (HB) ISBN 1-4020-3163-7 (e-book) Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. Sold and distributed in North, Central and South America by Springer, 101 Philip Drive, Norwell, MA 02061, U.S.A. In all other countries, sold and distributed by Springer, P.O. Box 322, 3300 AH Dordrecht, The Netherlands.
The camera ready text was prepared by Lawrence A. Orr, Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604, U.S.A.
Printed on acid-free paper
All Rights Reserved © 2004 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed in the Netherlands.
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SPECIAL PRAISE FOR BEING A ROLE MODEL FOR MY LIFE AND WELL BEING /UR DAUGHTER !NITA 'OVINDJEE AND HER HUSBAND -ORTEN #HRISTIANSEN PROVIDED FACILITIES AT THE TIME THIS BOOK WAS BEING PREPARED FOR PUBLICATION !UGUST 'OVINDJEE 3ERIES %DITOR !DVANCES IN 0HOTOSYNTHESIS AND 2ESPIRATION 5NIVERSITY OF )LLINOIS AT 5RBANA #HAMPAIGN $EPARTMENT OF 0LANT "IOLOGY 3OUTH 'OODWIN !VENUE 5RBANA ), 53! % MAIL GOV UIUCEDU 52, HTTPWWWLIFEUIUCEDUGOVINDJEE
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*RYLQGMHH IN THE ELECTRON AND PROTON TRANSPORT THERMOLUMINES CENCE DELAYED AND PROMPT mUORESCENCE PARTICULARLY LIFETIMES AND THEIR USE IN UNDERSTANDING ELECTRON TRANSPORT AND PHOTOPROTECTION AGAINST EXCESS LIGHT (E HAS COAUTHORED @0HOTOSYNTHESIS AND HAS ED ITED OR CO EDITED @"IOENERGETICS OF 0HOTOSYNTHESIS @0HOTOSYNTHESIS IN VOLUMES ,IGHT %MISSION BY 0LANTS AND "ACTERIA AMONG OTHER BOOKS (E IS A MEMBER OF THE !MERICAN 3OCIETY OF 0LANT "IOLOGY !MERICAN 3OCIETY FOR 0HOTOBIOLOGY "IOPHYSICAL 3OCIETY OF !MERICA AND THE )NTERNATIONAL 3OCIETY OF 0HOTOSYNTHESIS 2ESEARCH )302 'OVIN DJEES SCIENTIlC INTEREST NOW INCLUDES &LUORESCENCE ,IFETIME )MAGING -ICROSCOPY &,)- WITH 2OBERT #LEGG AND REGULATION OF EXCITATION ENERGY TRANSFER IN OSCILLATING LIGHT WITH ,ADA .EDBAL )N ADDITION 'OVINDJEE IS INTERESTED IN @(ISTORY OF 0HOTOSYNTHESIS 2ESEARCH AND IN @0HOTOSYNTHESIS %DUCATION (IS PERSONAL BACKGROUND APPEARS IN 6OLUME EDITED BY " 'REEN AND 7 0ARSON AND CONTRIBUTIONS TO PHOTOSYNTHESIS AND mUORESCENCE IN ALGAE IN 6OLUME ! ,ARKUM * 2AVEN AND 3 $OUGLAS EDITORS OF THE !DVANCES IN 0HOTOSYNTHESIS AND 2ESPIRATION !)0( (E SERVES AS THE 3ERIES %DITOR OF !)0( AND AS THE @(ISTORICAL #ORNER %DITOR OF @0HOTOSYNTHESIS 2ESEARCH &OR FURTHER INFORMATION SEE HIS WEB PAGE AT HTTPWWWLIFEUIUCEDUGOVINDJEE
'OVINDJEE IS 0ROFESSOR %MERITUS OF "IOCHEMISTRY "IOPHYSICS AND 0LANT "IOLOGY AT THE 5NIVERSITY OF )LLINOIS AT 5RBANA #HAMPAIGN 5)5# )LLINOIS 53! (E RECEIVED HIS 0H$ IN "IOPHYSICS FROM THE 5NIVERSITY OF )LLINOIS AT 5RBANA #HAMPAIGN IN WITH A THESIS ON THE @!CTION 3PECTRA OF THE %MERSON %NHANCEMENT %FFECT IN !LGAE UNDER %UGENE 2ABI NOWITCH &ROM HE SERVED AS A 5NITED 3TATES 0UBLIC (EALTH 530( 0OSTDOCTORAL &ELLOW FROM AS !SSISTANT 0ROFESSOR OF "OTANY FROM AS !SSOCIATE 0ROFESSOR OF "IOPHYS ICS AND "OTANY AND FROM AS 0ROFESSOR OF "IOPHYSICS AND 0LANT "IOLOGY ALL AT THE 5)5# )N HE BECAME 0ROFESSOR %MERITUS OF "IOCHEMISTRY "IOPHYSICS AND 0LANT "IOLOGY AT 5)5# *ULIAN %ATON 2YE 0RASANNA -OHANTY 'EORGE 0APAGEORGIOU !LAN 3TEMLER 4HOMAS 7YDRZYNSKI *IN 8IONG #HUNHE 8U AND "ARBARA :ILINSKAS ARE AMONG HIS MORE THAN 0H$ STUDENTS (IS HONORS INCLUDE &ELLOW OF THE !MERICAN !SSOCIATION OF !DVANCEMENT OF 3CIENCE $ISTINGUISHED ,ECTURER OF THE 3CHOOL OF ,IFE 3CIENCES 5)5# 0RESIDENT OF THE !MERICAN 3OCIETY OF 0HOTOBIOLOGY AND &ULBRIGHT 3ENIOR ,ECTURER 'OVINDJEES RESEARCH HAS FOCUSED ON THE FUNCTION OF @0HOTOSYSTEM )) WATER PLASTOQUINONE OXIDO REDUCTASE PARTICULARLY ON THE PRIMARY PHOTOCHEMISTRY ROLE OF BICARBONATE
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1SFGBDF 'RAM POSITIVES #HAPTER THE PATHOGENIC MICRO AEROPHILIC GENERA (ELICOBACTERR AND #AMPYLOBACTER #HAPTER ACETIC ACID BACTERIA #HAPTER NITROGEN lXING SYMBIONTS AND FREE LIVING BACTERIA #HAPTER OXYGENIC PHOTOTROPHS #YANOBACTERIA #HAPTER AND ANOXYGENIC PURPLE NON SULFUR PHOTOTROPHS #HAPTER )N VIEW OF THE EXTENSIVE ON GOING RESEARCH ON THE FUNCTIONAL ANNOTATION OF MICROBIAL GENOMES REVEALING NEW INSIGHTS INTO RESPIRATORY METABOLISM )M CONSCIOUS THAT A FEW OF THE TOPICS SELECTED IN THIS BOOK MIGHT REQUIRE SOME REAPPRAISAL WITHIN A YEAR OR SO 4O PARTIALLY OVERCOME THIS PROBLEM ) HAD RECOMMENDED TO ALL THE CONTRIBUTING AUTHORS TO MAKE A SERIOUS EFFORT TO DESCRIBE NOT ONLY WHAT WE KNOW AT PRESENT BUT ALSO TRY TO INDICATE THOSE ASPECTS DESERVING OUR FUTURE ATTENTION ) HOPE THAT THESE TWO VOLUMES WILL BE A LONG TERM SOURCE OF INFORMATION FOR 0H$ STUDENTS RESEARCHERS AND UNDERGRADUATES FROM DISCIPLINES SUCH AS MICROBIOLOGY BIOCHEMIS TRY CHEMISTRY AND ECOLOGY STUDYING BASIC SCIENCES MEDICINE AND AGRICULTURE !S EXPLAINED IN THE @0REFACE OF THE PRECEDING 6OL UME THERE ARE MANY PERSONS THAT ) WOULD LIKE TO THANK SINCE THEY HAVE BEEN CRUCIAL FOR THE COMPLETION OF THIS WORK 0RIMARILY ) WISH TO REMEMBER 0ROFESSOR ! +RÚGER WHO TRAGICALLY PASSED AWAY A FEW MONTHS AFTER THE CONCLUSION OF HIS CHAPTER SEE HIS OBITUARY WRITTEN BY 0ROFESSOR - +LINGENBERG SECONDLY ) LIKE TO THANK ALL THE CONTRIBUTING AUTHORS AND THE SERIES %DITOR OF THE !DVANCES IN 0HOTOSYNTHESIS AND 2ESPI RATION 'OVINDJEE WHO PROVIDED ME ENCOURAGEMENT AND USEFUL SUGGESTIONS TO COMPLETE A PROJECT OF SUCH A MAGNITUDE )N THE END )M GLAD TO THANK ,ARRY /RR FOR HIS FRIENDLY AND WONDERFUL ASSISTANCE IN PRODUCING THE PAGE LAYOUT OF THE TWO VOLUMES 4HIS WORK IS DEDICATED TO MY FAMILY AND MY PARENTS
$URING THE PAST THREE DECADES THE MICROBIAL PHYSIOL OGY HAS BEEN SIGNIlCANTLY CHANGED BY THE ADVENT OF MOLECULAR BIOLOGY )NDEED THE WAY TO APPROACH THE DEGREE OF METABOLIC SOPHISTICATION OF PROKARYOTES REmECTING THE TYPE OF HABITAT IN WHICH THEY EXIST WAS GREATLY ENHANCED BY THE POSSIBILITY TO STUDY GENE EXPRESSION AND REGULATION 4HE CREATION OF THE PROKARYOTIC !RCHAEAN DOMAIN MAINLY BASED ON THE PIONEERING MOLECULAR STUDIES OF #ARL 7OESE AT THE 5NIVERSITY OF )LLINOIS IN THE EARLY S NOT ONLY HAS MODIlED THE BACTERIAL TAXONOMY BUT RESULTED IN A TREMENDOUS RISE IN EFFORTS AIMING TO DElNE THE SO CALLED @MICROBIAL DIVERSITY )N THIS BOOK ENTITLED 2ESPIRATION IN !RCHAEA AND "ACTERIA ) HAVE ATTEMPTED TO PROVIDE AN EXTENSIVE COVERAGE OF ONE OF THE MOST ANCIENT BIOCHEMICAL PRO CESSES RESPIRATION 4HE BOOK HAS BEEN OPERATIONALLY DIVIDED INTO TWO VOLUMES OF THE SERIES !DVANCES IN 0HOTOSYNTHESIS AND 2ESPIRATION 'OVINDJEE %D 4HE lRST VOLUME HAS RECENTLY BEEN PUBLISHED 6OL AND IS SUB TITLED $IVERSITY OF 0ROKARYOTIC %LECTRON 4RANS PORT #ARRIERS )T INCLUDES A GROUP OF CHAPTERS COVER ING ALL MOLECULAR FUNCTIONAL AND STRUCTURAL ASPECTS OF THE MEMBRANE BOUND BACTERIAL REDOX COMPLEXES AND RESPIRATORY CO FACTORS SUCH AS PYRROLO QUINOLINE QUINONES AND HEMOGLOBINS 6OLUME ALSO INCLUDES A CHAPTER ILLUSTRATING THE EVOLUTION OF RESPIRATION IN THE PROKARYOTIC WORLD ALONG WITH TWO CHAPTERS ON TOP ICS SUCH AS CYTOCHROME C BIOGENESIS AND MEMBRANE OXIDASES AS REDOX SENSORS REGULATING THE EXPRESSION OF PHOTOSYNTHETIC GENES IN FACULTATIVE PHOTOTROPHS 4HE CURRENT VOLUME 6OL OF THE SERIES IS SUB TITLED $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS AND CONTAINS CHAPTERS 4HIS BOOK IS CONCEIVED AS A COMPREHENSIVE ACCOUNT OF RESPIRATORY SYSTEMS IN SELECTED GENERA OF THE DOMAIN "ACTERIA ALONG WITH AN EXTENSIVE CHAPTER #HAPTER ON THE REDOX CHAINS AND BIOENERGETICS OF EXTREMOPHILES BELONGING TO THE !RCHAEAN DOMAIN 4HE PRESENT VOLUME CONTAINS SIX CHAPTERS DEALING WITH METABOLIC FEATURES HAVING IMPORTANT MICROBIOLOGICAL AND ECOLOGICAL RELEVANCE SUCH AS THE USE OF AMMONIUM #HAPTER METHANE #HAPTER IRON #HAPTER SULFUR #HAPTER AND HYDROGEN #HAPTER AS RESPIRATORY SUBSTRATES OR NITROUS COMPOUNDS IN DENITRIlCATION PROCESSES #HAPTER 4HE REMAINING CHAPTERS ARE DEDICATED TO RESPIRATION OF SELECTED GROUPS OF BACTERIA SUCH AS
$AVIDE :ANNONI
$EPARTMENT OF "IOLOGY 5NIVERSITY OF "OLOGNA "O )TALY DAVIDEZANNONI UNIBOIT
XV
'DYLGH=DQQRQL IN AND HE VISITED DIFFERENT %URO PEAN LABORATORIES $EPARTMENT OF "IOCHEMISTRY AND -ICROBIOLOGY 3T !NDREWS 5NIVERSITY 3T !NDREWS 3COTLAND 5+ $ÏPARTEMENT DE "IOLOGIE #ELLULAIRE ET -OLÏCULAIRE #.23 52! #%! 3ACLAY 'IF SUR 9VETTE &RANCE $EPARTMENT OF -ICROBIOLOGY 5NIVER R SITY OF 'ÚTTINGEN 'ÚTTINGEN 'ERMANY TO INVESTIGATE THE STRUCTURE OF ELECTRON TRANSPORT REDOX COMPLEXES IN VARIOUS GENERA AND SPECIES OF FACULTATIVE PHOTOTROPHS AND AEROBES 4HE ROLE PLAYED BY $ :ANNONI ON THE DISCOVERY AND DESCRIPTION OF THE MEMBRANE ANCHORED CYTOCHROME CY OF 2B CAPSULATUS WAS PUBLISHED IN 0HOTOSYNTH 2ES :ANNONIS SCIENTIlC INTEREST NOW INCLUDES A RESEARCH LINE ON THE BIOENER GETICS OF MICROBIAL REMEDIATION OF METALS AND METAL LOIDS )N ADDITION $ :ANNONI IS PRESENTLY #HAIRMAN OF THE )TALIAN 3OCIETY FOR 'ENERAL -ICROBIOLOGY AND -ICROBIAL "IOTECHNOLOGIES
$AVIDE :ANNONI IS 0ROFESSOR OF 'ENERAL -ICROBIOLOGY IN THE $EPARTMENT OF "IOLOGY AT THE 5NIVERSITY OF "OLOGNA )TALY (E RECEIVED THE @,AUREA IN "IOLOGI CAL 3CIENCES FROM THE 5NIVERSITY OF "OLOGNA IN WITH A THESIS ON THE BIOENERGETICS OF THE FACULTATIVE PHOTOTROPH 2HODOBACTER 2B CAPSULATUS )N HE REPORTED &%"3 ,ETTERS THE lRST EXAMPLE OF A HIGH POTENTIAL B TYPE HEME CYTOCHROME OXIDASE TODAY RECOGNIZED AS CBB TYPE OXIDASE &ROM .O VEMBER TO .OVEMBER HE WAS A RESEARCH FELLOW OF THE .ORTH !TLANTIC 4REATY /RGANIZATION .!4/ AT THE 3T ,OUIS 3CHOOL OF -EDICINE $EPART MENT OF "IOCHEMISTRY 3T ,OUIS -/ 53! UNDER THE SUPERVISION OF 0ROFESSOR "ARRY , -ARRS )N HE WAS APPOINTED ,ECTURER IN 0LANT "IOCHEMISTRY AT THE 5NIVERSITY OF "OLOGNA AND PROMOTED TO !SSOCI ATE 0ROFESSOR IN !S A RESEARCH FELLOW OF THE %UROPEAN -OLECULAR "IOLOGY /RGANIZATION %-"/
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ARE ADDRESSED 3EVERAL ARCHAEAL GENOMES HAVE BEEN COMPLETELY ANALYZED BUT PROTEIN CHEMICAL VERIlCATION OF THE GENOMIC DATA ON ELECTRON TRANSPORT SYSTEMS LACKS BEHIND !CCORDING TO PRESENT KNOWLEDGE AEROBIC !RCHAEA CAN OXIDIZE .!$( BY A TYPE )) .!$( DEHYDROGENASE WHILE AN EQUIVALENT TO COMPLEX ) HAS NOT BEEN VERIlED )N SEVERAL REACTIONS THE FUNCTION OF .!$ IS REPLACED BY FERREDOXINS /THER AUXILIARY COFACTORS ARE SMALL COPPER PROTEINS WHICH MAY REPLACE THE FUNCTION OF C TYPE CYTOCHROMES AS WELL AS UNUSUAL THIO PHENO BENZOQUINONES WHICH ACT AS MEMBRANE RESIDING POOLS FOR REDUCING EQUIVALENTS &URTHER MOST A TYPE CYTOCHROMES CONTAIN AN ARCHETYPICAL HEME !3 WHICH ALSO REPLACES HEME B IN SEVERAL RESPIRATORY COMPLEXES OF THERMOACIDOPHILIC !RCHAEA !RCHAEAL COMPLEX )) EQUIVALENTS CONTAIN MODIlED 3 &E3 CLUSTERS AS WELL AS UNUSUAL CYSTEINE RICH MOTIFS IN THEIR SMALL SUBUNITS WHICH HOST A NOVEL ;&E3= CLUSTER AND IN ADDITION CAN BE DEVOID OF THEIR TYPICAL MEMBRANE ANCHORS ! NOVEL TYPE OF 2IESKECYTOCHROME COMPLEX EQUIVALENT TO RESPIRATORY COMPLEX ))) HAS BEEN DETECTED IN THERMOACIDOPHILIC !RCHAEA -INIMAL RESPIRATORY CHAINS CAN CONSIST SIMPLY OF A QUINOL OXIDASE AND ARE CONTRASTED BY SUPERCOMPLEXES COMPRISING A GENETIC AND STRUCTURAL FUSION OF A WHOLE RESPIRATORY CHAIN INTO ONE UNIQUE PROTEIN ASSEMBLY ! NOVEL REGULATORY ADAPTATION TO ACIDIC ENVIRONMENTS IS PROPOSED BASED ON THE UNIQUE PROPERTIES OF A #U! CONTAINING TERMINAL OXIDASE FROM AN EXTREME THERMOACIDOPHILE ! REVISED VIEW ON THE STRUCTURAL BASIS FOR PROTON PUMPING CAPABILITIES OF TER MINAL OXIDASES IS PROVIDED 4HE #ONCLUSIONS PARAGRAPH ADDRESSES THE NECESSARY EXPERIMENTAL APPROACHES REQUIRED TO INVESTIGATE THE UNSOLVED QUESTIONS ON ARCHAEAL RESPIRATORY SYSTEMS **OUSPEVDUJPO !RCHAEA ARE REPRESENTING THE SO CALLED THIRD DOMAIN OF LIFE FORMING A DISTINCT BRANCH OF THE UNIVERSAL PHY LOGENETIC TREE BASED ON THE EVOLUTIONARY DISTANCES OF S RIBOSOMAL 2.! 4HIS NOVEL DOMAIN WAS IDENTIlED lRST BY # 7OESE 7OESE AND &OX 7OESE ET AL 7HEELIS ET AL $OOLITTLE NAMED AS !RCHAEA FORMERLY ARCHAEBACTERIA ILLUSTRATING THE EVOLUTIONARY DISTANCE AND FUNDAMENTAL DIFFER ENCES OF LIFESTYLES $ESPITE THE LARGE EVOLUTIONARY DISTANCE TO "ACTERIA AND %UKARYA !RCHAEA CANNOT BE CONSIDERED AS LIVING FOSSILS INDEED THE ARCHAEAL DOMAIN REVEALS THE SHORTEST DISTANCE TO A HYPOTHETICAL COMMON ANCESTOR PRECEDING THE SPLIT INTO THE THREE MAJOR DOMAINS )NITIALLY A FEW GENERA OF METHANOGENS AND HALOBACTERIA ALONG WITH A RESTRICTED NUMBER OF THERMOACIDOPHILIC SULFUR METABOLIZING MICROBES WERE IDENTIlED TO BELONG TO THE ARCHAEAL DOMAIN )N THE PAST TEN YEARS HOWEVER THE LARGE DIVERSITY OF ARCHAEAL ORGANISMS BECAME EVIDENT "OONE ET AL "ARNS ET AL (ERSHBERGER ET AL 4AKAI AND 3AKO 3MITH WITH A CONTINUOUSLY INCREAS ING NUMBER OF NEWLY DETECTED SPECIES !RCHAEA STAND OUT ALSO PHENOTYPICALLY BECAUSE MOST OF THEM ARE IN HABITING UNUSUAL ENVIRONMENTS AND THEREFORE ARE REAL !BBREVIATIONS &DX n FERREDOXIN ()0)0 n HIGH POTENTIAL IRON SULFUR PROTEIN (1./ n HYDROXYQUINOLINE . OXIDE )&0 n IRON mAVOPROTEIN .$( n TYPE .!$( DEHYDROGENASE .$( )) n TYPE )) .A$( DEHYDROGENASE 1&2 n QUINOLFUMARATE REDUCTASE 3$( n SUCCINATE DEDYDROGENASE 312 n SUCCINATEQUINONE REDUCTASE 4&2 n THIOL FUMARATE REDUCTASE
EXTREMOPHILES %SPECIALLY THE LATTER FEATURES AS FOR EXAMPLE TO GROW OPTIMALLY AT TEMPERATURES CLOSE TO THE BOILING POINT OF WATER AT EXTREMELY ACIDIC OR ALKALINE P(S OR AT ALMOST SATURATING .A#L CONCENTRATION OR A COMBINATION OF THESE PROPERTIES CHALLENGED BOTH THE BASIC RESEARCH ON THE MOLECULAR ASPECTS OF LIFE UNDER EXTREME CONDITIONS AND THE BIOTECHNOLOGICAL APPLICA TIONS OF THESE ORGANISMS &OR A DETAILED DISCUSSION ON TAXONOMY METABOLISM MEMBRANE STRUCTURE AND MOLECULAR BIOLOGY OF !RCHAEA THE READER IS REFERRED TO MORE SPECIALIZED REVIEWS $ANSON 3CHÚNHEIT AND 3CHÊFER "LÚCHL ET AL 3TETTER $ANSON AND (OUGH 4HE BIOENERGETIC SYSTEMS OF !RCHAEA HAVE BEEN RECENTLY REVIEWED EXTENSIVELY 3CHÊFER ET AL #LEARLY TWO METABOLIC ASPECTS ARE RESTRICTED TO THE !RCHAEA DOMAIN BESIDES OTHER MOLECULAR PROPER TIES LIKE MEMBRANE STRUCTURE AND LIPID COMPOSITION NAMELY A THE USE OF LIGHT DRIVEN RHODOPSIN SYSTEMS IN CELLULAR ENERGETICS AND B THE REDUCTIVE FERMENTA TION OF LOW MOLECULAR CARBON COMPOUNDS AS THE SOLE CELLULAR ENERGY SOURCE TO FORM METHANE )N CONTRAST THE BASIC PRINCIPLES OF AEROBIC OR ANAEROBIC RESPIRA TIONS APPEAR TO BE SHARED BETWEEN !RCHAEA "ACTERIA AND %UCARYA MITOCHONDRIA AT LEAST TO A CERTAIN EXTENT .EVERTHELESS FUNDAMENTAL DIFFERENCES HAVE BEEN DISCOVERED AND A MERELY PARTIAL HOMOLOGY BETWEEN BACTERIAL MITOCHONDRIAL AND ARCHAEAL RESPIRATORY COMPLEXES HAVE RAISED THE DISCUSSION WHETHER OR NOT THE FORMERLY ANAEROBIC !RCHAEA MIGHT HAVE GAINED THEIR CAPABILITY TO COPE WITH OXYGEN FROM EXTENSIVE LATERAL GENE TRANSFER )NDEED A FREQUENT EXCHANGE OF
$IBQUFS "SDIBFBM3FTQJSBUJPO GENETIC MATERIAL BETWEEN PRIMORDIAL ORGANISMS HAS BEEN ASSUMED TO HAVE OCCURRED PRIOR TO BRANCHING INTO THE PRESENTLY EXISTING KINGDOMS OF LIFE 7OESE /N THE OTHER HAND SIGNIlCANT MOLECULAR DATA SUPPORT THE IDEA OF AN ORIGIN OF OXYGEN REDUCING HEMECOPPER OXIDOREDUCTASES #ASTRESANA AND 3ARASTE SEE ALSO #HAPTER BY #ASTRESANA 6OL AS PRECEDING THE EVOLUTIONARY ADVENT OF OXYGENIC PHOTOSYNTHESIS WHICH INCIDENTALLY IS COMPLETELY ABSENT FROM THE ARCHAEAL DOMAIN )N THIS CHAPTER THE DIFFERENT TYPES OF ANAEROBIC RESPIRATIONS ARE NOT DISCUSSED EMPHASIS IS PUT ON THE RESPIRATORY SYSTEMS OF !RCHAEA USING OXYGEN REDUC TION COMPULSORY OR FACULTATIVELY AS AN ENERGY SOURCE 4HOUGH THIS ISSUE HAS BEEN INCLUDED IN A RATHER RECENT REVIEW 3CHÊFER ET AL NOVEL RESULTS MAKE NEC ESSARY A REVISION OF THE PREVIOUSLY PROPOSED MODELS )N PARTICULAR THE QUESTION WHETHER OR NOT THE UNUSUAL ARCHAEAL TERMINAL OXIDASES ARE PROTON PUMPS AS WELL AS THE ISSUE WHETHER !RCHAEA DO HAVE A REDOX COMPLEX EQUIVALENT TO THE ORTHODOX RESPIRATORY COMPLEX ))) ARE ADDRESSED BELOW !LSO NEW STRUCTURAL DATA ON THE FUNCTION AND COMPOSITION OF ELECTRON TRANSPORT SUPER COMPLEXES AND HIGH RESOLUTION STRUCTURES OF INTERMEDIATE ELECTRON CARRIERS ARE PRESENTED **"FSPCJD"SDIBFBBOE$PNQPOFOUTPG &MFDUSPO5SBOTQPSU !EROBIC !RCHAEA WITH WIDELY DIFFERENT LIFESTYLES HAVE BEEN FOUND "ESIDES ORGANOTROPHIC ORGANISMS LITHO AUTOTROPHIC SPECIES EXIST AND MOREOVER OBLIGATE AS WELL AS FACULTATIVE AEROBES ARE AMONG THESE !N EXEMPLARY COMPILATION IS CONTAINED IN A PREVIOUS REVIEW 3CHÊFER ET AL 4HEREIN THOSE !RCHAEA WERE LISTED WHICH CAN BE GROWN AS LABORATORY CULTURES /NE HAS TO REALIZE HOWEVER THAT A MUCH HIGHER NUM BER OF SPECIES EXISTS WHICH HAVE ONLY BEEN IDENTIlED BY 2.! EXTRACTION AND ANALYSIS OF SOIL AND WATER SAMPLES TAKEN FROM A LARGE VARIETY OF ENVIRONMENTS "ARNS ET AL 5NFORTUNATELY SOME SPECIES ARE RESTRICTED TO SYMBIOTIC COMMUNITIES AND THEREFORE THEY REMAIN INACCESSIBLE TO GROWTH AS PURE CULTURES IN VITRO !T LEAST ONE HAS TO ACCEPT THAT ONLY A RELATIVELY SMALL NUMBER OF AEROBIC !RCHAEA CAN BE GROWN IN SUFlCIENT AMOUNTS TO ALLOW EXTENSIVE BIOCHEMICAL AND BIOENERGETIC STUDIES .EVERTHELESS RESPIRATORY SYSTEMS COULD BE STUDIED FROM HALOBACTERIA ACTUALLY THE lRST ARCHAEAL GENUS INVESTIGATED BIOENERGETICALLY FROM HYPERTHERMOPHILIC ACIDOPHILIC ALKALIPHILIC
OR NEUTROPHILIC GENERA INCLUDING #/ lXATING SULFUR METABOLIZERS AS WELL AS ORGANOTROPHIC SPECIES 4HE BEST INVESTIGATED SPECIES ARE (ALOBACTERIUM (B SALINARUM (ALOFERAX (F VOLCANII AND ( MEDITERA NEI .ATRONOMONAS . PHARAONIS 4HERMOPLASMA 4P ACIDOPHILUM 3ULFOLOBUS ACIDOCALDARIUS 3 SOLFATARICUS 3 TOKODAII STRAIN 3 METALLICUS AND !CIDIANUS !C AMBIVALENS )N ADDITION A NUMBER OF RESPIRATORY SYSTEMS CAN BE DEDUCED FROM FULLY SEQUENCED GENOMES AS FOR EXAMPLE FROM !EROPYRUM !E PERNIX +AWARABAYASHI OR 0YROBACULUM 0B AEROPHILUM &ITZ 'IBBON ET AL &ROM THE LATTER AND OTHERS WHICH ARE DIFlCULT TO CUL TIVATE A NUMBER OF REDOX PROTEINS RELATED TO BIOENER GETICS HAS BEEN OBTAINED BY HETEROLOGOUS EXPRESSION OF SINGLE GENES *ANSSEN ET AL 3CHMIDT ET AL (ENNINGER ET AL *ANSSEN ET AL +OMOROWSKI AND 3CHÊFER 'ENERALLY THE EXPERIMENTAL APPROACH TO !RCHAEAL RESPIRATORY COMPONENTS WAS CHARACTERIZED BY A SEARCH FOR ANALOGIES TO COMPONENTS OF KNOWNFOR EXAMPLE MITOCHONDRIALRESPIRATORY CHAINS IE THE PRESENCE OF COMPLEX ) TO )6 AS CLASSICAL ELECTRON TRANSPORT AND PROTON PUMPING DEVICES (OWEVER WHEREAS SPEC TROSCOPY OF INTACT AND SOLUBILIZED PLASMA MEMBRANES CLEARLY REVEALED THE OCCURRENCE OF A AND B TYPE CYTOCHROMES AS WELL AS OF CYANIDE SENSITIVE TERMINAL OXIDASES OF THE AA TYPE C TYPE CYTOCHROMES WERE OFTEN ABSENT EG 3ULFOLOBALES )NSTEAD UNUSUAL BANDS IN REDUCED OXIDIZED DIFFERENCE SPECTRA INDICATED NOVEL INTERMEDIATE ELECTRON CARRIERS LIKE THE CYTOCHROME A OF 3ULFOLOBUS ACIDOCALDARIUS -OREOVER ARCHAEAL RESPIRATION APPEARED IN MOST CASES INSENSITIVE TO ROTENONE PIERICIDINE DERIVATIVES ANTIMYCIN ! OR STIGMATELLIN WHICH ARE ALL SPECIlC INHIBITORS OF @OR THODOX RESPIRATORY COMPLEXES REVIEWED IN 3CHÊFER ET AL 4HIS INSENSITIVITY LARGELY HAMPERED THE USE OF ROUTINE APPROACHES FOR THE RESOLUTION OF ELECTRON TRANSPORT SYSTEMS ESPECIALLY IN VIEW OF THE lNDING THAT !RCHAEA SIMILARLY TO MANY SPECIES OF "ACTERIA CAN POSSESS MULTIPLE TERMINAL OXIDASES AND BRANCHED RESPIRATORY PATHWAYS 4HEREFORE INVESTIGA TION OF THE DISTINCT PROPERTIES OF ARCHAEAL RESPIRATORY SYSTEMS IS STILL A CHALLENGE AND MANY DETAILS REMAIN TO BE RESOLVED )N THIS CONTEXT THE IDENTIlCATION OF PROTON PUMPING SITES IS AN IMPORTANT ASPECT /NE HAS TO REMEMBER THAT THERMOACIDOPHILIC !RCHAEA ARE MAINTAINING AN UNUSUALLY LARGE PROTON GRADIENT ACROSS THEIR PLASMA MEMBRANE OF UP TO * -OLL AND 3CHÊFER ,àBBEN AND 3CHÊFER 6AN DE 6OSSENBERG ET AL 4HOUGH RESPIRATION COUPLED
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ACTIVE PROTON EXTRUSION HAS BEEN SHOWN -OLL AND 3CHÊFER THE IDENTIlCATION OF INDIVIDUAL PROTON PUMPS AND ESPECIALLY OF THEIR MECHANISM IS STILL INSUFlCIENT 4ABLE SUMMARIZES AEROBIC !RCHAEA WHICH HAVE BEEN INVESTIGATED IN CULTURE !MONG THESE SEVERAL THERMOPHILES AND HYPERTHERMOPHILES AS WELL AS EX TREMELY ACIDOPHILES WERE IDENTIlED HOWEVER THE FULL GENOME SEQUENCES HAVE BEEN DETERMINED IN A FEW SPECIES AND EVEN LESS HAS BEEN DONE WITH REGARD TO THE PROTEIN STRUCTURE OF THEIR RESPIRATORY SYSTEMS !MONG THE !RCHAEA DOMAIN THE HALOBACTERIAL MEM BERS BELONG TO THE MAJOR GROUP OF %URYARCHAEOTA AND THEY CAN USE ALSO THE LIGHT ENERGY EMPLOYING VARI OUS BACTERIORHODOPSIN BASED SYSTEMS )N THE DARK HOWEVER THEY HAVE TO RELY ON OXYGEN RESPIRATION )N ADDITION THEY ARE MESOPHILIC (ALOFERAX SPECIES CAN GROW ALSO WITH NITRATE AS TERMINAL ELECTRON AC CEPTOR 4HE SAME HOLDS FOR THE THERMOEXTREMOPHILIC CRENARCHAEON 0YROBACULUM AEROPHILUM WHICH IS A FACULTATIVE AEROBE /THER FACULTATIVE AEROBES INCLUDE SPECIES OF THE !CIDIANUS GENUS WHICH WAS DESCRIBED AS STRICT CHEMOLITHOAUTOTROPH SIMILARLY TO THE SPECIES 3ULFOLOBUS METALLICUS AND -ETALLOSPHERA SEDULA !LL OTHER LISTED !RCHAEA CAN GROW ORGANOTROPHICALLY AND OF THESE THE 3ULFOLOBALES AND THE HALOPHILES ARE OBLIGATE AEROBES 2ESPIRATORY COMPONENTS WHICH HAVE BEEN SPECTROS COPICALLY IDENTIlED OR WERE CHARACTERIZED PROTEIN CHEMICALLY FROM AEROBIC !RCHAEA ARE ALSO BRIEmY SUMMARIZED IN 4ABLE 3PECIALIZED DESCRIPTIONS OF ARCHAEAL CYTOCHROMES AND OF THE HOSTED HEMES HAVE BEEN PUBLISHED ELSEWHERE ,àBBEN AND -ORAND ,àBBEN !N INTERESTING DEVIATION FROM KNOWN A TYPE CYTOCHROMES IS THE ARCHAEAL HEME !3 WHICH HAS BEEN DESCRIBED lRST IN 3ULFOLOBALES 4HIS HEME IS PRESENT IN TERMINAL AA OXIDASES AS WELL AS IN THEIR AUXILIARY A TYPE CYTOCHROMES LIKE #YT A ! NUMBER OF COMPONENTS NOT INCLUDED IN THE TABLE WERE ONLY TENTATIVELY ASSIGNED AS RESPIRATORY ELECTRON CARRIERS IN VARIOUS ARCHAEAL GENOME PROJECTS BY SEQUENCE SIMILARITIES 4HESE INCLUDE C TYPE CYTOCHROMES AND CONSTITUENTS OF COMPLEX ) TYPE .!$( DEHYDRO GENASES )N MOST CASES HOWEVER IT IS YET UNKNOWN WHETHER OR NOT THE RESPECTIVE GENES ARE ACTUALLY EXPRESSED ANDOR LEADING TO FUNCTIONAL COMPLEXES /N THE OTHER HAND PROTEIN CHEMICAL ISOLATION AND CHARACTERIZATION OF SEVERAL FUNCTIONAL COMPLEXES RE VEALED THAT UNUSUAL ASSOCIATIONS OF REDOX COMPONENTS HAVE BEEN DETECTED AND THAT EVEN SUPERCOMPLEXES EXIST COMPRISING ALMOST AN ENTIRE RESPIRATORY CHAIN
AS IT WILL BE DISCUSSED BELOW !N OBVIOUS FACT IS THE CANONICAL lNDING OF SUC CINATEQUINONE REDUCTASES 312 EITHER BY ACTIVITY OR BY GENOME ANALYSIS 4HIS COMPLEX IN AEROBES CONSIDERED TO REPRESENT COMPLEX )) OF RESPIRATORY CHAINSREmECTS THE IMPORTANCE OF THE REVERSIBLE EQUILIBRIUM REACTION BETWEEN FUMARATE AND SUCCI NATE AS PART OF EITHER AN AEROBICALLY OR ANAEROBICALLY FUNCTIONING CITRIC ACID CYCLE !CTUALLY THE GENES OR OPERONS OF BOTH 312S AND QUINOLFUMARATE REDUC TASES 1&2S ORIGINATED PRESUMABLY FROM THE SAME ANCESTRAL GENES THUS UNDERLINING THE IMPORTANCE OF THIS REACTION IN THE EVOLUTION OF INTERMEDIARY METABOLISM ,IKEWISE .!$( OXIDIZING ACTIVITIES HAVE BEEN FREQUENTLY OBSERVED IN ARCHAEAL MEM BRANE PREPARATIONS 7AKAGI AND /SHIMA AND A CERTAIN NUMBER OF GENES HAS BEEN ATTRIBUTED TO .!$( DEHYDROGENASES IN THE ABOVE LISTED GENOME PROJECTS 4HIS IS IN CONTRAST HOWEVER TO THE LACK OF PROTEIN CHEMICAL PROOF FOR THE PRESENCE OF TYPICAL PROTON TRANSLOCATING .!$(QUINONE REDUCTASES AS KNOWN FROM MITOCHONDRIA AND BACTERIA 7EISS ET AL &RIEDRICH AN ISSUE TO BE DISCUSSED SEPARATELY IN DETAIL ***"VYJMJBSZ3FEPY'BDUPST )N GENERAL MEMBRANE RESIDING ELECTRON TRANSPORT COMPLEXES OF RESPIRATORY SYSTEMS REQUIRE BOTH SOLUBLE CYTOSOLIC HYDROGEN DONORS AND REDOX MEDIATORS #OMMONLY THE FORMER ARE PYRIDINE NUCLEOTIDES WHILE VARIOUS QUINONES AND CYTOCHROMES OF C TYPE ARE COUPLING THE ELECTRON mOW BETWEEN PRIMARY DONOR COMPLEXES AND THE TERMINAL OXIDASES )N ARCHAEA PARTICULAR DEVIATIONS FROM THIS CLASSICAL PICTURE HAVE BEEN DISCOVERED %SPECIALLY THE USE OF FERREDOXIN INSTEAD OF .!$ AS PRIMARY ELECTRON ACCEPTOR IS A TYPI CAL ARCHAEAL PROPERTY OF SEVERAL METABOLIC REACTIONS AS FOR EXAMPLE OXOACID DEHYDROGENASES ALDEHYDE DEHYDROGENASES OR EVEN GLYCERALDEHYDE DEHYDRO GENASE +ERSCHER ET AL 'EORGE ET AL -UKUND AND !DAMS "OCK ET AL :HANG ET AL )T HAS ALSO BEEN ASSUMED THAT .!$( FERREDOXIN REDUCTASES MAY PLAY A ROLE AS HYDROGEN DONORS TO THE RESPIRATORY CHAIN )WASAKI ET AL !NOTHER IS THE FUNCTION OF BLUE COPPER PROTEINS PRE SUMABLY REPLACING C TYPE CYTOCHROMES 3CHARF AND %NGELHARD +OMOROWSKI AND 3CHÊFER (IGH POTENTIAL &E3 PROTEINS ()0)0 AS DESCRIBED IN THE RESPIRATORY CHAIN OF THE MARINE BACTERIUM
4ABLE #OMPREHENSIVE LIST OF OBLIGATE AND FACULTATIVE AEROBIC ARCHAEA GROWTH GENOME P( RANGE CHARACTERIZED OR ISOLATED RESPIRATORY 3PECIES 4MAX # COMPLEXES ENZYMES SEQUENCED !CIDIANUS AMBIVALENS IN PEPARATION 312 AA QUINOL OXIDASE &DX .$( )) n n !CIDIANUS BRIERLEYI !CIDIANUS INFERNUS n !EROPYRUM PERNIX N M N (ALOBACTERIUM SACCHAROVORUM (ALOBACTERIUM SALINARUM N S '2" M SP 312 #YTB #YTC2IESKE &E3 C OXIDASE &DX .$( )) (ALOFERAX MEDITERANEI M N M N CYTOCHROME AA (ALOFERAX VOLCANII (ALOFERAX DENITRIlCANS M N n -ETALLOSPHERA SEDULA n .ATRONOMONAS PHARAONIS 312 (ALOCYANIN #YTC #YTB C OXIDASE 0ICROPHILUS OSHIMAE n n 0ICROPHILUS TORIDUS n 0YROBACULUM AEROPHILUM 312 2IESKE &E3 HEME#U OXIDASE
3ULFOLOBUS ACIDOCALDARIUS n $3- 312 3OX!"#$ 3OX- #YTB&DX 3ULFOCYANIN 3OX,. .$( )) n n 3ULFOLOBUS ISLANDICUS 3ULFOLOBUS METALLICUS n .$( TYPE 312 CYTB CYTA #YTA 2IESKE &E3 ()0)0 &DX 3ULFOLOBUS SHIBATAE n 3ULFOLOBUS SOLFATARICUS n 0 312 HEME#U QUINOLC OXIDASE #YTB #YT A 3ULFOLOBUS TOKODAII 312 #YTB #YTA 2IESKE &E3HEME#U QUINOL OXIDASE .$( ))&DX n STRAIN 4HERMOPLASMA ACIDOPHILUM n 312 #YTB HEME#U TERMINAL OXIDASE
4HERMOPLASMA VOLCANIUM n M MESOPHILIC N NEUTROPHILIC 2ESPIRATORY ELECTRON TRANSPORT COMPONENTS LISTED IN THE RIGHT HAND COLUMN WERE EITHER DETERMINED AS ENZYMATIC ACTIVITY OF MEMBRANES OR WERE STUDIED WITH ENRICHEDPURIlED COMPOUNDS AND COMPLEXES RESPECTIVELY )N SOME CASES THEY WERE ONLY PREDICTED FROM GENETIC DATA AS IDENTIlED BY ASTERISKS 312 SUCCINATEQUINONE REDUCTASE .$( )) .!$( DEHYDROGENASE TYPE )) &DX FERREDOXINS ()0)0 HIGH POTENTIAL IRON SULFUR PROTEIN INDICATES EXACT TYPE UNKNOWN
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2HODOTHERMUS MARINUS 0EREIRA ET AL MIGHT ASSUME A SIMILAR ROLE ALSO IN ARCHAEA "'FSSFEPYJOT )N SULFUR METABOLIZING ARCHAEA AN ABUNDANCE OF FER REDOXINS HAS BEEN OBSERVED 4EIXEIRA ET AL )WA SAKI AND /SHIMA 4HEIR HIGH THERMOSTABILITY !ONO ET AL MAY REmECT AN ADVANTAGE OVER .!$ AS A PRIMARY HYDROGEN TRANSDUCING COENZYME IN HYPERTHERMOPHILES 4HEY HAVE THE ADVANTAGE TO PROVIDE REDUCING EQUIVALENTS AT POTENTIALS NEGATIVE ENOUGH TO DRIVE HYDROGENASE CATALYZED REACTIONS % n M6 OR .!$0 REDUCTION % n M6 !LSO FROM THE ALKALIPHILIC .ATRONOMONAS PHARAONIS A FERREDOXIN WITH % n M6 HAS BEEN PURIlED 3CHARF ET AL 4HE PRIMARY STRUCTURES OF SEVERAL ARCHAEAL FERREDOXINS ARE KNOWN +ERSCHER ET AL -INAMI ET AL 0FEIFER ET AL *ANSSEN ET AL AND RECENT GENETIC AND SEQUENCING DATA SUGGEST THAT IN 3ULFOLOBALES MULTIPLE GENES MAY ENCODE HIGHLY SIMILAR FERREDOXINS ! PECULIARITY lRST DEMONSTRATED FROM THE $ STRUCTURE OF CRYSTALLIZED 3ULFOLOBUS FER REDOXIN &UJII ET AL IS THE PRESENCE OF :N IN ADDITION TO THE ;&E 3= AND ;&E 3= CLUSTERS 4HE ALIGNMENT OF SEQUENCES FROM THERMOPHILIC !RCHAEA REVEALS THAT THE PRESENCE OF A :N CHELATING DOMAIN IS THE PREVAILING CASE ONLY TWO OF THE SEQUENCES ARE DEVOID OF THIS :N BINDING MOTIF &IG 4HE ROLE OF ZINC HAS BEEN EMPHASIZED AS A THERMOSTABILITY MEDI ATING FACTOR )WASAKI ET AL +OJOH ET AL )WASAKI AND /SHIMA 4HIS IS IN CONTRADICTION HOWEVER TO OTHER OBSERVATIONS AND IT CAN NOT BE USED AS A GENERAL RULE &OR EXAMPLE THE TWO FERREDOXINS FROM !CIDIANUS AMBIVALENS HAVE BEEN SHOWN TO EXHIBIT ALMOST IDENTICAL THERMOSTABILITY AND THERMAL UNFOLDING CHARACTERISTICS 7ITTUNG 3TAFSHEDE ET AL *ANSSEN ET AL HOWEVER ONLY ONE OF THESE CONTAINS THE BINDINGS SITE FOR :N AS DEPICTED FROM &IG !CTUALLY FOR THE :N FREE FERREDOXIN FROM ! AMBIVALENS A MELTING TEMPERATURE OF # HAS BEEN EXTRAPOLATED FROM 'D(#L INDUCED UNFOLDING ISOTHERMS AT VARIOUS TEMPERATURES &IGURE SHOWS THE %02 SPECTRUM OF THE DITHIONITE REDUCED FERREDOXIN AND THE %02 REDOX TITRATION YIELDING A HALF OXIDATION REDUCTION POTENTIAL OF n M6 WHICH IS VERY CLOSE TO THAT OF PYRIDINE NUCLEOTIDES !LSO 3 METALLICUS EXPRESSES TWO FERREDOXINS WITH AND WITHOUT :N EX HIBITING PRACTICALLY IDENTICAL THERMOSTABILITY 'OMES ET AL !S AN ALTERNATIVE HYPOTHESIS FOR THE ROLE OF :N WE HAVE PROPOSED THAT SUCH FERREDOX
INS MIGHT DIFFER IN THEIR SPECIlCITY AGAINST VARIOUS ELECTRON DONORS OR ACCEPTORS RESPECTIVELY *ANSSEN ET AL A PROPOSAL WHICH REQUIRES FURTHER IN VESTIGATION !NOTHER OPEN ISSUE IS THE PHYSIOLOGICAL SIGNIlCANCE OF A REVERSIBLE REDOX LINKED PROTONATION EQUILIBRIUM WHICH WE DETERMINED WITH THE PURIlED FERREDOXIN FROM 3ULFOLOBUS ACIDOCALDARIUS "RETON ET AL !LSO FROM 3ULFOLOBUS A FERREDOXIN RE OXIDIZING IRON mAVOPROTEIN )&0 HAS BEEN DESCRIBED % n M6 TO BE INVOLVED IN AN ELECTRON TRANSPORT PATHWAY FERREDOXIN A )&0 A &-. A 8 WHERE 8 REPRESENTS AN UNKNOWN ACCEPTOR PUTATIVELY MEDIATING ELECTRON ENTRY INTO THE RESPIRATORY CHAIN )WASAKI ET AL 7HEREAS THE FERREDOXINS OF THERMOACIDOPIHILIC ARCHAEA &IG ARE CLUSTERING WITHIN A VERY HO MOLOGOUS GROUP HALOBACTERIAL L FERREDOXINS .G ET AL 0FEIFFER AND /ESTERHELT FORM A MORE DISTANT CLUSTER WITH LOW HOMOLOGY TO THOSE FROM 3ULFOLOBALES #2VJOPOFT !RCHAEAL RESPIRATORY CHAINS DO NOT CONTAIN UBI QUINONE OR DERIVATIVES THEREOF ! COMPREHENSIVE ANALYSIS ON ARCHAEAL QUINONES WAS lRST PERFORMED WITH ACIDOPHILIC !RCHAEA #OLLINS AND ,ANGWORTHY 4HESE WERE FOUND TO CONTAIN UNUSUAL BENZO ;B= THIOPHENO QUINONES AS FOR EXAMPLE IN 3 ACIDOCALDARIUS .ICOLAUS ET AL 4HEIR STRUCTURE AND PROPERTIES HAVE BEEN EXTENSIVELY DISCUSSED IN A PRECEDING REVIEW 3CHÊFER ET AL 5NUSUAL IS THE OCCURRENCE OF TRICYCLOQUINONE WHICH IS PRESENT ONLY IN TRACES HOWEVER .ICOLAUS ET AL 4RINCONE ET AL 4HE MOST ABUNDANT COMPOUND APPEARS TO BE CALDARIELLA QUINONE lRST DETECTED IN #ALDARIELLA ACIDOPHILA WHICH INTERESTINGLY WAS NOT DElNITELY CLASSIlED AS AN ARCHAEON BUT IS AN EXTREME THERMO PHILE $E 2OSA ET AL )T IS THE ONLY ARCHAEAL QUINONE OF WHICH EXACT ELECTROCHEMICAL PARAMETERS HAVE BEEN DETERMINED !NEMàLLER AND 3CHÊFER AND WHICH CAN BE ISOLATED FROM THE GENUINE MEM BRANES IN SUFlCIENT AMOUNTS FOR USE AFTER REDUCTION AS A SUBSTRATE FOR SEVERAL ARCHAEAL QUINOL OXIDASES )N 3ULFOLOBUS ABOUT OF THE TOTAL QUINONE POOL IS FORMED BY CALDARIELLA QUINONE 4HERMOPLASMA QUINONE WAS ISOLATED FROM 4HER R MOPLASMA ACIDOPHILUM 3HIMADA ET AL AND HAS CLOSE SIMILARITY TO MENAQUINONES 4HE LATTER ARE THE MAJOR QUINONES OF HALOPHILIC ARCHAEA PRE DOMINANTLY -+ WITH AN UNSATURATED ISOPRENOID
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&IG !LIGNMENT OF FERREDOXIN SEQUENCES FROM !RCHAEA 3HADED PLUS BOXED BINDING DOMAINS FOR THE TWO &E CLUSTERS SHADED PLUS ARROWS METAL LIGANDING RESIDUES IN :N CONTAINING FERREDOXINS BOXED OPEN REPLACEMENTS IN THE CORRESPONDING :N FREE FERREDOXINS 3EQUENCE DATA WERE TAKEN FROM GENE BANKS !BBREVIATIONS DENOTE !CID AMBI ! AMBIVALENS 3ULF ACID 3 ACIDOCALDARIUS 3ULF TOKO 3 TOKODAII 3ULF METAL 3 METALLICUS 4HPL ACID 4HERMOPLASMA ACIDOPHILUM !CI INF ! INFERNUS THOSE SPECIES EXPRESSING BOTH THE :N CONTAINING AND THE :N FREE FERREDOXINS ARE LABELED WITH ! AND " RESPECTIVELY
SIDE CHAIN #OLLINS AND ,ANGWORTHY #OLLINS )N CONTRAST THE QUINONES OF 3ULFOLOBUS AND HYPERTHERMOPHILIC ANAEROBIC !RCHAEA EXHIBIT FULLY REDUCED SIDE CHAINS $#MVF$V1SPUFJOT )N PLANTS AND BACTERIA A LARGE NUMBER OF SMALL BLUE COPPER PROTEINS HAS BEEN FOUND WHICH PLAY AN IMPOR TANT ROLE IN ELECTRON TRANSFER REACTIONS 3YKES 5SUALLY THESE ARE SOLUBLE PROTEINS WITH MOLECULAR MASSES FROM n K$A WHICH FOLD INTO STACKED ` SHEETS WITH ONE #U ION LIGATED IN A TRIGONAL EQUATO RIAL COORDINATION 4HE TYPICAL #U LIGANDS INCLUDE HISTIDINES AND A CYSTEINE IN THE EQUATORIAL PLAIN AND ADDITIONALLY ONE AXIALLY BOUND METHIONINE TYPE ) #U PROTEIN 4HE lRST ARCHAEAL BLUE COPPER PROTEIN WAS DETECTED IN .ATRONOMONAS PHARAONIS 3CHARF AND %NGELHARD AND NAMED @HALOCYANIN )TS 56VIS #$ AND %02 SPECTRAL PROPERTIES ARE IN GOOD AGREEMENT WITH
TYPICAL TYPE ) #U PROTEINS AND ARE PARTIALLY RESEM BLING THOSE OF PLASTOCYANIN )N CONTRAST TO SOLUBLE MEMBERS OF THIS FAMILY IT COULD BE REMOVED FROM THE PLASMA MEMBRANE ONLY BY MILD DETERGENTS 4HE PRIMARY STRUCTURE INDICATES INDEED A LIPID ANCHOR FOR MEMBRANE lXATION -ATTAR ET AL OF THE PROTEIN AND A TOTAL MOLECULAR MASS OF K$A 4HE ACTUAL MASS OF THE MATURE PROTEIN K$A INDICATES POST TRANSLATIONAL PROCESSING AS FOUND WITH OTHER PROKARY OTIC LIPOPROTEINS 3PECTRO ELECTROCHEMICAL STUDIES IN THE 56 AND )2 REGION REVEALED AN OXIDO REDUCTION MIDPOINT POTENTIAL OF %M M6 AT NEUTRAL P( "RISCHWEIN ET AL (OWEVER THE %M CHANGED DRAMATICALLY WITH P( FROM M6 AT P( TO M6 AT P( INDICATING A REVERSIBLE PROTON ATION COUPLED TO THE REDOX EQUILIBRIUM &4)2 SPECTRA REVEALED SIGNIlCANT STRUCTURAL ALTERATIONS ALONG WITH THE REDOX PROCESS AND SUGGESTED THE PARTICIPATION OF THE HISTIDINE LIGANDS IN THE PROTONATION EQUILIBRIUM AS WELL AS THE FUNCTION OF A CARBOXYL !SP OR 'LU AS A POSSIBLE lFTH LIGAND 4HE P( DEPENDENCE OF &4)2
&IG %02 SPECTRUM OF THE RECOMBINANTLY PRODUCED :N FREE IRON FERREDOXIN FROM 3 ACIDOCALDARIUS 4HE SPECTRUM OF THE DITHIONITE REDUCED FORM IS SHOWN TOGETHER WITH A REDOX TITRATION INDICATING A MIDPOINT POTENTIAL AROUND n M6 $ATA FROM *ANSSEN ET AL
SPECTRA AND MIDPOINT POTENTIAL WERE NOT REmECTED IN THE 2ESONANCE 2AMAN SPECTRA OF THE PROTEIN (IL DEBRANDT ET AL 4HUS IT WAS CONCLUDED THAT THE REDOX POTENTIAL IS NOT EXCLUSIVELY CONTROLLED BY THE GEOMETRY OF THE COORDINATION SPHERE THE REDOX POTENTIAL FALLS INTO THE RANGE OF C TYPE CYTOCHROMES AND THE MEMBRANE RESIDING HALOCYANINE MIGHT AS SUME A SIMILAR FUNCTION IN THE RESPIRATORY SYSTEM OF . PHARAONIS 4HE COMPLETION OF THE GENOME FROM ( SALINARUM STRAIN 2 REVEALED GENETIC EVIDENCE FOR AT LEAST SEVEN SMALL BLUE COPPER PROTEINS WITH HOMOLOGY TO HALOCYANIN FROM . PHARAONIS /NE OF THESE EXHIBITS TWO COPPER BINDING SITES AND MIGHT HAVE ARISEN FROM GENE DUPLICATION 3OME ARE CARRYING A MEMBRANE ANCHOR 0FEIFFER AND /ESTERHELT !LSO IN ( SALINARUM BLUE COPPER PROTEINS MIGHT BE FUNCTION ING IN REPLACEMENT FOR CYTOCHROME C !N AZURIN HAS BEEN ANNOTATED IN THE GENOME OF !E PERNIX +AWARABAYASHI !NOTHER ARCHAEAL BLUE #U PROTEIN WAS INITIALLY PREDICTED FROM GENETIC DATA OF 3 ACIDOCALDARIUS #ASTRESANA ET AL 4HE SOX% % GENE WITHIN A LARGE GENE CLUSTER ENCODING THE 3OX- SUPERCOMPLEX SEE
(ÛOUFS4DIÅGFS BELOW EXHIBITS THE SIGNATURE OF A TYPICAL #U BIND ING PROTEIN OF TYPE ) AND WAS NAMED @SULFOCYANIN 4HOUGH TRANSCRIPTION OF THE GENE HAD BEEN SHOWN IT COULD NOT BE IDENTIlED PROTEIN CHEMICALLY UNTIL LATELY )T WAS THOUGHT TO BE ABSENT FROM THE ISOLATED 3OX- COMPLEX THUS CAUSING ITS CATALYTIC INACTIVITY 3CHÊFER ET AL 2ECENTLY WE SUCCEEDED TO EXPRESS THIS PROTEIN IN % COLI CELLS AND TO STUDY ITS PROPERTIES IN DETAIL +OMOROWSKI +OMOROWSKI AND 3CHÊFER &ROM THE AMINO ACID SEQUENCE A NATIVE MOLECULAR MASS OF K$A IS CALCULATED AND ACCORDING TO HYDROPATHY PLOTS THE PROTEIN RE VEALS A TYPICAL MEMBRANE ANCHOR AT ITS . TERMINUS 3EQUENCE SIMILARITIES CAN BE DERIVED TO RUSTICYANIN FROM 4 FERROOXIDANS WHICH WAS TAKEN AS A SCAFFOLD FOR TENTATIVE HOMOLOGY MODELING +OMOROWSKI 4HE SEQUENCE HAS ONLY LOW HOMOLOGY TO ANY OF THE HALOCYANINS .EVERTHELESS NEITHER RUSTICYANIN NOR ANY OTHER BLUE #U PROTEIN WAS ABLE TO REPLACE 3ULFOCYANIN AS ELECTRON DONOR TO THE 3OX- RESPIRATORY COMPLEX -OREOVER THE EXPRESSION IN % COLI FAILED WITH THE GENUINE 3ULFOLOBUS GENE FOR EFlCIENT PRODUCTION AT LEAST IN SMALL AMOUNTS THE GENE HAD TO BE FULLY SYNTHESIZED ACCORDING TO THE OPTIMUM CODON USAGE OF % COLI &OR SIMPLIlCATION OF HANDLING THE RECOMBINANT PROTEIN WAS TRUNCATED OF THE MEMBRANE ANCHOR AND FURTHER INVESTIGATED AS 6 SULFOCYANIN 7ITH THAT IN HAND ITS FUNDAMENTAL PROPERTIES COULD BE DETERMINED &IGURE SHOWS THE VIS SPECTRUM IN THE OXIDIZED STATE TOGETHER WITH AN %02 SPECTRUM CLEARLY EXHIBITING THE TYPICAL #U HYPERlNE SPLITTING OF A TYPE ) COPPER LIGATION G\\ G AND THE HYPERlNE SPLIT TING IS SMALL !\\ Xn CM Mn )T APPEARS NEARLY AXIAL 4HE #$ SPECTRUM SHOWS A MINIMUM AT NM AND THE RELATIVE ` RATIO IS CALCULATED AS IN LINE WITH MOST OTHER BLUE COPPER PROTEINS 2EMARKABLY THE ABSORPTION SPECTRUM REMAINS UNCHANGED EVEN ON EXPOSURE TO TEMPERATURES ABOVE # REmECTING THE HIGH THERMOSTABILITY OF THE SULFOCYANIN FOLD 3ULFOCYANIN CAN BE REVERSIBLY REDUCED WITH ASCOR BATE DITHIONITE OR REDUCED CYTOCHROME C 2EDUCTION RESULTS IN THE LOSS OF THE NM ABSORPTION BAND !N EQUILIBRIUM TITRATION OF REDUCED CYTOCHROME C WITH OXIDIZED SULFOCYANIN ALLOWED THE DETERMINATION OF THE EQUILIBRIUM CONSTANT AT P( AS EQUIVALENT TO A HALF REDUCTION POTENTIAL OF % M6 3IMILAR TO HALOCYANIN THIS POTENTIAL SUGGESTS A CYTOCHROME C LIKE FUNCTION OF SULFOCYANIN 4HE P( DEPENDENCE OF THE REDOX POTENTIAL COULD NOT BE DETERMINED WITH THE APPLIED METHOD THOUGH IT APPEARS OF SIGNIlCANT
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TIES HAS BEEN EXPERIMENTALLY VERIlED SO FAR !CTUALLY THE SEQUENCE OF SULFOCYANIN REVEALS A NUMBER OF POSSIBLE GLYCOSYLATION DOMAINS &OR COMPARISON AN HIGH DEGREE OF GLYCOSYLATION HAS BEEN SHOWN FOR CYTOCHROME B ANOTHER ELECTRON CARRIER EXPOSED TO THE OUTER SURFACE OF THE 3ULFOLOBUS PLASMA MEMBRANE (ETTMANN ET AL :ÊHRINGER ET AL %)FNPQSPUFJOT
&IG /PTICAL TOP AND %02 BOTTOM SPECTRA OF THE OXIDIZED BLUE COPPER PROTEIN SULFOCYANIN FROM 3 ACIDOCALDARIUS 3PECTRA WERE PRODUCED WITH THE TRUNCATED 6 FORM OF THE RECOMBINANT PROTEIN 4HE DATA ARE TAKEN WITH PERMISSION FROM , +OMOROWSKI $ISSERTATION 5NIVERSITY OF ,UEBECK AND +OMOROWSKI AND 3CHÊFER
IMPORTANCE BECAUSE IT IS ASSUMED TO BE EXPOSED TO THE OUTER SURFACE OF THE PLASMA MEMBRANE )N MEMBRANE DENATURATING GELS SULFOCYANIN SHOWS A MOLECULAR MASS ^ K$A WHICH IS LIKELY TO RESULT FROM GLYCOSYLATION OR LIPID ASSOCIATION +OMOROWSKI AND 3CHÊFER ALTHOUGH NONE OF THESE POSSIBILI
-ANY THERMOACIDOPHILIC ARCHAEA WERE FOUND TO LACK C TYPE CYTOCHROMES "ESIDES BLUE COPPER PROTEINS ALSO NOVEL HEMOPROTEINS MIGHT TAKE ON THE FUNCTION OF CYTOCHROME C AS A LINKER BETWEEN LARGER ELECTRON TRANSPORT COMPLEXES !T LEAST TWO CANDIDATES COME INTO QUESTION /NE IS THE AS YET FUNCTIONALLY UNAS SIGNED CYTOCHROME B FROM 3 ACIDOCALDARIUS 4HIS HIGHLY GLYCOSYLATED MEMBRANE BOUND MONO HEME CYTOCHROME IS FACING THE OUTER SURFACE OF THE PLASMA MEMBRANE (ETTMANN ET AL 0REVIOUSLY IT WAS DISCUSSED THAT IT COULD BE INVOLVED IN REDOX REACTIONS OF A HYPOTHETICAL PERIPLASMATIC METABO LISM -ORE LIKELY HOWEVER CYTOCHROME B MAY FUNCTION AS A MEMBRANE lXED INTERMEDIATE ELECTRON CARRIER WITH A MOVABLE HEAD )N FACT BY %02 STUD IES IT HAS BEEN DEMONSTRATED THAT THE HEME BEARING EXTRINSIC DOMAIN OF THIS CYTOCHROME CAN ASSUME TWO DISTINCT LIMIT ORIENTATIONS IN THE NATIVE MEMBRANE 3CHOEPP #OTHENET ET AL OF 3 ACIDOCALDARIUS 4HE IMPLICATIONS FOR THE FUNCTION OF A BC ANALOGOUS NOVEL COMPLEX IS DISCUSSED BELOW !NOTHER CANDIDATE MIGHT BE THE MONO HEME CY TOCHROME B DESCRIBED AS MEMBER OF A RESPIRATORY SUPERCOMPLEX IN 3ULFOLOBUS SP STRAIN )WASAKI ET AL A )WASAKI AND /SHIMA NOW CLASSIlED AS 3ULFOLOBUS TOKODAII +AWARABAYASHI !S IT IS A MONO HEME CYTOCHROME A CYTOCHROME B LIKE FUNCTION IN A PROTON TRANSDUCING 1 CYCLE AS ORIGINALLY PROPOSED APPEARS UNCONCEIVABLE *7*T$PNQMFY*1SFTFOUJO"SDIBFB 4HE PROTON PUMPING .!$( DEHYDROGENASE OF MITO CHONDRIA AND MANY AEROBIC BACTERIA WAS PREVIOUSLY POSTULATED AS MISSING IN ARCHAEA 3CHÊFER ET AL 3CHÊFER !FTER THE FULL RESOLUTION OF SEV ERAL ARCHAEAL GENOMES THE ABOVE QUESTION HAS TO BE TAKEN UP AGAIN !CTUALLY THE lRST REPORTS ON RESPIRA TORY PROPERTIES OF HALOBACTERIA AND THERMOACIDOPHILIC 3ULFOLOBALES DEMONSTRATED THE PRESENCE OF .!$(
OXIDIZING ACTIVITIES IN THEIR MEMBRANES ,ANYI /SHIMA 7AKAGI AND 7AKAO ET AL (OWEVER LATER STUDIES USING %02 SPECTROSCOPY AND SITE SPECIlC INHIBITORS FAILED TO DEMONSTRATE THE PRES ENCE OF ANY OF THE IRON SULFUR SIGNALS TYPICAL FOR ENERGY TRANSDUCING ORTHODOX TYPE ) .!$( DEHYDROGENASES #OMPLEX ) FURTHERMORE IT HAS BEEN SHOWN THAT ARCHAEAL .!$( OXIDIZING SYSTEMS ARE INSENSITIVE TO ROTENONE AMYTAL OR PIERICIDINES )T IS NOTEWORTHY THAT HYBRIDIZATION ATTEMPTS MADE IN OUR LABORATORY WITH ARCHAEAL $.! USING .$( ) DIRECTED PROBES FAILED AS WELL UNPUBLISHED RESULTS 4HE PRESENT DAY SITUATION IS UNCHANGED AND THEREFORE ONE HAS TO ASSUME THAT INDEED A PROTON PUMPING COMPLEX ) IS ABSENT FROM ARCHAEAL RESPIRATORY SYSTEMS 4HIS MAY APPEAR UNLIKELY IN VIEW OF THE POSITIVE HITS OF TENTATIVE GENE ASSIGNMENTS IN RECENTLY SEQUENCED ARCHAEAL GENOMES (OWEVER IN NONE OF THESE CASES THE PROTEIN CHEMICAL OR FUNCTIONAL EQUIVALENT OF A COMPLEX ) HAS BEEN DEMONSTRATED )N THAT CONTEXT ONE HAS TO KEEP IN MIND THAT A SURPRISING STRUCTURAL AND TOPOLOGICAL RELATION OR EVEN HOMOLOGY EXISTS BETWEEN COMPONENTS OF COMPLEX ) AND HYDROGENASES !LBRACHT AND (EDDERICH &RIEDRICH -OREOVER ONLY SINGLE HOMOLOGOUS GENES HAVE BEEN ASSIGNED BUT IN NO CASE THE PRESENCE OF A FULL SET OF UP TO &RIEDRICH GENES FOR COMPLETION OF A FUNCTIONAL COMPLEX ) HAVE BEEN IDENTIlED )N EVOLUTION THE MODULAR ASSOCIATION OF INITIALLY SEPA RATE MEMBRANE REDOX COMPONENTS AND PERIPHERAL DEHYDROGENASE MODULES WITH TRANSPORT MODULES MAY HAVE GIVEN RISE TO THE FORMATION OF BOTH HYDROGENASES AND .!$( DEHYDROGENASES !LSO THE TYPICAL OPERON STRUCTURE OF BACTERIAL COMPLEX ) GENES IS NOT VERIlED IN !RCHAEA 4HUS THE ABOVE STATEMENT STANDS AS LONG AS THE FUNCTION OF THE TENTATIVELY ASSIGNED GENES HAS NOT BEEN SHOWN ! HINT TO THE FUNCTION OF PROTEINS ENCODED BY THE NEWLY DETECTED GENES MAY BE DERIVED FROM OUR KNOWL EDGE ON THE MODULAR DESIGN OF COMPLEX ) &RIEDRICH 4HREE MODULES ARE ESSENTIAL OF WHICH THE TWO MEMBRANE INTEGRAL FRAGMENTS CONTAIN THE IRON SULFUR HOSTING DEVICE AND COUPLING THE REDOX PROCESS TO PROTON TRANSLOCATION AND THE MODULE TRANSFERRING ELECTRONS TO A QUINONE (OMOLOGUES TO THOSE HAVE BEEN IDENTIlED FOR EXAMPLE IN ( SALINARUM .G ET AL 0FEIFFER AND /ESTERHELT 7HAT GEN ERALLY IS MISSING IS THE PERIPHERAL MODULE RECEIVING ELECTRONS FROM .!$( 4HUS IT MAY BE PROPOSED AS A WORKING HYPOTHESIS THAT THIS MODULE COULD BE REPLACED IN THE RESPECTIVE ARCHAEA BY A DIFFERENT REDOX DEVICE
(ÛOUFS4DIÅGFS USING OTHER DONORS THAN .!$( &ERREDOXINS MIGHT BE SUITABLE CANDIDATES 4HE ABUNDANCE OF FERREDOXINS IN ARCHAEA SHOULD CHALLENGE FURTHER EXPERIMENTAL INVESTIGATION ON THIS ISSUE ESPECIALLY BECAUSE IRON SULFUR CENTERS CAN LINK THE ELECTRON mOW FROM THE SUBSTRATE OXIDIZING MODULE TO THE MEMBRANE CORE OF THE WHOLE MACHINERY ALSO IN THE KNOWN COMPLEX ) ARCHITECTURES )N CONTRAST THE ABILITY OF AEROBIC ARCHAEA TO OXIDIZE .!$( BY TYPE )) DEHYDROGENASES WITHOUT COUPLED PROTON TRANSLOCATION HAS BEEN REPEATEDLY DEMON STRATED AND SOME OF THE RESPECTIVE ENZYMES COULD BE CHARACTERIZED 4HIS IMPLIES HOWEVER THAT ARCHAEAL RESPIRATORY CHAINS IN GENERAL ARE LACKING ONE POSSIBLE ENERGY COUPLING SITE !N EARLY REPORT DESCRIBES THE ISOLATION OF AN .!$(ACCEPTOR OXIDOREDUCTASE FROM 3ULFOLOBUS SP STRAIN NOW 3 TOKODAII 7AKAO ET AL 4HE WATER SOLUBLE DIMERIC ENZYME K$A CONTAINS TWO &!$MOLECULE AND MAY REPRESENT THE PERIPHERAL COMPONENT OF A LARGER MEMBRANE BOUND TYPE ) .!$( DEHYDROGENASE IE WITHOUT COUPLING THE REACTION TO PROTON PUMPING 4HIS PREPARATION HAD VIRTUALLY NO AC TIVITY WITH CALDARIELLA QUINONE AS HYDROGEN ACCEPTOR ! SIMILAR ENZYME K$A WAS lRST DESCRIBED FROM !CIDIANUS AMBIVALENS 'OMES AND 4EIXEIRA AND HAS INITIALLY BEEN CONSIDERED TO ACT AS AN .!$( FERREDOXIN OXIDOREDUCTASE )N THIS LATTER ORGANISM THE SAME LABORATORY REPORTED RECENTLY THE IDENTIlCATION OF A NEW TYPE )) .!$( DEHYDROGENASE WHICH WAS RECONSTITUTED IN LIPOSOMES TO FORM AN ARTIlCIAL RESPIRA TORY CHAIN TOGETHER WITH THE TERMINAL AA TYPE QUINOL OXIDASE 'OMES ET AL B 4HE ENZYME K$A HAS NO IRON SULFUR CLUSTERS IT REACTS RAPIDLY WITH CALDARIELLA QUINONE NMOLMINMG PROTEIN AND IT CAN TRANSFER ELECTRONS ALSO TO SYNTHETIC QUINONES WITH A +- FOR .!$( OF ^ - .OTABLY THIS IN VITRO SYSTEM RESEMBLES THE PREVIOUSLY POSTULATED MINIMAL RESPIRATORY CHAIN OF PRIMORDIAL AEROBIC ORGANISMS 3CHÊFER ET AL A (YDROXYQUINOLINE . OXIDE (1./ IS AN INHIBITOR OF TYPE )) .!$( DEHYDROGE NASES 4HE .!$( OXIDIZING ACTIVITY OF MEMBRANES FROM (ALOBACTERIUM SALINARUM HAS BEEN SHOWN TO BE DUE TO A TYPE )) .!$( DEHYDROGENASE WHICH IS SENSITIVE TO THIS INHIBITOR 3REERAMULU ET AL )N CONTRAST TO 3ULFOLOBUS THE MEMBRANE BOUND ENZYME IS CAPABLE TO TRANSFER ELECTRONS TO CYTOCHROME C AS AN ARTIlCIAL ACCEPTOR )N CONCLUSION TO THE BEST OF OUR KNOWLEDGE AEROBIC ARCHAEA POSSESS ONLY TYPE )) .!$( DEHYDROGENASES WHICH NOT LINKED TO PROTON PUMPING ! PECULIARITY
$IBQUFS "SDIBFBM3FTQJSBUJPO IS THE EXISTENCE OF A (/ (/ FORMING .!$( OXIDASE IN THE OBLIGATE ANAEROBE 0YROCOCCUS FURIOSUS 7ARD ET AL WHICH PRESUMABLY FUNCTIONS AS AN OXYGEN SCAVENGER 7"SDIBFBM$PNQMFY**&RVJWBMFOUT 423 2'3
2EVERSIBLE SUCCINATE DEHYDROGENASE 3$( ACTIVITIES HAVE BEEN UBIQUITOUSLY DETECTED IN ORGANISMS FROM THE THREE DOMAINS OF LIFE 4HEY REPRESENT CONSTITUENTS EITHER OF RESPIRATORY COMPLEX )) IN AEROBES OR OF FUMARATE REDUCTASES COMPLEXES IN ANAEROBES !R CHAEAL SUCCINATEQUINONE OXIDOREDUCTASES HAVE BEEN COMPARATIVELY ANALYZED EXTENSIVELY IN A RECENT REVIEW 3CHÊFER ET AL THEREFORE ONLY THE MOST NOTABLE PECULIARITIES OF !RCHAEAL 312S WILL BE DISCUSSED HERE 4HESE ARE THE PRESENCE OF UNUSUAL IRON SULFUR CLUSTERS IN SUBUNIT " OF SUCCINATE DEHYDROGENASES FROM THER MOACIDOPHILIC CREN ARCHAEOTA THE OCCURRENCE OF AN UNUSUAL CYSTEINE RICH SEQUENCE MOTIF IN SUBUNIT # AND THE LACK OF TYPICAL MEMBRANE ANCHORS OR MEM BRANE SPANNING HELICES IN THE SMALL SUBUNITS 4HESE OBSERVATIONS TAKEN TOGETHER JUSTIFY AN ASSIGNMENT AS A NOVEL TYPE OF SUCCINATE DEHYDROGENASES 4HE OPERON STRUCTURE OF !RCHAEAL 3$(S USUALLY FOLLOWS THE ORDER OF GENES FOR THE FOUR SUBUNITS SDH! SDH" SDH# SDH$ WITH EXCEPTION OF .ATRONOMONAS PHARAONIS RESEMBLING THE % COLI ORDER # $ ! " AND ( SALINARUM WITH SDH$ PRECEDING SDH# 0OLYPEPTIDE ! IS THE CENTRAL SUBSTRATE AND PRIMARY ACCEPTOR BINDING LARGE SUBUNIT CONTAINING THE CANONI CAL &!$ IN ALL 312 1&2 COMPLEXES 0OLYPEPTIDE " CONTAINS &E3 CLUSTERS AS A LINK TO FURTHER ELEC TRON ACCEPTORS OR DONORS 4HE SMALL POLYPEPTIDES # AND $ USUALLY SERVE AS MEMBRANE ANCHORS FOR THE COMPLEX AND IN SOME CASES CONTAIN FUNCTIONALLY IMPORTANT HISTIDINE RESIDUES 7HEN PRESENT THESE SERVE AS HEME LIGANDING RESIDUES FOR B TYPE CYTO CHROMES BURIED BETWEEN THE MEMBRANE SPANNING _ HELICES 3UCH HEME BINDING SITES WERE IDENTIlED IN COMPLEX )) FROM 4 ACIDOPHILUM ( SALINARUM 0 AEROPHILUM . PHARAONIS BUT ARE MISSING FROM THE HYPERTHERMO ACIDOPHILIC ARCHAEAL SPECIES 3ULFOLOBUS AND !CIDIANUS 4HE RECENTLY ACHIEVED HIGH RESOLUTION $ STRUCTURE OF 1&2 FROM 7OLINELLA SUCCINOGENES ,ANCASTER ET AL CAN SERVE AS A VERY GENERAL AND BASIC STRUC TURAL MODEL ALSO FOR COMPLEX )) DUE TO THEIR COMMON EVOLUTIONARY ORIGIN AND THE OVER ALL SIMILARITY OF BOTH
MEMBRANE RESIDING REDOX SYSTEMS SEE ALSO #HAPTER BY ,ANCASTER 6OL "5IF'F4$MVTUFST &ROM !RCHAEAL SPECIES WITH 312 ACTIVITY OR GENETIC EVIDENCE FOR A COMPLEX )) ONLY PARTIALLY OR ENTIRELY PURIlED PREPARATIONS HAVE BEEN DESCRIBED -OLL )WASAKI ET AL B *ANSSEN ET AL 3CHARF ET AL ,EMOS ET AL )N VIVO THE ABOVE MENTIONED !RCHAEAL QUINONES SERVE AS A TERMINAL ELECTRON ACCEPTOR AND ALSO THE REDUC TION OF B CYTOCHROMES BY SUCCINATE HAS BEEN SHOWN VERY EARLY IN !RCHAEAL MEMBRANES (ALLBERG AND (EDERSTEDT !NEMàLLER ET AL 3URPRIS INGLY A QUINONE REDUCTASE ACTIVITY WAS NOT PRESERVED IN ALL PURIlED PREPARATIONS AS FOR EXAMPLE FROM 3 ACIDOCALDARIUS -OLL )N SEVERAL !RCHAEAL MEMBRANES 312 IS SO ABUN DANT THAT THE TYPICAL IRON SULFUR CENTERS 3 3 AND 3 COULD BE STUDIED BY %02 SPECTROSCOPY ALREADY IN THE INTACT MEMBRANE !NEMàLLER ET AL 'OMES ET AL 3PECIlC !RCHAEAL DEVIATIONS FROM THE USUAL CLUSTER STRUCTURE WERE IDENTIlED IN 3 ACIDO CALDARIUS *ANSSEN ET AL AND ! AMBIVALENS 'OMES ET AL ,EMOS ET AL 'ENERALLY IT WAS ASSUMED THAT SUBUNIT " HOSTS ;&E 3= ;&E 3= AND ;&E 3= CLUSTER !S SHOWN BY THE SEQUENCE ALIGNMENT OF &IG IN A NUMBER OF CASES INCLUDING THE RELATED THIOL FUMARATE REDUCTASE FROM -ETHANOCOCCUS JANNASHII AN ADDITIONAL CYSTEINE WAS FOUND AT THE BINDING DOMAIN OF CLUSTER 3 )N FACT %02 SPECTRA OF 312S FROM 3 ACIDOCALDARIUS AND ! AMBIVALENS REVEALED THE PRESENCE OF AN ADDITIONAL ;&E 3= CLUSTER REPLACING THE CLASSICAL 3 CLUSTER 4HIS ALTERED MOTIF IS ALSO FOUND IN - THERMOAUTO TROPHICUM AS WELL AS IN BACTERIAL FUMARATE REDUCTASE SUBUNITS &RD" FROM !QUIFEX AEOLICUS 3YNECHOCYSTIS SP AND #AMPYLOBACTER JEJUNI 3CHÊFER ET AL &OR FURTHER DETAILS THE READER IS REFERRED TO THE SPECIAL IZED REVIEW 3CHÊFER ET AL #"/PWFM4FRVFODF.PUJG !NOTHER IRREGULARITY AS COMPARED TO @CLASSICAL COM PLEX )) IS THE APPEARANCE OF AN UNUSUAL CYSTEINE MOTIF IN SUBUNIT # OF 312S FROM THERMOACIDOPHILIC 3ULFOLOBALES )T HAS NO EQUIVALENT IN ANY OTHER 312 OR 1&2 BUT HAS BEEN FOUND ALSO IN POLYPEPTIDES " OF THIOL FUMARATE REDUCTASES 4&2 FROM METHANOGENIC !RCHAEA AND ALSO IN A SMALL NUMBER OF COMPLETELY
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&IG 3EQUENCE ALIGNMENTS OF THE THREE IRON BINDING SITES ) )) AND ))) IN SUBUNIT " FROM ARCHAEAL 3121&2 COMPLEXES 4HE BOXED RESIDUES INDICATE THE ADDITIONAL CYSTEINE OF THE SECOND ;&E 3= CLUSTER 3PECIES ABBREVIATIONS 3ACID 3 ACIDOCALDARIUS 3SOLF 3 SOLFATARICUS !AMBI ! AMBIVALENS 3TOKO 3 TOKODAII -JANN -ETHANOCOCCUS JANNASCHII 4HE LATTER SEQUENCE REFERS TO SUBUNIT " OF THE THIOL FUMARATE REDUCTASE OF - JANNASCHII 3EQUENCES ARE TAKEN FROM GENE BANK
DIFFERENT AND FUNCTIONALLY UNRELATED OXIDOREDUCTASES INCLUDED IN THE ALIGNMENT OF &IG 4HIS MOTIF OCCURS AS A TANDEM WITH THE GENERAL SIGNATURE n'# 8 ##'0 8 #XX# REPEAT ) 8 '0 # 8 ##' 8 #88# REPEAT )) THE LATTER EXAMPLE IS TAKEN FROM 3 ACIDOCALDARIUS4HE SECOND REPEAT WITHIN THIS TANDEM CONTAINS A STRICTLY CONSERVED GLYCIN PRECEDING THE ##' MOTIF BY RESIDUES %02 SPECTROSCOPY OF ISOLATED 3 ACIDOCALDARIUS COMPLEX )) GAVE NO HINT TO THE EXISTENCE OF ADDITIONAL IRON CLUSTERS INVOLVING THESE CYSTEINES 4HEREFORE A POSSIBLE INVOLVEMENT IN DISULlDE BONDS OR IN REDOX REACTIONS WITH PARTICIPATION OF THIOL RADICALS HAS BEEN DISCUSSED *ANSSEN ET AL !CTUALLY IT SHOULD BE EMPHASIZED THAT ISOLATED 3$( FROM 3 ACIDOCALDARIUS WHICH BELONGS TO THIS NOVEL 312 TYPE IS STRONGLY INHIBITED BY THE STABLE RADICAL FORMING TETRA CHLORO BENZOQUINONE -OLL SUGGESTING DIRECT INTERACTION WITH THE INHIBITOR OF A FREE RADICAL INVOLVED IN THE ELECTRON TRANSPORT REACTION ! RECENT STUDY WITH SUBUNIT # FROM 3 TOKODAII PRODUCED RECOMBINANTLY IN % COLI REVEALED HOWEVER THAT IT CONTAINS A NEW ;&E3= CLUSTER COMPLETELY LIGANDED BY CYSTEINES AS WELL AS AN ISOLATED ZINC SITE )WASAKI ET AL 4HESE NEW METAL CENTERS WERE CHARACTERIZED BY %02 AND %8!& SPECTROSCOPY 4HE ;&E3= CENTER IS THOUGHT TO REPRESENT AS A NOVEL ELEC TRON ENTRY SITE FROM 3$( TO THE RESPIRATORY CHAIN OF THE ORGANISM )N THIS CONTEXT IT IS OF INTEREST THAT THE CHEMICALLY DETERMINED &E CONTENT OF ISOLATED COM PLEX )) FROM 3 ACIDOCALDARIUS SIGNIlCANTLY EXCEEDED THE AMOUNT DETERMINED BY %02 QUANTIlCATION -OLL
4HUS IN THESE PREPARATIONS THE NOVEL CENTER # MIGHT HAVE LOST ITS INTEGRITY THAT COULD ALSO EXPLAIN THE OBVIOUS INACTIVITY OF THESE PREPARATIONS WITH THE NATURAL QUINONE AS ELECTRON ACCEPTOR ! PHYLOGENETIC ANALYSIS HAS SUGGESTED A COEVO LUTION OF SUBUNITS ! AND " OF 312S 3CHÊFER ET AL )NTERESTINGLY IN AN UNROOTED TREE OF THE IRON SULFUR HOSTING SUBUNIT " &IG THE ARCHAEAL DOMAIN ORIGINATES FROM A COMMON BRANCH POINT SPLITTING INTO SUBDOMAINS SHADED AREA 4HESE SUBDOMAINS CLEARLY REmECT THE DIVISION INTO @CLASSICAL COMPLEXES BEARING THE HEME LIGANDING HISTIDINES IN THEIR SMALL SUBUNITS AND THE @NOVEL TYPE SHARING THE UNUSUAL CYSTEINE MOTIF AS WELL AS THE ;&E 3= CLUSTER INSTEAD OF A REGULAR 3 7*%PFTB CD$PNQMFY&YJTUJO"SDIBFB 'ENERALLY QUINOLCYTOCHROME C OXIDOREDUCTASES COMPLEX ))) OF RESPIRATORY CHAINS ARE FUNCTIONING AS EFlCIENT REDOX DRIVEN PROTON PUMPS BASED ON A SOPHISTICATED MECHANISM TERMED AS 1 CYCLE -ITCH ELL 4RUMPOWER 4ALFOURNIER ET AL 4HE ESSENTIAL COMPONENTS ARE THE 2IESKE ;&E 3= IRON SULFUR PROTEIN B TYPE CYTOCHROMES AND AN INTERMEDIATE ELECTRON ACCEPTOR #YT C BETWEEN THIS CORE COMPLEX AND CYTOCHROME C 4HUS ANSWERING THE ABOVE QUESTION HAS TO RELY ON THE PROOF OF WHETHER OR NOT THE RESPECTIVE CONSTITUENTS ARE PRESENT IN !RCHAEA !CTUALLY THE ANSWER CAN ONLY BE BOTH @YES AND @NO FOR SEVERAL REASONS @YES BECAUSE FOR A NUMBER OF
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&IG 0ARTIAL SEQUENCE ALIGNMENT OF POLYPEPTIDES BEARING THE UNUSUAL CYSTEINE MOTIF /NLY THE lRST REPEAT IS SHOWN 3EQUENCES ARE FROM SUCCINATE DEHYDROGENASES SUBUNIT # 3DH# HETERODISULlDE REDUCTASES SUBUNIT " (DR" THIOL FUMARATE REDUCTASE SUBUNIT "4FR" 'LYCEROL PHOSPHATE DEHYDROGENASE AND GLUCOSE OXIDASE THE LATTER TWO SEQUENCES ARE FROM % COLI FOR COMPARISON 4HE AR CHAEAL SEQUENCES ARE FROM 3ACID 3 ACIDOCALDARIUS 3SOLF 3 SOLFATARICUS !AMBI ! AMBIVALENS -THER - THERMOAUTOTROPHICUM -JANN - JANNASCHII 3TOKO 3 TOKODAII !FULG !FULGIDUS 4HE SEQUENCES WERE EXTRACTED FROM GENE BANK .UMBERS IN COLUMN INDICATE THE STARTING POSITION WITHIN THE RESPECTIVE SEQUENCE
ARCHAEA THE EXISTENCE OF ONE OR EVEN TWO RESPIRATORY 2IESKE &E3 PROTEINS HAS BEEN DEMONSTRATED AND @NO BECAUSE THE ASSOCIATION OF THESE 2IESKE PROTEINS WITH CYTOCHROMES DEVIATES SIGNIlCANTLY FROM ORTHODOX BC COMPLEXES 4HOUGH IN #RENARCHRAEOTA LIKE THE THERMOACIDOPHILIC 3ULFOLOBALES 2IESKE PROTEINS WERE DETECTED SPECTROSCOPICALLY !NEMàLLER ET AL ,àBBEN ET AL A 'OMES ET AL THE COM PLETE ABSENCE OF C TYPE CYTOCHROMES IS INCOMPATIBLE WITH THE EXISTENCE OF A REGULAR BC COMPLEX &URTHER THE USUALLY ASSOCIATED DI HEME CYTOCHROME IS HOST ING HEME !3 !LSO THE TERMINAL ELECTRON ACCEPTOR C IS APPARENTLY REPLACED FOR EXAMPLE BY A BLUE COPPER PROTEIN OR ANOTHER CYTOCHROME !ND lNALLY THE SEN SITIVITY AGAINST SPECIlC INHIBITORS LIKE ANTIMYCIN ! OR MYXOTHIAZOL COMMONLY CONSIDERED AS DISCRIMINATING INHIBITORS FOR 2IESKEBC COMPLEXES IS ABSENT 4HE SITUATION APPEARS LESS DIVERGENT WITH EURYARCHAEOTA FOR EXAMPLE THE (ALOBACTERIALES SINCE BOTH B (ALLBERG 'RADIN AND "ALTSCHEFFSKY (ALLBERG 'RADIN AND #OLMSJÚ +UHN AND 7ARD 4ANAKA ET AL AND C TYPE CYTOCHROMES HAVE BEEN FOUND AS WELL AS %02 SPECTROSCOPIC EVIDENCE FOR A 2IESKE PROTEIN HAS BEEN DEMONSTRATED 3REERAMULU ET AL &IGURE SHOWS SPECTRA OF A PARTIALLY ENRICHED MEMBRANE FRACTION FROM ( SALINARUM
STRAIN *7 STRONGLY SUGGESTING THE PRESENCE OF A BC LIKE COMPLEX ADDITIONAL SUPPORT COMES FROM THE REPORTED SENSITIVITY TO ANTIMYCIN &ROM NONE OF THE ARCHAEAL ORGANISMS A FUNCTIONAL COMPLEX COULD BE ISOLATED SO FAR (OWEVER A FUNCTIONAL EQUIVALENT TO A RESPIRATORY COMPLEX ))) IS OBVIOUSLY INCLUDED IN THE SUPERCOMPLEX 3OX- AS TO BE DISCUSSED IN A SUBSE QUENT PARAGRAPH )N THE FOLLOWING SINGLE CONSTITUENTS OF POSSIBLE ARCHAEAL EQUIVALENTS TO COMPLEX ))) ARE DISCUSSED AS WELL AS THEIR FUNCTIONAL SIGNIlCANCE " "SDIBFBM3JFTLF1SPUFJOT 4HE UNIQUE ELECTRONIC STRUCTURE OF THE TYPICAL &E3 CLUSTER IN RESPIRATORY 2IESKE PROTEINS IS GENERATED BY THE MIXED LIGANDATION OF TWO &E IONS BY TWO HISTIDINES ON ONE SIDE AND TWO CYSTEINES ON THE OTHER WITH TWO SULFUR IONS BRIDGING THE &E IONS 4HIS COORDINATION GIVES RISE TO A CHARACTERISTIC %02 SPECTRUM WHICH IS STRONGLY DIFFERENT FROM THOSE OF OTHER IRON SULFUR CLUSTERS 4HE HIGHLY RESOLVED 8 RAY STRUCTURE OF MITOCHONDRIAL BC COMPLEXES COULD BE DETERMINED 8IA ET AL "ERRY ET AL (UNTE REVEALING TWO OR EVEN THREE -ONTOYA ET AL QUINOLQUINONE BINDING SITES AND THE SURPRISING FACT THAT THE PRIMARY ELECTRON ACCEPTOR FOR QUINOL
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&IG 5NROOTED PHYLOGENETIC TREE OF THE mAVIN HOSTING POLYPEPTIDES ! OF 312S AND 1&2S 4HE !RCHAEA ARE CLUSTERING IN TWO MAJOR BRANCHES SHADED ONE COMPRISING THOSE ORGANISMS WITH REGULAR LIGANDS FOR THE 3 ;&E 3= CLUSTER THE OTHER COMPRISING THE NOVEL TYPE WITH A SECOND ;&E 3= CLUSTER !LL OTHER NON ARCHAEAL SEQUENCES CONTAIN ALSO THE @REGULAR TYPE !LL SEQUENCE DATA EXTRACTED FROM GENE BANK AND THE ARCHAEAL GENOME PROJECTS
OXIDATION THE 2IESKE PROTEIN ACTS AS A REORIENTING REDOX DOMAIN BETWEEN THE SITE OF ELECTRON INPUT AND THE ACCEPTOR CYTOCHROME C 3NYDER ET AL 4HE SECOND ELECTRON OF A QUINOL IS TRANSFERRED TO THE B TYPE CYTOCHROME FOR FURTHER MECHANISTIC DETAILS SEE 8IA ET AL 9U ET AL $ARROUZET ET AL 'UTIERREZ #IRLOS AND 4RUMPOWER SEE ALSO #HAPTER BY #OOLEY ET AL 6OL 4HE lRST ARCHAEAL RESPIRATORY 2IESKE PROTEIN WAS PURIlED FROM 3 ACIDOCALDARIUS 3CHMIDT ET AL 3URPRISINGLY A SECOND 2IESKE PROTEIN WAS DETECTED IN THE SAME ORGANISM AS A CONSTITUENT OF THE 3OXRESPIRATORY COMPLEX #ASTRESANA ET AL 3CHMIDT ET AL 4HESE THE 3OX, AND 3OX& 2IESKE
PROTEINS WERE NAMED ACCORDING TO THEIR RESPECTIVE GENES "OTH COULD BE PRODUCED HETEROLOGOUSLY AND THEIR REDOX POTENTIALS REDOX LINKED P+A VALUES AND DETAILED SPECTROSCOPIC PROPERTIES WERE DETERMINED !NEMàLLER ET AL B "OEKEMA ET AL 3HIMA ET AL &ROM THE CLONED GENE ALSO THE 2IESKE PROTEIN FROM THE NEUTROPHILIC AND HYPERTHERMOPHILIC CRENARCHAEON 0YROBACULUM AEROPHILUM COULD BE PRODUCED (ENNINGER ET AL 4HE ONLY OTHER ARCHAEAL 2IESKE PROTEINS IDENTIlED IN NATIVE MATERIAL ARE THAT FROM 3ULFOLOBUS SP STRAIN NOW RENAMED 3 TOKODAII )WASAKI ET AL A AND THAT FROM (ALOBACTERIUM SALINARUM $3- STRAIN *7 IN A PARTIALLY ENRICHED FORM 3REERAMULU ET AL
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&IG 3PECTROSCOPIC EVIDENCE FOR A BC COMPLEX IN ( SALINA RUM 4HE SPECTRA WERE PRODUCED WITH A PARTIALLY PURIlED FRACTION OF SOLUBILIZED PLASMA MEMBRANES 4OP %02 SPECTRUM OF THE ASCORBATE REDUCED PREPARATION CLEARLY INDICATING THE PRESENCE OF A 2IESKE LIKE &E3 CENTER "OTTOM &IRST DERIVATIVE OPTICAL SPECTRUM OF THE SAME ASCORBATE REDUCED FRACTION INDICATING THE PRESENCE OF BOTH B AND C TYPE CYTOCHROMES $ETAILS ARE GIVEN IN THE TEXT AND 3REERAMULU ET AL
!LSO IN MEMBRANE PREPARATIONS FROM 3 METALLICUS CLEAR EVIDENCE FOR A 2IESKE TYPE PROTEIN EMERGED FROM %02 SPECTRA WHICH IN ADDITION INDICATE A NOVEL &E3 CENTER WITH HIGH REDUCTION POTENTIAL M6 REMINISCENT OF ()0)0 IRON SULFUR PROTEINS 'OMES ET AL 4ABLE GIVES A SURVEY OF EXPERIMENTAL DATA FOR ARCHAEAL 2IESKE PROTEINS ! NUMBER OF FURTHER !R CHAEAL 2IESKE PROTEINS IS ALSO SUGGESTED FROM THE RECENTLY COMPLETED AND ONGOING GENOME PROJECTS INCLUDING ( SALINARUM ( MARISMORTUI !E PERNIX AND ! AMBIVALENS ! COMPARISON OF ARCHAEAL 2IESKE PROTEINS WITH THEIR BACTERIAL AND EUKARYAL COUNTERPARTS REVEALS THAT !RCHAEAL 2IESKE PROTEINS ARE USUALLY MUCH LARGER AND ARE BEARING AN EXTENDED INSERTION BETWEEN THE IRON CLUSTER BINDING SITES &OR ILLUSTRA
TION A TYPICAL PARTIAL ALIGNMENT IS SHOWN IN &IG ! COMPREHENSIVE REVIEW ON SEQUENCE FEATURES AND THE PHYLOGENETIC RELATION OF 2IESKE PROTEINS 2IESKE TYPE PROTEINS AND 2IESKE LIKE FERREDOXINS IS GIVEN IN 3CHMIDT AND 3HAW 4HE ONLY $ STRUCTURE WITH ATOMIC RESOLUTION OF AN !RCHAEAL 2IESKE PROTEIN HAS RECENTLY BEEN DETERMINED FOR 3OX& FROM 3 ACIDOCALDARIUS THE . TERMINAL MEMBRANE ANCHOR HAS BEEN TRUNCATED "ÚNISCH ET AL "ÚNISCH ET AL 4HIS ¯ STRUCTURE GIVES A HINT ON THE STRUCTURAL DIFFERENCES IMPOSED BY THE LARGE SEQUENCE INSERTION SEE &IG AS DEMONSTRATED IN &IG !N ADDITIONAL ` SHEET `# IS INSERTED BETWEEN NORMALLY PRESENT ` AND ` AS WELL AS AN ADDITIONAL DISULlDE BRIDGE BETWEEN #YS AND #YS 4HIS ENABLES A lRM lXATION OF THE # TERMINUS TOGETHER WITH THE ( BOND INTERACTION BETWEEN ` ` AND MAY CONTRIBUTE TO THE ENORMOUS HEAT STABILITY )MPORTANTLY THE . TERMINUS AND THE ADJACENT HINGE REGION NECESSARY FOR A LARGE SCALE DOMAIN MOVEMENT )WATA ET AL ARE NOT lXED IN THE SAME WAY &URTHER A HELIX NOT FOUND IN OTHER KNOWN 2IESKE PROTEINS )WATA ET AL 8IA ET AL (UNTE IS PRESENT IN THIS !RCHAEAL PROTEIN &OR A DETAILED STRUCTURAL ALIGNMENT THE READER IS REFERRED TO $ARROUZET ET AL )N COMPARISON TO THE !RCHAEAL 2IESKE PROTEIN THOSE FROM SPINACH B F OR BOVINE F BC COMPLEXES DISPLAY A MUCH LOWER PACKING DENSITY OF THEIR $ STRUCTURE #"TTPDJBUFEC$ZUPDISPNFTBOEB/PWFM $PNQMFY &OR A DECISIVE ANSWER TO THE ABOVE QUESTION THE ARCHAEAL 2IESKE PROTEINS HAVE TO BE SEEN IN A MORE GENERAL CONTEXT ALONG WITH THEIR PROTEIN CHEMICAL AND GENETIC ENVIRONMENT !GAIN THE BEST INVESTIGATED EXAMPLE IS THE THERMOACIDOPHILIC ARCHAEON 3ULFOLOBUS ACIDOCALDARIUS WHERE GENETIC TRANSCRIPTIONAL PRO TEIN CHEMICAL AND FUNCTIONAL STUDIES ARE SUPPORTING EACH OTHER %ARLIER A MONO HEME B TYPE CYTOCHROME FROM 3 ACIDOCALDARIUS ;#YT B= HAS BEEN DESCRIBED TOGETHER WITH ITS GENES CBS! AND CBS" (ETTMANN ET AL :ÊHRINGER ET AL 4HE FUNCTION OF THIS HIGHLY GLYCOSYLATED PROTEIN WAS NOT KNOWN 4HIS CYTOCHROME IS AN INTEGRAL MEMBRANE PROTEIN WITH ITS HEME BEARING DOMAIN FACING THE OUTER SURFACE OF THE PLASMA MEMBRANE IT IS ABUNDANT IN 3ULFOLOBUS MEMBRANES GROWING UNDER LIMITED OXYGEN SUPPLY 4HE DISCOVERY THAT IT IS PART OF A GENE CLUSTER ALSO
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4ABLE #OMPARATIVE LIST OF AUTHENTIC AND RECOMBINANT ARCHAEAL 2IESKE PROTEINS INCLUDING A FEW CASES OF 2IESKE PROTEINS IDENTIlED ONLY IN SITU BY %02 SPECTROSCOPY 4HE DATA WERE EXTRACTED FROM THE LITERATURE QUOTED IN THE TEXT % Mn P+A %02 CHARACTERISTICS /RGANISM 0ROTEIN AMINO -W AUTHRECOMB ACIDS K$A M6 GZ GY GX 3 ACIDOCALDARIUS 3OX& AUTH 3 ACIDOCALDARIUS
3OX, MATURE PRECURSOR
3 ACIDOCALDARIUS
3OX& RECOMB
n
n
3 ACIDOCALDARIUS
3OX& RECOMB 6n
n
3 ACIDOCALDARIUS
3OX, RECOMB
n
3 METALLICUS
IN MEMBRANE
n
n
n
0 AEROPHILUM
0AR2 AUTH
n
n
ND
0 AEROPHILUM
0AR2 RECOMB 6
0 AEROPHILUM
0A2 RECOMB 6n&7
n
n
n
n
( SALINARUM
IN MEMBRANE
n
n
n
n
ND
3 TOKODAII
IN MEMBRANE
n
n
ND
ND
n
n
3 TOKODAII
SOL FRAGMT
&IG #OMPARATIVE PARTIAL ALIGNMENT OF THE CLUSTER BINDING DOMAINS FROM ARCHAEAL 2IESKE PROTEINS 3EVERAL EXHIBIT A LONG INSERTION AS COMPARED TO THE MITOCHONDRIAL OR CHLOROPLAST 2IESKE PROTEINS 4HE SEQUENCES FROM 0 AEROPHILUM AND ( SALINARUM HAVE AN ADDITIONAL LONG INSERTION 8 AND 8 BETWEEN THE SECOND CLUSTER BINDING MOTIF AND THE PROLINE LOOP 3OX, AND 3OX& ARE BOTH FROM 3 ACIDOCALDARIUS SEE TEXT 5NUSUAL IS THE PRESENCE OF AN ASPARTATE IN CLUSTER BINDING MOTIF OF THE 3OX, PROTEIN )NTERESTINGLY THE SEQUENCE FROM ! ACIDIANUS REVEALS OVER ALL IDENTITY TO 3OX, FROM 3ULFOLOBUS
ENCODING THE 2IESKE PROTEIN 3OX, TOGETHER WITH A NOT YET PURIlED B TYPE CYTOCHROME 3OX. AND A SMALL SOLUBLE PROTEIN STRONGLY SUGGESTS A FUNCTIONAL COORDINATION OF THE RESPECTIVE GENE PRODUCTS INTO A NOVEL COMPLEX TENTATIVELY TERMED THE 3OX,. COM PLEX &IGURE ILLUSTRATES THE GENE ORDER TOGETHER WITH HYDROPHOBICITY PROlLES #YTOCHROME 3OX. CLEARLY
REVEALS HEME BINDING SITES BETWEEN THE PUTATIVE TRANSMEMBRANE HELICES )) AND )6 THUS PERFECTLY RE SEMBLING CYTOCHROME B OF KNOWN BC COMPLEXES )T HAS A # TERMINAL EXTENSION OF ABOUT AMINO ACID RESIDUES PROVIDING TWO ADDITIONAL TRANSMEMBRANE HELICES 6ERY RECENT STUDIES (ILLER (ILLER ET AL HAVE SHOWN THAT ALL GENES ARE TRANSCRIBED
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&IG 3TEREO REPRESENTATION OF THE 3OX& 2IESKE PROTEIN FROM 3 ACIDOCALDARIUS STRUCTURE VIEW WITH CROSSED EYES SEE ALSO #OLOR 0LATE 4HE STRUCTURE WAS RESOLVED WITH ¯ 4HE ADDITIONAL DISULlDE BRIDGE # AND THE lXATION OF THE # TERMINUS ONTO AN ADDITIONAL ` SHEET ARE PRESUMABLY IMPORTANT FOR THE EXTREME THERMOSTABILITY &OR DETAILS SEE TEXT
PARTLY INDIVIDUALLY AND PARTLY AS MULTICYSTRONIC TRANSCRIPTS )T IS JUSTIlED THEREFORE TO PROPOSE THE EXISTENCE OF A NOVEL B&E3 COMPLEX EQUIVALENT TO CLASSICAL BC COMPLEXES IN THESE THERMOACIDOPHILIC ARCHAEA )N PARTICULAR IT IS SUGGESTED THAT CYTOCHROME B PRODUCT OF THE CBS! GENE REPLACES THE FUNCTION OF CYTOCHROME C AND POSSIBLY EVEN THAT OF CYTOCHROME C 4HE POSITIVE REDOX POTENTIAL OF CYTOCHROME B IS WELL IN LINE WITH THIS ASSUMPTION -ORE IMPORTANT IT HAS BEEN SHOWN BY %02 SPECTROSCOPY OF NATIVE 3ULFOLOBUS MEMBRANES THAT ITS HEME BEARING HEAD GROUP CAN mUCTUATE BETWEEN TWO LIMIT ORIENTATIONS 3CHOEPP #OTHENET ET AL THEORETICALLY ENABLING THE SHUFmING OF ELECTRONS BETWEEN DISTANT DONOR AND ACCEPTOR SITES RESPECTIVELY 4HE OCCURRENCE OF SIMILAR COMPLEXES IN 3 SOL FATARICUS AND 3 TOKODAII IS VERY LIKELY DUE TO THE OBVIOUS PRESENCE OF HOMOLOGOUS GENES +AWARA BAYASHI 3HE ET AL .EVERTHELESS THE NATURE OF THE OXIDIZING ELECTRON ACCEPTOR FOR THE NEW COMPLEX IS UNCLEAR AT PRESENT 0OSSIBLE CANDIDATES ARE THE 3OX!"#$ COMPLEX OR AN ADDITIONAL AS YET UNDETECTED TERMINAL OXIDASE (OWEVER NO INDICATIONS FOR THE LATTER CAN BE EXTRACTED FROM THE COMPLETED 3 SOLFATARICUS GENOME FOR EXAMPLE 3IMILARLY TO 3 ACIDOCALDARIUS A GENE CLUSTER HAS BEEN FOUND IN ( SALINARUM STRAIN 2 FORMING
A TRANSCRIPTIONAL UNIT OF THREE GENES 0FEIFFER AND /ESTERHELT ONE ENCODING FOR AN IRON SULFUR PROTEIN HOMOLOGOUS TO 2IESKE PROTEINS AND ANOTHER WHICH ENCODES A B TYPE CYTOCHROME 4HUS THE CORE ELEMENTS FOR A COMPLEX ANALOGOUS TO 3OX,. ARE ALSO PRESENT IN ( SALINARUM )T SHOULD BE EMPHASIZED HOWEVER THAT NO CYTOCHROME C HOMOLOGUE HAS BEEN FOUND IN THE GENOME 4HIS DOES NOT EXCLUDE THE PRESENCE OF HEME # !CTUALLY MEMBRANES FROM ( SALINARUM STRAIN *7 DISPLAY AN ABSORPTION MAXIMUM AT NM IN REDUCED OXIDIZED DIFFERENCE SPECTRA INDICATIVE OF HEME # -OLL ET AL )N D THE SAME STUDY THE OCCURRENCE OF CYTOCHROME D WAS ALSO SUGGESTED WHICH AGREES WITH RECENT GENOMIC DATA 0FEIFFER AND /ESTERHELT !CIDIANUS AMBIVALENS IS A SPECIAL CASE 4HOUGH NO RESPECTIVE SPECTROSCOPIC OR PROTEIN CHEMICAL DATA FROM LABORATORY GROWN CULTURES ARE AVAILABLE THE ONGOING GENOME PROJECT REVEALED BOTH A GENE CODING FOR A 2IESKE &E3 PROTEIN WITH IDENTITY TO 3OX, AS WELL AS A B TYPE CYTOCHROME HOMOLOGOUS TO 3OX# OR 3OX. RESPECTIVELY ! +LETZIN PERSONAL COMMUNICATION 4HEREFORE THE FUNCTIONAL FORMATION OF A 3OX,. LIKE COMPLEX APPEARS POSSIBLE UNDER CONDITIONS WHEN THESE GENES ARE EXPRESSED )N SUMMARY THE EXISTENCE OF ANCIENT COMPLEX ))) PRECURSORS CONSISTING OF A CYTOCHROME B;&E3= PROTEIN ASSEMBLY IS EVIDENT AND ALSO INDICATES THAT
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&IG 'ENES AND TRANSCRIPTS OF THE 3OX,. COMPLEX FROM 3 ACIDOCALDARIUS 4HE NARROW ARROWS INDICATE TRANSCRIPT LENGTHS THE LIGHT GREY TRANSCRIPT OCCURS ONLY IN LOW ABUNDANCE 4HE GENES ARE INDICATED BY THE BOLD ARROWS BELOW CBS! AND CBS" ARE ENCODING CYTOCHROME B WITH A MONO HEME BINDING SITE IN CBS! 3OX, ENCODES THE 2IESKE PROTEIN EXHIBITING A TARGETING PEPTIDE AND A MEMBRANE ANCHOR ILLUSTRATED IN THE HYDROPATHY PROlLE 3OX. ENCODES AN ANALOG TO DIHEME . B TYPE CYTOCHROMES WITH A LONG # TERMINAL EXTENSION AS INDICATED IN THE HYDROPATHY PLOT &OR FUNCTIONAL ATTRIBUTION SEE TEXT
ARCHAEAL COMPLEXES ARE MUCH LESS SOPHISTICATED THAN THOSE FROM HIGHER ORGANELLES LIKE MITOCHONDRIA OR CHLOROPLASTS 7**3FTQJSBUPSZ$IBJOTBOE4VQFSDPN QMFYFT 4HE ORGANIZATION OF ELECTRON TRANSPORT COMPONENTS FROM AEROBIC !RCHAEA INTO STRUCTURED ENERGY TRANS DUCING REDOX CHAINS HAS BEEN RESOLVED FOR ONLY VERY FEW SPECIES )N MOST CASES A NUMBER OF POSSIBLE CONSTITUENTS HAS BEEN STUDIED WHICH BASED ON THEIR REDOX POTENTIALSCAN BE PUT INTO A LIKELY ORDER AS FOR . PHARAONIS 3CHARF ET AL /THER CONSTITUENTS LIKE TERMINAL OXIDASES FROM EXTREME HALOPHILES COULD BE ISOLATED IN CATALYTICALLY INACTIVE FORM &UJIWARA ET AL $ENDA ET AL 3CHARF ET AL ! RECENT STUDY CHARACTERIZES AN AA TYPE OXIDASE FROM ( VOLCANII 4ANAKA ET AL WITH CYTOCHROME C OXIDIZING ACTIVITY 4AKING INTO ACCOUNT THAT AN EQUIVA LENT TO RESPIRATORY COMPLEX ) HAS NOT BEEN VERIlED IN !RCHAEA IT BECOMES EVIDENT THAT THE OXYGEN REDUCING REDOX CHAINS ARE LIMITED TO QUINOL REOXIDIZING DEVICES
AND THUS ALSO TO A MAXIMUM OF TWO ENERGY COUPLING SITES PROVIDED A COMPLEX ))) EQUIVALENT AND AN ION TRANSLOCATING TERMINAL OXIDASE ARE FUNCTIONING 4HE USE OF PROTONS AS COUPLING IONS HAS BEEN SHOWN -OLL AND 3CHÊFER AND IS LIKELY TO BE THE GENERAL CASE )T IS NOT KNOWN WHETHER ALSO SODIUM TRANSLOCATING RESPIRATORY COMPLEXES EXIST IN !RCHAEA AS THEY HAVE BEEN SHOWN FOR % COLI !VETISYAN ET AL OR 6 ALGINOLYTICUS 3TEUBER ET AL AND + PNEUMONIAE +REBS ET AL FOR REVIEWS SEE $IMROTH 3KULACHEV FOR EXAMPLE THOUGH IT MIGHT BE USEFUL TOOLS FOR ALKALIPHILIC ORGANISMS 3O FAR ARCHAEAL SODIUM BASED BIOENERGETICS ARE KNOWN ONLY IN METHA NOGENS REVIEWED IN 3CHÊFER ET AL )NTERESTINGLY THE TWO OUTMOST EXTREMES OF RE SPIRATORY CHAINS HAVE BEEN REPORTED FOR !RCHAEA A MINIMUM RESPIRATORY SYSTEM ON THE ONE HAND AND ELECTRON TRANSPORTING SUPERCOMPLEXES ON THE OTHER AS TO BE ILLUSTRATED BELOW "5IF.JOJNBM4ZTUFNPG"DJEJBOVTBNCJWB MFOT )N PREVIOUS REVIEWS WE HAVE ALREADY POSTULATED A
$IBQUFS "SDIBFBM3FTQJSBUJPO MINIMAL RESPIRATORY CHAIN FOR THE EXTREMELY THER MOACIDOPHILIC ARCHAEON !CIDIANUS AMBIVALENS !NEMàLLER ET AL A 3CHÊFER 3CHÊFER ET AL A HOWEVER WITH A NUMBER OF QUESTION MARKS 4HE IDEA RELIES ON SPECTROSCOPIC DATA WHICH IN PLASMA MEMBRANES OF AEROBICALLY GROWN ! AM BIVALENS SHOWED ONLY ONE TYPE OF HEME ! CONTAINING CYTOCHROMES OTHER CYTOCHROMES WERE NOT DETECTED )N ADDITION THE CHROMOPHORE BEARING COMPLEX AN AA TYPE QUINOL OXIDASE COULD BE ISOLATED AND CHARACTERIZED BIOCHEMICALLY AND GENETICALLY 'IUF F FRE ET AL 0URSCHKE ET AL 4HE ENZYME USES CALDARIELLAQUINONE AS SUBSTRATE /N THE OTHER HAND A CALDARIELLAQUINONE REDUCING TYPE )) .!$( DEHYDROGENASE HAS BEEN PURIlED FROM THIS ORGANISM AND COULD BE RECONSTITUTED WITH THE TERMINAL OXIDASE INTO LIPOSOMES THUS RESEMBLING THE MINIMAL RESPIRA TORY CHAIN OF ! AMBIVALENS IN VITRO 'OMES ET AL B 4OGETHER WITH THE SUCCINATE DEHYDROGENASE ,EMOS ET AL A RESPIRATORY ELECTRON mOW CAN BE POSTULATED AS ILLUSTRATED IN THE SCHEME OF &IG 4HE UNIQUE FEATURE OF THIS MINIMAL SYSTEM IS THE PRESENCE OF ONLY ONE ENERGY COUPLING SITE IE THE TERMINAL OXIDASE WHICH WAS SHOWN TO ACT AS A PROTON PUMP 'ILDERSON ET AL )T APPEARS CONCEIVABLE THAT ADDITIONALLY @CHEMICAL VECTORIAL PROTONS ARE CONTRIBUTING TO THE LARGE PROTON GRADIENT ACROSS THE MEMBRANE OF THIS EXTREME ACIDOPHILE BECAUSE THE RELEASE OF PROTONS FROM REDUCED QUINOL MIGHT OCCUR ON THE OUTSIDE OF THE PLASMA MEMBRANE DURING 1( OXIDATION (OWEVER AN EXACT ANALYSIS OF THE PROTON STOICHIOMETRY IN WHOLE CELLS IS STILL AN OPEN ISSUE ! AMBIVALENS IS AN OBLIGATE CHEMOLITHOAUTOTROPH AND SULFUR OXIDIZER 4HOUGH THE OXIDATION OF ELEMENTAL SULFUR TO SULFURIC ACID 3 (/ / A (3/ PROCEEDS WITH A LARGE CHANGE OF FREE ENERGY 6' n K*MOL THE EFlCIENCY OF THE RESPIRATORY SYS TEM PROVIDING ONLY ONE ENERGY CONSERVATION SITE IS SURPRISINGLY LOW 7HEREAS THE mUX OF REDUCING EQUIVALENTS FROM .!$( OR SUCCINATE TO OXYGEN IS THE USUAL CASE IN MOST ORGANISMS THE RECENT DETECTION OF A MEM BRANE BOUND THIOSULFATEQUINONE REDUCTASE IN ! AMBIVALENS IS AN IMPORTANT NOVELTY +LETZIN ET AL )T PROVIDES NEW INSIGHT HOW OXIDATIVE SULFUR METABOLISM IS DIRECTLY LINKED TO OXYGEN RESPIRATION /F SIGNIlCANT INTEREST IS THE CO PURIlCATION OF THE ENZYME WITH SUBUNIT )) 3OX! FROM THE TERMINAL AA TYPE OXIDASE SUGGESTING THE EXISTENCE OF A FUNC TIONAL METABOLIC UNIT 4HE TERMINAL OXIDASE WHICH BY ITSELF IS THE FULL
&IG 4HE MINIMAL RESPIRATORY CHAIN OF ! AMBIVALENS 4HE ONLY QUINOL OXIDIZING COMPLEX IS $OX!"#$%& )T IS NAMED AFTER $ESULFUROLOBUS AMBIVALENS THE PREVIOUS NAME OF !CIDIANUS AM BIVALENS 4HE QUINONE POOL IS REDUCED BY .!$( AND SUCCINATE HOW ELECTRONS FROM OXIDATION OF ELEMENTAL SULFUR ARE TRANSFERRED TO THE QUINONE POOL IS STILL UNKNOWN EXCEPT FOR THE THIOSULFATE QUINONE REDUCTASE +LETZIN ET AL
RESPIRATORY @CHAIN IS COMPOSED OF AT LEAST lVE DIFFERENT POLYPEPTIDES OF AND K$A 4HESE ARE ENCODED WITHIN TWO OPERONS $OX! AND $OX" 0URSCHKE ET AL WHICH ARE BOTH PRESENT IN DUPLICATE ON DISTANT LOCI OF THE ! AMBIVALENS GENOME /NLY THE K$A POLYPEPTIDE PRODUCT OF DOX" GENE EXHIBITS CLEAR HOMOLOGY TO HEME#U OXIDASES IT REPRESENTS THE HEME BEARING SUBUNIT ) WITH TWO HEME BINDING MOTIFS 0OLYPEPTIDE DOX! WAS CLASSIlED AS PSEUDOSUBUNIT )) WITH A POS SIBLE QUINONE BINDING SITE AND POLYPEPTIDE DOX# AS A PSEUDOSUBUNIT ))) BY WEAK ANALOGIES 4HE ENZYME SHOWS HIGHEST ACTIVITY WITH CALDARIEL LAQUINOL 5NMOL HEME A AND IS EFFECTIVELY INHIBITED BY QUINOLONE ANALOGS ^ AT - AS REPORTED ALSO FOR % COLI QUINOL OXIDASE -EUNIER ET AL )NTERESTINGLY ALSO STIGMATELLIN IS AN INHIBI TOR BUT WITH MUCH WEAKER ACTIVITY ^ AT - 4HE lNDING OF AN ESSENTIALLY NON COMPETITIVE TYPE OF INHIBITION +OMOROWSKI WAS INTERPRETED AS INDICATIVE OF TWO DIFFERENT QUINOL BINDING SITES )T WAS PROPOSED THAT A TIGHTLY BOUND QUINONE MAY REPLACE THE FUNCTION OF #U! OF REGULAR CYTOCHROME C OXIDASES SERVING AS THE PRIMARY ELECTRON ACCEPTOR AND CAPABLE OF MEDIATING THE STEPWISE TRANSFER OF TWO ELECTRONS INTO THE ELECTRON ACCEPTING HEME A SITE OF THE ENZYME )NDEPENDENT SPECTROSCOPIC AND KINETIC STUDIES CAME TO THE SAME CONCLUSION )WASAKI AND /SHIMA THAT A TIGHT QUINONE MAY REPRESENT THE FOURTH REDOX CENTER OF TYPICAL TERMINAL OXIDASES 4HE SAME STUDIES REPORT ON THE MIDPOINT POTENTIALS OF THE HEME CENTERS AT P( AND ON THE AFlNITY VERSUS
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#/ +A = -n WHICH IS SIGNIlCANTLY LOWER THAN WITH MAMMALIAN ENZYMES 4HE REDOX POTENTIALS OF THE HEME CENTERS ARE M6 HEME A AND M6 HEME A IN VERY GOOD AGREEMENT WITH OTHER TERMINAL HEME#U OXIDASES )N CONTRAST TO THE SULFUR OXIDIZING PATHWAY NEITHER THE GENETICALLY PREDICTED 2IESKE &E3 PROTEIN NOR THE PUTATIVE B TYPE CYTOCHROME HAVE BEEN INCLUDED IN THE SCHEME OF &IG BECAUSE NEITHER ONE COULD BE DETECTED IN LABORATORY GROWN CULTURES SO FAR 4HIS DOES NOT IMPLY THAT UNDER AS YET UNKNOWN GROWTH CONDITIONS ! AMBIVALENS MIGHT EXPRESS A MORE COMPLICATED RESPIRATORY SYSTEM #5IF4VQFSDPNQMFYFTPGG 4VMGPMPCVT 5NUSUAL AND LARGE RESPIRATORY COMPLEXES HAVE BEEN lRST DETECTED IN 3ULFOLOBALES COMPRISING THE 3OX !"#$ COMPLEX AND THE 3OX- COMPLEX FROM 3 ACIDOCALDARIUS AS WELL AS THE SUPERCOMPLEX DESCRIBED FROM 3 TOKODAII FORMERLY 3ULFOLOBUS SP STRAIN 7HETHER A PREVIOUSLY DESCRIBED TERMINAL OXIDASE FROM 3 SOLFATARICUS WITH CYTOCHROME C OXIDIZING ACTIVITY 7AKAGI ET AL IS A FRAGMENT ALSO OF A SUPERCOMPLEX IS NOT CLEAR HOWEVER BY COMPARING THE GENOME DATA OF 3 SOLFATARICUS 3HE ET AL THIS CONCLUSION IS NOT FAR FROM BEING LIKELY 5IF4PY"#$%$PNQMFY $ITHIONITE REDUCED 3 ACIDOCALDARIUS CELLS EXHIBIT A RATHER UNUSUAL REDUCED OXIDIZED DIFFERENCE SPEC TRUM IN THE VISIBLE REGION WITH THE MOST PROMINENT ABSORPTION BAND AT n NM 4HIS BAND HAS lRST BEEN CONSIDERED TO INDICATE AN A TYPE CYTOCHROME !NEMàLLER ET AL WHICH COULD NEVER BE ISO LATED INDIVIDUALLY HOWEVER )NSTEAD IT TURNED OUT TO BE DIFlCULT TO GET RID OF THIS HEME COMPOUND WHEN ATTEMPTING TO PURIFY THE PUTATIVE AA TYPE TERMINAL OXIDASE DISPLAYING A TYPICAL ABSORPTION AT NM )NITIALLY A @SINGLE SUBUNIT AA TYPE OXIDASE WAS DE SCRIBED !NEMàLLER AND 3CHÊFER !NEMàLLER AND 3CHÊFER !NEMàLLER ET AL CAPABLE TO OXIDIZE CALDARIELLA QUINONE IN VITRO WITH HIGH ACTIVITY AND AS A PSEUDO SUBSTRATE 4-0$ ,ATER AFTER THE RESPECTIVE GENE LOCUS HAD BEEN SEQUENCED ,àBBEN ET AL ,àBBEN ET AL B THIS PREPARATION TURNED OUT TO BE A KIND OF @OVER PURIlCATION NAMELY A FRAGMENT OF A THUS FAR UNSEEN TERMINAL OXIDASE SU PERCOMPLEX CONTAINING lVE REDOX CENTERS HEMES !3
AND #U" 4HIS COMPLEX SEEMS TO COMBINE FEATURES OF RESPIRATORY COMPLEX ))) AND COMPLEX )6 ,àBBEN ET AL !CCORDING TO THE RESPECTIVE GENES THE COMPLEX WAS ASSIGNED AS 3OX!"#$ IT IS COMPOSED OF FOUR POLYPEPTIDES AND HAS VERY HIGH IN VITRO ACTIVITY AS A CALDARIELLA QUINOL OXIDASE &IGURE GIVES A SKETCH OF ITS COMPOSITION 3UBUNIT 3OX" REPRESENTS THE AA TYPE HEME#U OXIDASE WITH PUTATIVE MEMBRANE SPANNING HELICES SUBUNIT 3OX! IS A TYPICAL HOMOLOG TO SUBUNIT )) OF CYTOCHROME C OXIDASES BUT LACKING A #U! BINDING MOTIF 0OLYPEPTIDE 3OX# IS BY SEQUENCE ANALOGY A HOMOLOG TO B TYPE CYTOCHROMES WITH TWO HEME BINDING SITES IN THIS CASE HOSTING HEMES !3 )T IS RESPONSIBLE FOR THE PROMINENT ABSORPTION BAND AT NM 4HE FOUR HEME !3 CENTERS COULD BE ALSO CHARACTERIZED BY UNIQUE RESONANCE 2AMAN SPECTRA WHICH DIFFER SIGNIlCANTLY FROM THOSE OF KNOWN AA TYPE CYTOCHROME C OXIDASES 'ERSCHER ET AL !N UNUSUAL FEATURE IS THE OCCURRENCE OF A PENTA CO ORDINATED HIGH SPIN SIGNATURE FOR THE A HEME ! COMPILATION OF SPECTRAL AND KINETIC DATA COM PARING THE SINGLE ENTITY FRAGMENT 3OX" AND THE ENTIRE 3OX!"#$ COMPLEX IS GIVEN IN 4ABLE 4HE KINETIC DATA ON #/ BINDING AND REDUCTIONREOXIDA TION KINETICS WERE VERIlED WITH INTACT MEMBRANES OF 3ULFOLOBUS ACIDOCALDARIUS 'IUFFRÒ ET AL !S AN IMPORTANT OBSERVATION IT HAS BEEN DETECTED THAT AT LEAST TWO DIFFERENT POOLS OF CYTOCHROMES ABSORBING AT n NM ARE PRESENT IN MEMBRANES AS WELL AS A SECOND #/ BINDING SITE WITH PROPERTIES UNUSUAL FOR TERMINAL AA TYPE OXIDASES "OTH THE SINGLE ENTITY FRAGMENT 3OX" OR 3OX"! AND THE INTACT 3OX!"#$ COMPLEX COULD BE RECONSTI TUTED INTO LIPOSOMES AND WERE SHOWN TO GENERATE A PROTON MOTIVE FORCE UPON REDUCTANT PULSES $RIESSEN 'LEISSNER ET AL 4HE MECHANISM OF PRO TON TRANSLOCATION WAS INITIALLY DISCUSSED TO POSSIBLY INVOLVE A 1 CYCLE BECAUSE A ( En RATIO SLIGHTLY ABOVE ONE WAS MEASURED 2ECENT RESULTS ON TERMINAL OXI DASES OF THE 3OX" TYPE 'OMES ET AL A SUGGEST HOWEVER THAT ALSO OR PROBABLY ONLY THE 3OX" MOIETY OF THIS COMPLEX REPRESENTS A PROTON PUMP !S TO THE ROLE OF CYTOCHROME A 3OX# IT AP PEARS CONCEIVABLE TO FUNCTION AS A ELECTRON ACCEPTOR OR AN INTERMEDIATE ELECTRON STORAGE SITE ACCORDING TO THE SCHEME ELECTRON DONOR A #YT A A #YT A A #YT A#U" A /
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&IG 3CHEMATIC REPRESENTATION OF THE 3OX!"#$ SUPERCOMPLEX OF 3 ACIDOCALDARIUS 4HE MEMBRANE RESIDING POLYPEPTIDES ARE NOT DRAWN TO SCALE &OR FUNCTIONAL DETAILS SEE TEXT 4HIS COMPLEX WAS THE lRST EXAMPLE OF A PROTON PUMPING HEME#U OXIDASE LACKING THE $ CHANNEL AND THE 'LU 4HE PRESENCE OF A NATIVE QUINOL OXIDIZING SITE IS QUESTIONABLE FOR DETAILS SEE TEXT
7HETHER OR NOT THE 3OX!"#$ COMPLEX IS REALLY A QUINOL OXIDASE IN VIVO IS AN OPEN TOPIC TO BE AD DRESSED FURTHER BELOW
4HIS ALTERNATIVE TERMINAL OXIDASE TO THE 3OX!"#$ COMPLEX WAS lRST DESCRIBED FOR 3 ACIDOCALDARIUS ,àBBEN ET AL A AND WITH RESPECT TO ITS CONSTITU ENTS HAS SIMILARITIES TO A RESPIRATORY SUPERCOMPLEX DESCRIBED FROM 3 TOKODAII FORMERLY 3ULFOLOBUS SP STRAIN )WASAKI ET AL A 4HE INITIALLY ISOLATED COMPLEX NAMED 3OX- HAD VERY LITTLE CATA LYTIC ACTIVITY OBVIOUSLY DUE TO THE LOSS OF ESSENTIAL CONSTITUENTS 7ITH THE AID OF THE GENOMIC INFORMATION #ASTRESANA ET AL AND THE STUDY OF HETEROLOGOUS EXPRESSED SINGLE CONSTITUENTS 3CHÊFER ET AL B +OMOROWSKI ET AL IT WAS RECENTLY POSSIBLE TO ISOLATE AND CHARACTERIZE THE ENTIRE SUPERCOMPLEX IN A CATALYTICALLY FULLY COMPETENT FORM +OMOROWSKI +OMOROWSKI ET AL 4HE COMPLEX CON TAINS A COMPLETE RESPIRATORY CHAIN BETWEEN QUINOL AND OXYGEN ! SKETCH OF ITS COMPOSITION AND FUNCTION IS ILLUSTRATED IN &IG 4HE SUPERCOMPLEX CONSISTS OF TWO FUNCTIONAL SUB COMPLEXES AN !RCHAEAL HOMOLOGUE TO BC COMPLEXES AND A TERMINAL OXIDASE SUBCOMPLEX "OTH ARE LINKED BY THE BLUE COPPER PROTEIN 3ULFOCYANIN SEE ALSO SEC TION )))# !CCORDING TO A STOICHIOMETRY OF ITS CONSTITUENTS THE COMPLEX CONTAINS EIGHT METAL REDOX CENTERS WITH A TOTAL OF MOL &E AND MOL #U PER MOL OF COMPLEX )N CONTRAST TO THE 3OX!"#$ COMPLEX 3OX- IS HOSTING HEMES B AND THUS IS A BB OXIDASE
4ABLE #OMPARISON OF MOLECULAR PROPERTIES BETWEEN THE ISOLATED AA TYPE TERMINAL OXIDASE MOIETY 3OX" AND THE INTACT QUINOL OXIDASE COMPLEX 3OX!"#$ FROM 3ULFOLOBUS ACIDOCALDARIUS $ATA FROM THE REFERENCES QUOTED IN THE TEXT 0ARAMETER 3OX" SINGLE ENTITY OXIDASE 3OX!"#$ COMPLEX MOL MASS $.! DERIVED K$A POLYPEPTIDE SUBUNITS SOX" SOX! SOX" SOX# SOX$ APP MASS ON DENAT GELS n n SUBUNIT STOICHIOMETRY n COFACTORS MOL HEME A HEME A #U HEME A HEME A #U VIS ABSORPTION BAMDS RED OXID NM REDOX POTENTIALS OPTICAL TITR M6 A A A A A REDOX POTENTIALS %02 TITR M6 ND G VALUES ,3 HEME GZ GY GX GZ GY GX #YT A GZ GY GX #YT A G VALUES (3 HEME GY GX GZ GY ELECTRON DONORS CALDARIELLA QUINOL 4-0$ CALDARIELLA QUINOL 3OX, &E3 ACTIVATION ENERGY K*MOL ND TURNOVER Sn # INHIBITORS #/ #.n .n 3n #/ #.n .n 3n #/ BINDING K - n S n K - n S n +A
IN ADDITION IT HAS A #U! CENTER IN ITS SUBUNIT )) 3OX( LIKE TYPICAL CYTOCHROME C OXIDASES "ECAUSE C TYPE CYTOCHROMES ARE ABSENT FROM 3 ACIDOCALDARIUS AN ALTERNATE NATURAL REDUCTANT HAS TO BE USED THIS IS SULFOCYANIN WHICH IS ANCHORED IN THE MEMBRANE BY A LONG HYDROPHOBIC . TERMINAL SEGMENT 4HE EXPOSED PERIPHERAL DOMAIN BEARING THE BLUE COPPER CENTER IS THOUGHT TO EXERT PIVOTING mUCTUATIONS BETWEEN THE TWO SUBCOMPLEX MOIETIES 4HUS THE BLUE COPPER PROTEIN SIMULTANEOUSLY SERVES AS THE ELECTRON ACCEPTOR FOR THE 2IESKE &E33OX' SUBCOMPLEX 3OX' IS A STRUC TURAL ANALOG TO B TYPE CYTOCHROMES OF BC COMPLEXES BUT IN THIS CASE IS BEARING TWO HEMES !3 WHICH ARE RESPONSIBLE FOR THE PROMINENT ABSORPTION AT NM IN REDUCED OXIDIZED DIFFERENCE SPECTRA 4HE PHYSI COCHEMICAL PROPERTIES OF ALL REDOX CENTERS COULD BE DETERMINED EITHER WITH THE INTACT COMPLEX OR IN PART WITH ITS HETEROLOGOUS EXPRESSED CONSTITUENTS AND ARE SUMMARIZED IN 4ABLE 4HOUGH HIGHEST CATALYTIC ACTIVITIES ARE REPORTED WITH ITS NATURAL REDUCTANT CALDARIELLA QUINOL THE
(ÛOUFS4DIÅGFS ISOLATED COMPLEX CAN USE CYTOCHROME C AS A PSEU DOSUBSTRATE 4HIS IS PRESUMABLY DUE TO THE ABILITY TO REDUCE EITHER SULFOCYANIN AS WELL AS #U! IN THE ISOLATED FORM OF SUBUNIT )) +OMOROWSKI ET AL )T MAY ALSO BE RESPONSIBLE FOR AN EARLIER OBSERVED CYTOCHROME C OXIDASE ACTIVITY IN CRUDE MEMBRANE PREPARATIONS!NEMàLLER AND 3CHÊFER 4HE COMPLEX IS FULLY INHIBITED BY CYANIDE BUT NOT BY TYPICAL COMPLEX ))) INHIBITORS LIKE ANTIMYCIN ! 4HUS CLEAR STRUCTURAL DIFFERENCES EMERGE OF THE 2IESKE3OX' SUBCOMPLEX COMPARED TO THE KNOWN BC COMPLEXES (OWEVER A NUMBER OF AURACHIN # AND $ DERIVATIVES WITH AN ATTACHED HYDROPHOBIC SIDE CHAIN WAS SHOWN TO EXERT STRONG INHIBITION WITH P) VALUES AS HIGH AS +OMOROWSKI ET AL 4HESE INHIBITORS ARE QUINONE ANALOGS WHICH PRESUM ABLY HAVE HIGH AFlNITY TO THE QUINOL BINDING SITES OF THE SUBCOMPLEX "ASED ON THE SEQUENCE ANALYSES OF THE CONSTITUENTS AND ITS OVERALL COMPOSITION THE 3OX- SUPERCOMPLEX SHOULD INCLUDE TWO PUTATIVE PROTON PUMPING SITES
&IG 3CHEMATIC REPRESENTATION OF THE 3OX- SUPERCOMPLEX OF 3 ACIDOCALDARIUS ! GENE ORDER AND ARRANGEMENT ON OPPOSITE STRANDS OF THE $.! " &UNCTIONAL ARRANGEMENT OF THE CONSTITUENT POLYPEPTIDES NOT DRAWN TO SCALE AND THE PUTATIVE PROTON TRANSLOCATING SITES 4HE 3OX'&% MOIETY FUNCTIONS AS A BC ANALOGOUS SUBCOMPLEX 3OX% AS A REORIENTING INTERMEDIATE ELECTRON CARRIER IS THOUGHT TO REDUCE THE TERMINAL OXIDASE SUBCOMPLEX 3OX(- #YTOCHROME C CAN SERVE IN VITRO AS A PSEUDOSUBSTRATE &OR FURTHER DETAILS SEE TEXT
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ONE IN THE 2IESKE3OX' SUBCOMPLEX AND ONE IN THE TERMINAL OXIDASE SUBCOMPLEX 0ROTON PUMPING IN A RECONSTITUTED VESICLE SYSTEM REMAINS TO BE VERIlED HOWEVER 4HE DElCIENCY OF CATALYTIC ACTIVITY AS PREVIOUSLY REPORTED CAN BE EXPLAINED BY THE UNEXPECTED PROPERTY OF SUBUNIT )) TO DISSOCIATE EASILY FROM THE COMPLEX 4WO REASONS MAY BE INVOLVED /NE IS THE RELATIVELY SHORT HYDROPHOBIC . TERMINAL ANCHOR OF THIS POLY PEPTIDE AND THE PRESENCE OF A NUMBER OF POLAR AMINO ACID RESIDUES IN THIS DOMAIN ANOTHER IS AN OBVIOUS PROTEOLYTIC CLEAVAGE WHEN MEMBRANES WERE PREPARED AT NEAR NEUTRAL P( ITS LOSS CAN BE TOTALLY AVOIDED BY A NOVEL ISOLATION PROTOCOL +OMOROWSKI *T4PY"#$%B2VJOPM0YJEBTFJOWJWP 3EVERAL REASONS SUPPORT THE PROPOSAL THAT THE QUINOL
OXIDIZING FUNCTION OF DETERGENT ISOLATED 3OX!"#$ MIGHT BE AN ARTIFACTUAL IN VITRO ACTIVITY )NSTEAD AN ALTERNATE PROPOSAL FOR THE ELECTRON TRANSPORT CHAIN OF 3 ACIDOCALARIUS IS DEPICTED IN &IG !CCORDING TO THIS NOVEL SCENARIO 3OX!"#$ IS THE OXIDANT OF THE REDUCED 3OX,. COMPLEX CF 3ECTION 6)" " IN VIVO WITH CYTOCHROME B SERVING AS A LINK REPLACING THE FUNCTION OF CYTOCHROME CC OF @CLASSICAL RESPIRATORY CHAINS OR OF SULFOCYANIN OF THE 3OX- SUPERCOMPLEX #YTOCHROME B APPEARS AS AN IDEAL CANDIDATE DUE TO ITS POSITIVE REDOX POTENTIAL AND ITS HIGH STABILITY ESPECIALLY AT ACIDIC P( ALSO ITS HIGH DEGREE OF GLY COSYLATION SUGGESTS A TOPOLOGICAL ORIENTATION TO THE OUTER SURFACE OF THE MEMBRANE LIKE CYTOCHROME C 4HE REASONS SUPPORTING THIS HYPOTHESIS ARE THE FOLLOWING I $ETERGENT SOLUBILIZED 3OX!"#$ CAN ACCEPT ELECTRONS FROM TOTALLY UNUSUAL DONORS AS FOR EXAMPLE THE PURIlED 2IESKE PROTEIN 3OX, II AT LOW
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&IG 2EVISED SCHEME OF THE BRANCHED RESPIRATORY CHAINS OF 3 ACIDOCALDARIUS 4HE UPPER BRANCH IS DEPICTING THE HYPOTHESIS ON THE ORGANIZATION OF ELECTRON mOW FROM CALDARIELLA QUINONE THROUGH THE NOVEL 3OX,. COMPLEXB AND 3OX!"#$ TO OXYGEN 4HE LOWER BRANCH IS DEPICTING THE FUNCTION OF THE 3OX- SUPERCOMPLEX AS DISCUSSED IN THE TEXT 5NDERLINED COMPONENTS ARE DISCUSSED TO EXERT REGULATORY FUNCTIONS IN ELECTRON mOW
P( THE QUINOL OXIDIZING ACTIVITY IS ONLY MARGINAL UNPUBLISHED III UNDER THE USUAL TEST CONDITIONS AT NEAR NEUTRAL P( CALDARIELLA QUINOL IS RATHER LABILE AU TOXIDABLE AND MAY UNSPECIlCALLY REDUCE CYTOCHROME A OF THE COMPLEX IV 3OX!"#$ HAS A PROMINENT 4-0$ OXIDASE ACTIVITY WHICH IS UNUSUAL FOR QUINOL OXIDASES V 3OX!"#$ IS PRACTICALLY INSENSITIVE TO 1 ANALOGS ACTING AS INHIBITORS OF QUINOL OXIDASES AND lNALLY VI THE FULLY SEQUENCED 3ULFOLOBUS GENOMES DO NOT ENCODE ANY ADDITIONAL TERMINAL OXIDASE BE SIDES 3OX!"#$ AND THE 3OX- COMPLEX PROPOSED TO SERVE AS AN ALTERNATE REOXIDANT FOR THE 3OX,. COMPLEX 4HIS LATTER ARGUMENT IS CERTAINLY THE MOST CONVINCING ONE 7***3FHVMBUPSZ"EBQUBUJPOT 4HE QUESTION WHETHER OR EVEN HOW ARCHAEAL SPECIES WITH A BRANCHED RESPIRATORY SYSTEM RESPOND TO ENVI RONMENTAL CHANGES HAS NOT BEEN ANSWERED IN ANY OF THE KNOWN CASES /UR OWN ATTEMPTS TO INmUENCE THE RELATIVE ABUNDANCE OF EITHER 3OX!"#$ OR THE 3OX- COMPLEX IN 3 ACIDOCALDARIUS BY VARIATIONS OF OXYGEN SUPPLY FAILED TO YIELD SIGNIlCANT EFFECTS 4HE ONLY OB VIOUS DIFFERENCE WAS FOUND FOR THE AMOUNT OF #YT B WHICH WAS EXPRESSED IN HIGHEST AMOUNTS AT LIMITING OXYGEN CONCENTRATIONS BUT ALSO WAS INmUENCED BY THE TYPE OF CARBON SOURCE (ETTMANN ET AL )F THAT CYTOCHROME OPERATES AS A LINK BETWEEN THE 3OX,. COMPLEX AND ITS PUTATIVE OXIDIZING COMPLEX 3OX!"#$ THE ADAPTATION OF ITS ABUNDANCE MIGHT INmUENCE THE mUX OF ELECTRONS THROUGH THE RESPECTIVE BRANCH OF THE RESPIRATORY CHAIN !N INTERESTING NOVEL CASE IS THE PROPOSED FUNCTION
OF SUBUNIT )) 3OX( OF THE 3OX- SUPERCOMPLEX AS A P( SENSOR AND REGULATOR OF ELECTRON mOW TO OXY GEN THROUGH THIS PATHWAY 7ITH THE RECOMBINANTLY PRODUCED SUBUNIT 3OX( A HITHERTO UNUSUAL P( DE PENDENCE OF THE 566IS AND THE %02 SPECTRA WAS OBSERVED +OMOROWSKI ET AL SHOWN IN THE SPECTRA GIVEN IN &IG )N A P( RANGE FROM ACIDIC TO ALKALINE A REVERSIBLE CHANGE OF SPECTRAL PROPERTIES OCCURS THE REVERSIBILITY OF THE PROCESS BETWEEN P( TO IS OF FUNDAMENTAL IMPORTANCE #ONVERSELY WHILE AT INTERMEDIATE P(S n A VISIBLE ABSORP TION SPECTRUM IS ASSOCIATED WITH #U! CENTERS MORE ACIDIC OR ALKALINE P( CONDITIONS GENERATE ABERRANT SPECTRA INDICATING VARIATIONS IN THE LIGAND lELD OF THE BINUCLEAR #U! CENTER 4HE %02 SPECTRA REmECT THESE CHANGES EQUALLY WELL 4HE MOLECULAR REARRANGEMENTS PRODUCING THE SPECTRUM SEEN AT ALKALINE P( ARE STILL UNKNOWN (OWEVER AT ACIDIC P( THE SPECTRUM SHOWS PROPERTIES OF MONO NUCLEAR #U CENTERS SIMILAR TO BLUE COPPER PROTEINS AS WELL AS HYPERlNE SPLITTING INDICATING FREE COPPER 3UCH DRAMATIC REARRANGE MENTS WILL CERTAINLY RESULT IN ALTERATIONS OF THE REDOX PROPERTIES ! TITRATION OF THE P( EFFECTS SUGGESTS THE PARTICIPA TION OF ONE OF THE CLUSTER LIGANDING HISTIDINES WITH A P+A OF 4HE P( OPTIMUM WAS FOUND AT ^ )NTERESTINGLY THIS COINCIDES PERFECTLY WITH THE P( OPTIMUM OF THE ACTIVITY PROlLE OF THE 3OX- COM PLEX 4ABLE )T APPEARS CONCEIVABLE THEREFORE THAT SUBUNIT )) 3OX( CAN ACT AS BOTH A P( SENSOR AND A REGULATOR OF ACTIVITY OF THE 3OX- SUPERCOMPLEX IN A PHYSIOLOGICAL P( RANGE BETWEEN P( n 4HE COMPELLING ASPECT OF THIS HYPOTHESIS IS THAT UNDER NORMAL ACIDIC CONDITIONS A BASIC RESPIRATORY SYSTEM FOR EXAMPLE 3OX!"#$ IS SUFlCIENT TO SUS
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&IG P( DEPENDENCE OF THE BINUCLEAR #U CENTER IN SUBUNIT )) 3OX( OF THE 3OX- SUPERCOMPLEX ! VISIBLE SPECTRA AND " %02 SPECTRA OF THE RECOMBINANT PROTEIN TAKEN AT A SERIES OF DIFFERENT P( CONDITIONS &OR DETAILS SEE +OMOROWSKI ET AL AND TEXT
TAIN THE ENERGY METABOLISM )F THE ENVIRONMENTAL P( INCREASES ABOVE THE 3OX- SUPERCOMPLEX IS ACTIVATED AND PROVIDES TWO ADDITIONAL PROTON PUMPS SUPPORTING A RAPID RESTORATION OF THE OPTIMUM AMBI ENT P( FOR 3 ACIDOCALDARIUS !T P( ABOVE NONE OF THE RESPIRATORY SYSTEMS IS ACTIVE ANY LONGER AND IN LINE WITH EXPERIMENTAL EXPERIENCE THE ORGANISM CAN NOT SURVIVE *9"SF"MM"SDIBFBM)FNF$V0YJEBTFT 1SPUPO1VNQT 7HEN THE lRST ARCHAEAL TERMINAL OXIDASE GENE HAD BEEN SEQUENCED FROM 3 ACIDOCALDARIUS AN OBVIOUS CONTRADICTION TO THE ESTABLISHED PARADIGM BECAME EVIDENT 4HAT WAS THE ABSENCE OF FUNCTIONALLY ESSEN TIAL AMINO ACID RESIDUES OF SUBUNIT ) WHICH BEARS THE HEME#U CENTER ,àBBEN ET AL )N ALL KNOWN ARCHAEAL SEQUENCES THIS SUBUNIT HAS OR MORE PU TATIVE TRANSMEMBRANE HELICES AS WELL AS ALL RESIDUES TO BIND HEMES AND #U LOCATED IN HELICES )) AND 8 LOW SPIN HEME SITE AND 6)6)) HEME#U CENTER RESPECTIVELY &ROM SITE DIRECTED AMINO ACID EXCHANGES IN THE TERMINAL OXIDASES FROM 2H SPHAEROIDES AA OXIDASE AND % COLI BO OXIDASE THE IDENTIlCATION OF TWO CHANNELS FOR PROTON DELIVERY TO THE BINUCLEAR OXYGEN REDUCTION SITE AND FOR ACTIVE PROTON TRANS
LOCATION WAS CONCLUDED THE $ AND THE + CHANNEL (OLLÊNDER ET AL 'ARCIA (ORSMAN ET AL (ELLWIG ET AL 0lTZNER ET AL 4HE $ CHANNEL PROVIDES A PROTON ENTRY SITE CONTAINING A TYPICAL . 8 $ 8 . MOTIF CONNECTED VIA TYROSIN AND SERINS TO AN ESSENTIAL GLUTAMATE RESIDUE % NUMBERING OF 0 DENITRIlCANS CLOSE TO THE BINUCLEAR REACTION CENTER 4HE + CHANNEL STARTING WITH + LEADS DIRECTLY TO THE BINUCLEAR SITE 4HE LIKELY IN VOLVEMENT OF THESE CHANNELS IN PROTON DELIVERY AND TRANSDUCTION WAS STRONGLY SUPPORTED BY THE lRST HIGH RESOLUTION STRUCTURES OF CYTOCHROME C OXIDASES FROM 0 DENITRIlCANS AND FROM BOVINE HEART MITOCHONDRIA &ETTER ET AL )WATA ET AL 7HEREAS IN THE NOVEL ARCHAEAL OXIDASES THE RESIDUES TO FORM THE + CHANNEL WERE CONSERVED AT IDENTICAL OR VERY CLOSE POSITIONS THE ESSENTIAL GLUTAMATE % AS WELL AS ASPARTATE $ 0 DENITR NUMBERING WERE MISSING #ONSEQUENTLY IT WAS PROPOSED THAT THE RESPECTIVE TERMINAL OXIDASES DO NOT PUMP PROTONS BUT MAY GEN ERATE A PROTON MOTIVE POTENTIAL SIMPLY BY CHEMICAL CHARGE SEPARATION 'LEISSNER ET AL 3CHÊFER ET AL A 0URSCHKE ET AL THAT OCCURS BY RELEASE OF SUBSTRATE PROTONS FROM 1( AT THE MEMBRANE OUTER SURFACE WHILE TAKING UP PROTONS FOR OXYGEN REDUCTION FROM THE MEMBRANE INSIDE !LSO ALTERNATE MECHANISMS HAVE BEEN DISCUSSED BASED ON THE PRINCIPLES OF A 1 CYCLE CATALYZED BY THE TERMINAL
1 OXIDASE COMPLEX ,àBBEN ET AL 3CHÊFER ET AL A BECAUSE IT WAS EVIDENT THAT !RCHAEA CAN ACIDIFY THE MEDIUM BY PROTON EXTRUSION VERY RAPIDLY UPON OXYGEN PULSES -OLL AND 3CHÊFER 4HE CONSTRUCTION OF SIMILARITY TREES FROM MULTIPLE SEQUENCE ALIGNMENTS SUGGESTED INDEED THE EVOLUTION OF TWO TYPES OF TERMINAL OXIDASES THOSE WITH AND THE OTHER WITHOUT THE $ CHANNEL SIGNATURES "OTH ORIGINATED PRESUMABLY FROM THE DUPLICATION OF ONE ANCESTRAL GENE ! CRITICAL ASPECT EMERGED HOWEVER FROM THE FACT THAT THESE BRANCHES DID NOT REmECT THE EVOLUTIONARY TREES BASED ON S R2.! SEQUENCES BUT RATHER DISCLOSED A SCATTERED OCCURRENCE OF BOTH !RCHAEA AND "ACTERIA IN EITHER GROUP WITH OR WITHOUT A $ CHANNEL -OREOVER SOME !RCHAEAL AND BACTE RIAL ORGANISMS WERE FOUND TO POSSESS BOTH TYPES OF TERMINAL OXIDASES 4HIS IS EXEMPLIlED BY THE PARTIAL SEQUENCE ALIGNMENT OF HELIX 6) OF SUBUNIT ) FROM !RCHAEAL AND TWO BACTERIAL SPECIES &IG DEPICT ING THE LOCUS OF THE @ESSENTIAL 'LU )N ADDITION IT HAS RECENTLY BEEN DEMONSTRATED THAT ALSO THOSE TERMINAL OXIDASES WHICH LACK THE ESSENTIAL 'LU AS WELL AS THE $ CHANNEL ;!SP= CAN ACTIVE LY TRANSLOCATE PROTONS LIKE THAT FROM 4H THERMOPHILUS BA +ANNT ET AL OR FROM ! AMBIVALENS AA WITH A ( En STOICHIOMETRY OF CLOSE TO 'OMES ET AL A 4HEREFORE THE PREVIOUS PARADIGM HAD TO BE ABANDONED AND ONE HAS TO ASSUME THAT ALL HEME#U OXIDASES CAN ACTIVELY TRANSLOCATE PROTONS /BVIOUSLY A VARIETY OF POSSIBLE PROTON PATHWAYS EXISTS WHICH CAN BE VERIlED ONLY BY FURTHER HIGH RESOLUTION 8 RAY STRUCTURES AND MUTAGENESIS STUDIES WITH THE RESPECTIVE ENZYMES !AGAARD ET AL 4AKING THESE RESULTS INTO ACCOUNT ALSO THE PREVI OUS INTERPRETATION OF A POSSIBLE 1 CYCLE 'LEISSNER ET AL HAS TO BE REVISED AND MOST LIKELY ONLY SUBUNIT ) 3OX" OF THE 3OX!"#$ OXIDASE FROM 3 ACIDOCALDARIUS IS RESPONSIBLE FOR PROTON PUMPING -OREOVER THE DETERMINED PROTON STOICHIOMETRY OF ( En IS CLOSER TO THAT OF A TYPICAL HEME#U PROTON PUMP ( En THAN TO A 1 CYCLE ( En 4HIS LATTER SCENARIO SUPPORTS THE ABOVE CONCLUSION THAT IN THE 3OX!"#$ OXIDASE 3OX# SERVES AS A TWO ELECTRON ACCEPTOR OR ELECTRON BUFFER ! DETAILED ANALYSIS OF THE FUNCTIONAL SUBUNITS ) AND )) OF HEME#U TERMINAL OXIDASES INCLUDING AMINO ACID SEQUENCES HAS BEEN RECENTLY COMPILED 0EREIRA ET AL )NTERESTINGLY OUT OF SEQUENCES REVEAL A LACK OF THE $ CHANNEL AND ONLY SEQUENCES CONTAIN THE VERY 'LU IN A n8'( 0%6n % MOTIF PREVIOUSLY ASSIGNED AS INDISPENSABLE
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&IG 0ARTIAL ALIGNMENT OF THE ESSENTIAL HELIX 6) MOTIF OF TERMINAL OXIDASES 4HE TOP GROUP COMBINES VARIOUS !RCHAEA AND 4THERM WHICH ARE LACKING THE ESSENTIAL GLUTAMATE IN THE $ CHAN NEL 4HE BOTTOM GROUP SHOWS ALTERNATE OXIDASES OF !RCHAEA AND 4 THERMOPHILUM WHICH HAVE THIS RESIDUE AS IT IS ALSO CONSERVED IN CYTOCHROME OXIDASES OF MITOCHONDRIA OR PURPLE BACTERIA OX AND OX REFER TO THE TWO TERMINAL OXIDASES OF !EROPHILUM PERNIX +AWARABAYASHI 4HE OTHER ORGANISMS ARE !CIDIANUS AM BIVALENS .ATRONOMONAS PHARAONIS 3ULFOLOBUS ACIDOCALDARIUS (ALOBACTERIUM SALINARUM AND 0YROBACULUM AEROPHILUM &OR DETAILED DISCUSSION SEE TEXT
FOR PROTON PUMPING &OR SEQUENCES AN ALTERNATE n93(086n MOTIF WAS SUGGESTED TO FUNCTIONALLY REPLACE THE FORMER &INALLY REMAINING SEQUENCES CONTAIN NEITHER ONE OF THESE MOTIFS ! PROTEIN BASED UNROOTED PHYLOGENETIC TREE SUPPORTS THE PROPOSAL OF THREE EVOLUTIONARY GROUPS GROUP ! HAVING BOTH THE $ AND + CHANNELS GROUP " HAVING AN ALTERNATE + CHANNEL BUT NO $ CHANNEL A NOVEL GROUP # WITH NO $ CHANNEL AND ONLY A RUDIMENTARY + CHANNEL AND ALSO THE 4YR 0 DENITRIlCANS NUMBERING MISSING WHICH IN ALL OTHER SUBUNITS ) IS COVALENTLY LINKED TO THE #U LIGANDING HISTIDINE !CTUALLY !RCHAEA AND "ACTERIA APPEAR AGAIN SCATTERED BETWEEN THE GROUPS ! AND " )N SUMMARY THIS ANALYSIS UNDERLINES THE DISCREPANCY BETWEEN 2.! BASED PHYLOGENY AND PHYLOGENETIC TREES BASED ON AMINO ACID SEQUENCES /NE SHOULD REMEMBER THAT THE LATTER DO NOT REPRESENT REAL EVOLUTIONARY PATHWAYS BUT SIMPLY REmECT DISTANCE MATRICES OF SEQUENCE SIMILARITIES 9$PODMVTJPOTBOE1FSTQFDUJWFT )N RECENT YEARS IT BECAME EVIDENT THAT THE CORE STRUCTURES OF ARCHAEAL RESPIRATORY ELECTRON TRANSPORT SYSTEMS ARE FOUND EVEN IN THE LOWEST BRANCHES OF THE PHYOLGENETIC TREE !N IMMEDIATE CONCLUSION WOULD SUGGEST THAT IN ESSENCE THESE AS WELL AS THE CHEMIOSMOTIC PRINCIPLE OF ENERGY CONSERVATION WERE
$IBQUFS "SDIBFBM3FTQJSBUJPO PRESENT ALREADY IN PROGENOTIC PRECURSORS PRIOR TO THE SPLIT INTO THE THREE KINGDOMS OF ORGANISMS AND PRIOR TO THE ORIGIN OF OXYGENIC PHOTOSYNTHESIS SEE ALSO #HAPTER BY #ASTRESANA 6OL 4HIS ISSUE IS STILL MATTER OF DEBATE AND OTHER HYPOTHESES ASSUME AN ACQUISITION OF HEME#U OXIDASES BY LATERAL GENE TRANSFER FROM "ACTERIA TO !RCHAEA 0EREIRA ET AL "UT AEROBIOSIS MAY ALSO BE SEEN AS AN ADAPTA TION SIMPLY BY CHANGES OF SPECIlCITIES WITH RESPECT TO ELECTRON DONORS AND nACCEPTORS 3TRONG EVIDENCE COMES FROM THE RELATION BETWEEN HYDROGENASES AND ELEMENTS OF .!$( DEHYDROGENASES !LBRACHT ET AL &RIEDRICH +ERSCHER ET AL OR OF THE FAMILY OF HEME#U OXIDOREDUCTASES INCLUDING BOTH ./ REDUCTASES AND OXYGEN REDUCTASES (ENDRIKS ET AL A (ENDRIKS ET AL B 6AN DER /OST ET AL 3IMILARLY THE SUCCINATE DEHYDROGENASES AND FUMARATE REDUCTASES ARE MEMBERS OF A FAMILY WITH COMMON ORIGIN AS ARE THE COMPONENTS OF 2IESKE &E3 CYTOCHROME COMPLEXES ACTING AS QUINOL OXIDOREDUC TASES AND REDOX DRIVEN PROTON PUMPS 3CHMIDT AND 3HAW ! COMPARISON OF PHYLOGENETIC TREES EVEN SUGGESTS A COEVOLUTION OF RESPIRATORY B CYTOCHROMES AND 2IESKE &E3 PROTEINS 4HE INVESTIGATION OF !RCHAEA BROUGHT ALSO INTO LIGHT THAT PRESUMABLY ALL TERMINAL HEME#U OXIDASES ARE ACTING AS PROTON PUMPS THUS RAISING THE QUESTION FOR OTHER PROTON CONDUCTING PATHWAYS THAN THOSE SUG GESTED FROM PREVIOUS PARADIGMS !NOTHER SURPRISING RESULT WAS THE DISCOVERY OF INTEGRATED SUPERCOMPLEXES IN RESPIRATORY SYSTEMS WHICH MIGHT BE AN ADAPTATION TO HIGH TEMPERATURES OR TO THE SPECIlC MEMBRANE ARCHITECTURE OF HYPERTHERMOPHILIC !RCHAEA (OWEVER A LARGE NUMBER OF PROBLEMS REGARDING STRUCTURE AND FUNCTION OF THE ARCHAEAL ELECTRON TRANSPORT MODULES REMAINS TO BE SOLVED IN FUTURE STUDIES /NE OF THESE IS THE FUNCTION OF GENES WHOSE PROD UCTS HAVE NEVER BEEN CHEMICALLY PROVED !RE THESE GENES ENCODING PUTATIVE RESPIRATORY ELECTRON TRANSPORT MODULES ACTUALLY TRANSCRIBED )F SO UNDER WHICH CONDITIONS &URTHERMORE THE OCCURRENCE OF MULTIPLE COPIES OF GENES LIKE THOSE OF THE TERMINAL OXIDASE FROM ! AMBIVALENS 0URSCHKE ET AL LEADS TO THE QUESTION OF THEIR REGULATION !LSO UNKNOWN OF ! AMBIVALENS AND OTHER SULFUR OXIDIZERS IS THE PATHWAY HOW THE SIX ELECTRONS ARE DELIVERED TO THE RESPIRATORY CHAIN DURING OXIDATION OF 3 TO (3/ !N AMBITIOUS GOAL WILL BE THE STRUCTURAL RESOLU TION OF THE SUPERCOMPLEXES 4O COME UP WITH THIS A MASSIVE PRODUCTION OF HOMOGENEOUS PREPARATIONS IS REQUIRED THIS POINT IS HOWEVER HAMPERED BY A
NUMBER OF DIFlCULTIES NAMELY A DISSOCIATION OF THE COMPLEXES OR LOSS OF SINGLE CONSTITUENTS UPON SOLU BILIZATION OF PLASMA MEMBRANES B THE EQUILIBRATION OF IONIC STRENGTH AND P( UPON CELL DISRUPTION CON VERSELY UNDER NATIVE CONDITIONS THESE COMPLEXES IE FROM THERMOACIDOPHILES ARE EXPOSED TO DRAMATICALLY ASYMMETRIC ENVIRONMENTS ON OPPOSITE MEMBRANE SURFACES 6P( * C THE GLYCOSYLATION OF SURFACE EXPOSED COMPONENTS MAY CAUSE A NON HOMOGENE ITY AND ALSO EXCLUDE THE HETEROLOGOUS EXPRESSION IN BACTERIAL SYSTEMS ALONG WITH A CORRECT ASSEMBLY OF THE SUPERCOMPLEXES &INALLY ALSO THE SPECIlC ARCHAEAL LIPID ENVIRONMENT IS A CRITICAL PARAMETER INmUENCING STRUCTURE AND FUNCTION OF ARCHAEAL RESPIRATORY COM PLEXES &UNCTIONAL AND REGULATORY PROPERTIES OF SINGLE ELECTRON TRANSPORT PATHWAYS FROM ORGANISMS WITH A BRANCHED RESPIRATORY SYSTEM COULD BE BEST INVES TIGATED IN SPECIALLY DESIGNED DELETION MUTANTS OR OVER PRODUCERS 4HEREFORE THE DEVELOPMENT OF AN EFlCIENT GENETIC MANIPULATION SYSTEMS IS A PREREQUISITE TO FURTHER FUNCTIONAL AND STRUCTURAL STUDIES )NDEED WHEREAS THE GENETIC MANIPULATION OF (ALOFERAX HAS BEEN ACHIEVED ALL ATTEMPTS FAILED SO FAR TO ESTABLISH A STABLE GENETIC SYSTEM FOR THERMOACIDOPHILIC 3UL FOLOBALES .EITHER USEFUL SHUTTLE VECTORS REPLICATING IN BOTH % COLI AND 3ULFOLOBUS NOR RELIABLE SELECTION MARKERS ARE PRESENTLY AVAILABLE 3UCCESSFUL GENETIC MANIPULATION OF EXTREME THERMOACIDOPHILIC !RCHAEA WOULD BE A MERITORIOUS CONTRIBUTION FOR THE ELUCIDA TION OF THE BASIC MOLECULAR MECHANISMS OF RESPIRATION IN PROKARYOTES "DLOPXMFEHNFOUT ) AM GRATEFUL TO $R 3 !NEMàLLER AND $R #, 3CHMIDT FOR CRITICAL ADVICE FRUITFUL DISCUSSIONS AND CAREFUL INSPECTION OF THE MANUSCRIPT AS WELL AS FOR PROVIDING SOME OF THEIR DATA PRIOR TO PUBLICATION ) AM INDEBTED ALSO TO $R & 0FEIFFER AND $R $ /ESTERHELT -AX 0LANCK )NSTITUTE -ARTINSRIED FOR GIVING ACCESS TO UNPUBLISHED GENOMIC DATA FROM ( SALINARUM ) AM GRATEFUL TO $R ! +LETZIN )NSTITUTE OF -ICROBIOLOGY 45 $AMSTADT FOR PROVIDING UNPUBLISHED DATA ON SUL FUR METABOLISM OF ! AMBIVALENS AND ALSO FOR INSIGHT INTO THE UNPUBLISHED ! ACIDIANUS D GENOME PROJECT ) THANK $R 0 0ALM -AX 0LANCK )NSTITUTE -ARTINSRIED FOR PROVIDING THE RHODOPSIN AND CAROTENOID DElCIENT ( SALINARUM STRAIN *7 FOR OUR EXPERIMENTS
3FGFSFODFT !AGAARD ! 'ILDERSON ' -ILLS $! &ERGUSON -ILLER 3 AND "RZEZ INSKI 0 2EDESIGN OF THE PROTON PUMPING MACHINERY OF CYTOCHROME C OXIDASE 0ROTON PUMPING DOES NOT REQUIRE 'LU) "IOCHEMISTRY n !LBRACHT 30* AND (EDDERICH 2 ,EARNING FROM HYDROG ENASES ,OCATION OF A PROTON PUMP AND OF A SECOND &-. IN BOVINE .!$( UBIQUINONE OXIDOREDUCTASE #OMPLEX ) &%"3 ,ETT n !LBRACHT 30* -ARIETTE ! AND $E *ONG 0 "OVINE HEART .!$(UBIQUINONE OXIDOREDUCTASE IS A MONOMER WITH &E 3 CLUSTERS AND &-. GROUPS "IOCHIM "IOPHYS !CTA n !NEMàLLER 3 AND 3CHÊFER ' #YTOCHROME AA FROM THE THERMOACIDOPHILIC ARCHAEBACTERIUM 3ULFOLOBUS ACIDOCALDARIUS &%"3 ,ETT n !NEMàLLER 3 AND 3CHÊFER ' #YTOCHROME AA FROM 3ULFOLO BUS ACIDOCALDARIUS %UR * "IOCHEM nn !NEMàLLER 3 ,àBBEN - AND 3CHÊFER ' 4HE RESPIRATORY SYSTEM OF 3ULFOLOBUS ACIDOCALDARIUS A THERMOACIDOPHILIC ARCHAEBACTERIUM &%"3 ,ETT n !NEMàLLER 3 "ILL % 3CHÊFER ' 4RAUTWEIN !8 AND 4EIXEIRA - %02 STUDIES OF CYTOCHROME AA FROM 3ULFOLOBUS ACIDO CALDARIUS%VIDENCE FOR A BINUCLEAR CENTER IN ARCHAEBACTERIAL TERMINAL OXIDASE %UR * "IOCHEM n !NEMàLLER 3 3CHMIDT #, 3CHÊFER ' AND 4EIXEIRA - %VIDENCE FOR A 2IESKE TYPE &E3 CENTER IN THE THERMOACIDO PHILIC ARCHAEBACTERIUM 3ULFOLOBUS ACIDOCALDARIUS &%"3 ,ETT n !NEMàLLER 3 3CHMIDT #, 0ACHECO ) 3CHÊFER ' AND 4EIXEIRA - A ! CYTOCHROME AA TYPE QUINOL OXIDASE FROM $ESUL FUROLOBUS AMBIVALENS THE MOST ACIDOPHILIC ARCHAEON &%-3 -ICROBIOL ,ETT n !NEMàLLER 3 3CHMIDT #, 3CHÊFER ' "ILL % 4RAUTWEIN !8 AND 4EIXEIRA - B %VIDENCE FOR A TWO PROTON DEPENDENT REDOX EQUILIBRIUM IN AN !RCHAEAL 2IESKE IRON SULFUR CLUSTER "IOCHEM "IOPHYS 2ES #OMMUN n !NEMàLLER 3 (ETTMANN 4H -OLL 2 4EIXEIRA - AND 3CHÊFER ' %02 CHARACTERIZATION OF AN !RCHAEAL SUCCINATE DEHYDROGENASE IN THE MEMBRANE BOUND STATE %UR * "IOCHEM n !ONO 3 "RYANT &/ AND !DAMS -77 ! NOVEL AND RE MARKABLY THERMOSTABLE FERREDOXIN FROM THE HYPERTHERMOPHILIC ARCHAEBACTERIUM 0YROCOCCUS FURIOSUS * "ACTERIOL n !VETISYAN !6 "OGACHEV !6 -URTASINA 2! AND 3KULACHEV 60 )NVOLVEMENT OF A DD TYPE OXIDASE IN THE .A MOTIVE RESPIRATORY CHAIN OF %SCHERICHIA COLI GROWING UNDER LOW 6 ( CONDITIONS &%"3 ,ETT n "ARNS 3- &UNDYGA 2% *EFFRIES -7 AND 0ACE .2 2EMARKABLE !RCHAEAL DIVERSITY DETECTED IN A 9ELLOWSTONE .ATIONAL 0ARK HOT SPRING ENVIRONMENT 0ROC .ATL !CAD 3CI 53! n "ARNS 3- $ELWICHE #& 0ALMER *$ AND 0ACE .2 0ER SPECTIVES ON !RCHAEAL DIVERSITY THERMOPHILY AND MONOPHYLY FROM ENVIRONMENTAL R2.! SEQUENCES 0ROC .ATL !CAD 3CI 53! n "ERRY %! (UANG ,3 :HANG :, AND +IM 3( 3TRUCTURE OF THE AVIAN MITOCHONDRIAL CYTOCHROME BC COMPLEX * "IOENERG
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$IBQUFS "SDIBFBM3FTQJSBUJPO MANAKA 4 -OLECULAR CLONING OF THE CYTOCHROME AA GENE FROM THE ARCHAEON !RCHAEBACTERIUM (ALOBACTERIUM HALOBIUM "IOCHEM "IOPHYS 2ES #OMMUN n $IMROTH 0 0RIMARY SODIUM ION TRANSLOCATING ENZYMES "IOCHIM "IOPHYS !CTA n $OOLITTLE 7& !T THE CORE OF THE !RCHAEA 0ROC .ATL !CAD 3CI 53! n $RIESSEN !*- (OW PROTEINS CROSS THE BACTERIAL CYTOPLASMIC MEMBRANE * -EMBR "IOL n &ETTER *2 1IAN * 3HAPLEIGH * 4HOMAS *7 'ARCIA (ORSMAN ! 3CHMIDT % (OSLER *0 "ABCOCK '4 'ENNIS 2" AND &ERGUSON -ILLER 3 0OSSIBLE PROTON RELAY PATHWAYS IN CYTOCHROME C OXIDASE 0ROC .ATL !CAD 3CI 53! n &ITZ 'IBBON 3 #HOI !* -ILLER *( 3TETTER +/ 3IMON -) 3WAN SON 2 AND +IM 5 ! FOSMID BASED GENOMIC MAP AND IDENTIlCATION OF GENES OF THE HYPERTHERMOPHILIC ARCHAEON 0YROBACULUM AEROPHILUM %XTREMOPHILES n &ITZ 'IBBON 3 ,ADNER ( +IM 5 3TETTER +/ 3IMON -) AND -ILLER *( 'ENOME SEQUENCE OF THE HYPERTHERMOPHILIC CRENARCHAEON 0YROBACULUM AEROPHILUM 0ROC .ATL !CAD 3CI 53! n &RIEDRICH 4 4HE .!$(UBIQUINONE OXIDOREDUCTASE COMPLEX ) FROM %SCHERICHIA COLI "IOCHIM "IOPHYS !CTA n &RIEDRICH 4 #OMPLEX ) ! CHIMAERA OF A REDOX AND CONFORMATION DRIVEN PROTON PUMP * "IOENER "IOMEMBR n &UJII 4 (ATA 9 7AKAGI 4 4ANAKA . AND /SHIMA 4 .OVEL ZINC BINDING CENTRE IN THERMOACIDOPHILIC !RCHAEAL FERREDOXINS .ATURE 3TRUCT "IOL n &UJIWARA 4 &UKUMORI 9 AND 9AMANAKA 4 0URIlCATION AND PROPERTIES OF (ALOBACTERIUM HALOBIUM @CYTOCHROME AA WHICH LACKS #U! AND #U" *OURNAL OF "IOCHEMISTRY n 'ARCIA (ORSMAN *! 0UUSTINEN ! 'ENNIS 2" AND 7IKSTRÚM 0ROTON TRANSFER IN CYTOCHROME BO UBIQUINOL OXIDASE OF %SCHERICHIA COLI 3ECOND SITE MUTATIONS IN SUBUNIT ) THAT RESTORE PROTON PUMPING IN THE MUTANT !SP A !SN "IOCHEMISTRY n 'EORGE '. 0RINCE 2# -UKUND 3 AND !DAMS -77 !L DEHYDE &ERREDOXIN /XIDOREDUCTASE FROM THE HYPERTHERMOPHILIC ARCHAEBACTERIUM 0YROCOCCUS FURIOSUS CONTAINS A TUNGSTEN OXO THIOLATE CENTER * !M #HEM 3OC n 'ERSCHER 3 $ÚPNER 3 (ILDEBRANDT 0 'LEISSNER - AND 3CHÊFER ' 2ESONANCE 2AMAN SPECTROSCOPY OF THE INTEGRAL QUINOL OXIDASE COMPLEX OF 3ULFOLOBUS ACIDOCALDARIUS "IOCHEMISTRY n 'ILDERSON ' !AGAARD ! 'OMES #- DELROTH 0 4EIXEIRA - AND "RZEZINSKI 0 +INETICS OF ELECTRON AND PROTON TRANSFER DURING / REDUCTION IN CYTOCHROME AA FROM ! AMBIVALENS !N ENZYME LACKING 'LU) "IOCHIM "IOPHYS !CTA n 'IUFFRÒ ! !NTONINI ' "RUNORI - $)TRI % -ALATESTA & .ICOLETTI & !NEMàLLER 3 'LEISSNER - AND 3CHÊFER ' 3ULFOLOBUS ACIDOCALDARIUS TERMINAL OXIDASE ! KINETIC INVESTIGATION AND ITS STRUCTURAL INTERPRETATION * "IOL #HEM n 'IUFFRÒ ! 'OMES # !NTONINI ' $)TRI % 4EIXEIRA - AND "RUNORI - &UNCTIONAL PROPERTIES OF THE QUINOL OXIDASE FROM !CIDIANUS AMBIVALENS AND THE POSSIBLE ROLE OF ITS ELECTRON DONOR 3TUDIES IN THE MEMBRANE INTEGRATED AND PURIlED ENZYME %UR * "IOCHEM n 'LEISSNER - %LFERINK -', $RIESSEN !*- +ONINGS 7. !NE
MàLLER 3 AND 3CHÊFER ' 'ENERATION OF PROTON MOTIVE FORCE BY AN !RCHAEAL TERMINAL QUINOL OXIDASE FROM 3ULFOLOBUS ACIDOCALDARIUS %UR * "IOCHEM n 'LEISSNER - +AISER 5 !NTONOPUOLOS % AND 3CHÊFER ' 4HE !RCHAEAL 3OX!"#$ COMPLEX IS A PROTON PUMP IN 3ULFOLOBUS ACIDOCALDARIUS * "IOL #HEM n 'OMES #- AND 4EIXEIRA - 4HE .!$( OXIDASE FROM THE THERMOACIDOPHILIC !RCHAEA !CIDIANUS AMBIVALENS )SOLATION AND PHYSICOCHEMICAL CHARACTERISATION "IOCHEM "IOPHYS 2ES #OMMUN n 'OMES #- (UBER ( 3TETTER +/ AND 4EIXEIRA - %VIDENCE FOR A NOVEL TYPE OF IRON CLUSTER IN THE RESPIRATORY CHAIN OF THE ARCHAEON 3ULFOLOBUS METALLICUS &%"3 ,ETT n 'OMES #- ,EMOS 23 4EIXEIRA - +LETZIN ! (UBER ( 3TETTER +/ 3CHÊFER ' AND !NEMàLLER 3 4HE UNUSUAL IRON SULFUR COMPOSITION OF THE !CIDIANUS AMBIVALENS SUCCINATE DEHYDROGE NASE COMPLEX "IOCHIM "IOPHYS !CTA n 'OMES #- 6ERKHOVSKAYA -, 4EIXEIRA - 7IKSTRÚM - AND 6ERKHOVSKY -) #ANONICAL PROTON PUMPING BY AN ENZYME THAT LACKS BOTH $ AND + CHANNELS %"%# 3HORT 2EPORTS 'OMES #- "ACKGREN # 4EIXEIRA - 0UUSTINEN ! 6ERVKHOVSKAYA -, 7IKSTRÚM - AND 6ERKHOVSKY -) A (EME COPPER OXIDASES WITH MODIlED $ AND + PATHWAYS ARE YET EFlCIENT PROTON PUMPS &%"3 ,ETT n 'OMES #- "ANDEIRAS 4- AND 4EIXEIRA - B ! NEW TYPE )) .!$( DEHYDROGENASE FROM THE ARCHAEON !CIDIANUS AMBIVALENS #HARACTERIZATION AND IN VITRO RECONSTITUTION OF THE RESPIRATORY CHAIN * "IOENERG "IOMEMBR n 'OMES # &ARIA ! #ARITA *# -ENDES * 2EGALLA - #HICAU 0 (UBER 2 3TETTER +/ AND 4EIXEIRA - $ICLUSTER SEVEN IRON FERREDOXINS FROM HYPERTHERMOPHILIC 3ULFOLOBALES * "IO )NORG #HEM n 'UTIERREZ #IRLOS %" AND 4RUMPOWER ", )NHIBITORY ANALOGS OF UBIQUINOL ACT ANTI COOPERATIVELY ON THE YEAST CYTOCHROME BC COMPLEX%VIDENCE FOR AN ALTERNATING HALF OF THE SITES MECHA NISM OF UBIQUINOL OXIDATION * "IOL #HEM n (ALLBERG 'RADIN # AND "ALTSCHEFFSKY ( 3OLUBILIZATION AND SEPARATION OF TWO B TYPE CYTOCHROMES FROM A CAROTENOID MUTANT OF (ALOBACTERIUM HALOBIUM &%"3 ,ETT n (ALLBERG 'RADIN # AND #OLMSJÚ ! &OUR DIFFERENT B TYPE CYTOCHROMES IN THE HALOPHILIC ARCHAEBACTERIUM (ALOBACTERIUM HALOBIUM "IOCHIM "IOPHYS !CTA n (ALLBERG 'RADIN # AND (EDERSTEDT , 3UCCINATE DEHY DROGENASE ACTIVITY AND THE SUCCINATE REDUCIBLE CYTOCHROME IN (ALOBACTERIUM HALOBIUN !CTA #HEMICA 3CANDIN " n (ELLWIG 0 "EHR * /STERMEIER # 2ICHTER /-( 0lTZNER 5 /DENWALD ! ,UDWIG " -ICHEL ( AND -ÊNTELE 7 )NVOLVEMENT OF GLUTAMIC ACID IN THE REDOX REACTION OF THE CYTOCHROME C OXIDASE FROM 0ARACOCCUS DENITRIlCANS INVESTI GATED BY &4)2 SPECTROSCOPY "IOCHEMISTRY n (ENDRIKS * 'OHLKE 5 AND 3ARASTE - A &ROM ./ TO // .ITRIC OXIDE AND DIOXYGEN IN BACTERIAL RESPIRATION * "IOENERG "IOMEMBR n (ENDRIKS * 7ARNE ! 'OHLKE 5 (ALTIA 4 ,UDOVICI # ,àBBEN - AND 3ARASTE - B 4HE ACTIVE SITE OF THE BACTERIAL NITRIC OXIDE REDUCTASE IS A DINUCLEAR IRON CENTER "IOCHEMISTRY n (ENNINGER 4 !NEMàLLER 3 &ITZ 'IBBON 3 -ILLER *( 3CHÊFER ' AND 3CHMIDT #, ! NOVEL 2IESKE IRON SULFUR PROTEIN FROM
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4VNNBSZ 4HE GROUP OF 'RAM POSITIVE BACTERIA IS A MAJOR PHYLUM OF PROKARYOTES INCLUDING SEVERAL TYPICAL SAPROPHYTIC AEROBES 4HEIR RESPIRATORY CHAINS ARE APPARENTLY SIMILAR TO THOSE OF EUKARYOTIC MITOCHONDRIA BUT IN SEVERAL POINTS ARE DIFFERENT FROM THEM 4HE RESPIRATORY CHAIN OF 'RAM POSITIVES LIKE MANY BACTERIA CONTAINS BRANCHED D ELECTRON TRANSFER PATHWAYS USUALLY HEME #U OXIDASES AND CYTOCHROME BD TYPE QUINOL OXIDASES -OST HEME #U OXIDASES ARE 3OX- TYPE AS IN THE MITOCHONDRIAL CYTOCHROME C OXIDASES BUT 3OX" TYPE CYTOCHROME C OXIDASES CYTOCHROME BAO ARE WORKING UNDER AIR LIMITED CONDITIONS IN THERMOPHILIC AND ALKALIPHILIC "ACILLUS SPECIES /N THE OTHER HAND 3OX- TYPE QUINOL OXIDASE CYTOCHROME AA IS MAINLY WORKING IN MESOPHILIC "ACIL LUS SUBTILIS 1UINOL CYTOCHROME C REDUCTASE OF LOW ' # CONTENT 'RAM POSITIVES &IRMICUTES IS COMPOSED OF A 2IESKE &E 3 SUBUNIT A SPLIT CYTOCHROME B B TYPE AND A NOVEL CYTOCHROME C SUBUNIT FORMED FROM @SUBUNIT )6 AND A KIND OF CLASS ) CYTOCHROME C WHILE THAT OF HIGH ' # CONTENT 'RAM POSITIVES !CTINOBACTERIA HAS A CYTOCHROME B A &E 3 PROTEIN WITH THREE TRANSMEMBRANE HELICES AND A NOVEL DIHEME CYTOCHROME CC WITHOUT ANY SMALL CYTOCHROMES C IN THE CELL 3OME OF THESE CHARACTERISTICS ARE DUE TO THE LACK OF OUTER MEMBRANE OF 'RAM POSITIVES IN WHICH ANY C TYPE CYTOCHROME SHOULD BE ANCHORED TO THE CELL MEMBRANE AND OTHERS ARE PROBABLY DUE TO THE EVOLUTIONARY HISTORY OF BACTERIA *0WFSWJFX "5XP%JTUJODU(SPVQTPG(SBN1PTJUJWF #BDUFSJB 4HE GROUP OF 'RAM POSITIVE BACTERIA IS A MAJOR PHYLUM OF THE EUBACTERIA WHICH INCLUDES MANY SPE CIES OF SCIENTIlC MEDICAL OR INDUSTRIAL IMPORTANCE -OLECULAR STUDIES OF THE BACTERIAL RESPIRATORY CHAIN HAS RELATIVELY BEEN FOCUSED ON PROTEOBACTERIA SUCH AS 0ARACOCCUS DENITRIlCANS 2HODOBACTER SPHAEROIDES AND %SCHERICHIA COLI SINCE THEIR MAIN RESPIRATORY ENZYMES ARE SIMILAR TO THOSE OF MITOCHONDRIA AND THAT THESE ORGANISMS ARE SUSCEPTIBLE TO GENETIC MANIPULA TION (OWEVER 'RAM POSITIVE BACTERIA IS ONE OF THE MAJOR GROUP OF PROKARYOTES COMPARABLE TO OR EVEN LARGER THAN PROTEOBACTERIA IN TERMS OF THE NUMBER OF !BBREVIATIONS #% n OCTO POLYOXYETHYLENE LAURYL ETHER #OM PLEX ) n .!$( DEHYDROGENASE TYPE ) AND )) #OMPLEX ))n 312 SUCCINATE DEHYDROGENASE #OMPLEX ))) n 1C2 QUINOLCYTO CHROME C REDUCTASE #OMPLEX )6 n #C/ CYTOCHROME C OXIDASE #YTn CYTOCHROME -!,$) 4/& n MATRIX ASSISTED LASER DESORPTION IONIZATION TIME OF mIGHT -+ n MENAQUINONE .$( ) n .!$( DEHYDROGENASE TYPE ) .$( )) n .!$( DEHYDROGENASE TYPE )) 0-& n PROTON MOTIVE FORCE 312 n SUCCINATEMENAQUINONE
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SPECIES INCLUDED 4HUS IT SHOULD BE FRUITFUL TO STUDY BIOCHEMISTRY AND MOLECULAR BIOLOGY OF THEIR RESPI RATORY CHAINS BOTH IN ORDER TO REVEAL THE VARIETY OF ENERGY METABOLISM OF THESE IMPORTANT ORGANISMS AND TO lND THE UNIVERSAL CORES OF ENERGETIC TRANSDUCING MECHANISMS 4HE 'RAM POSITIVE BACTERIA ARE SUBDIVIDED INTO TWO GROUPS BASED ON THE ' # CONTENT OF THE CHRO MOSOMAL $.! 4HE LOW ' # GROUP lRMICUTES INCLUDES ENDOSPORE FORMING RODS SUCH AS "ACILLUS SPECIES NONSPORE FORMING COCCI SUCH AS 3TAPHYLO COCCUS AUREUS AND %NTEROCOCCUS FAECALIS PHOTO SYNTHETIC BACTERIA SUCH AS (ELIOBACILLUS MOBILIS AND CELL WALL LESS BACTERIA MYCOPLASMAS WHILE THE HIGH ' # 'RAM POSITIVE BACTERIA ACTINOBACTERIA INCLUDES CORYNE FORM BACTERIA #ORYNEBACTERIUM ROD SHAPED MYCOLIC ACID CONTAINING ORGANISMS -YCOBACTERIUM AND lLAMENTOUS ACTINOBACTERIA OR !CTINOMYCES 4HE MOLECULAR COMPOSITION OF THE CELLS INCLUDING THE ENZYME COMPONENTS OF THE RESPIRATORY CHAIN IS SUBSTANTIALLY DIFFERENT IN THESE TWO GROUPS 4ABLE !CETOGENETIC LOW ' # 'RAM POSITIVE BACTERIA SUCH AS !CETOBACTERIUM WOODII AND #LOSTRIDIUM ACETICUM CAN GROW EITHER CHEMOORGANOTROPHICALLY BY FERMENTATION OF SUGAR OR CHEMOLITHOTROPHICALLY
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MULTI SUBUNIT .!$( DEHYDROGENASE TYPE ) .$( ) OR #OMPLEX ) )N THE HIGH ' # ACTINOBACTERIA COM PLEX ) IS PRESENT IN -YCOBACTERIUM TUBERCULOSIS AND 3TREPTOMYCES COELICOLOR BUT ABSENT IN -YCOBACTE RIUM LEPRAE AND # GLUTAMICUM 'ENES ENCODING SUCCINATE DEHYDROGENASE 312 OR #OMPLEX )) ARE FOUND IN THE GENOMES OF ALL THE SEQUENCED SPECIES BOTH IN THE LOW AND HIGH ' # GROUPS 4HE QUINONE USED BY THE 'RAM POSITIVE BACTERIA IS TYPICALLY MENA QUINONE -+ WITH THE ISOPRENOID SIDE CHAIN LENGTH VARYING BETWEEN SEVEN TO NINE UNITS AMONG THE SPE CIES -+S IN HIGH ' # 'RAM POSITIVES ARE PARTIALLY REDUCED 7HEN COMPARING THE RESPIRATORY CHAIN OF 'RAM POSITIVE BACTERIA TO THAT OF OTHER ORGANISMS USING UBIQUINONE IT IS IMPORTANT TO REMEMBER THAT THE REDOX POTENTIAL OF MENAQUINONE IS ALMOST M6 LOWER THAN THAT OF UBIQUINONE ,IKE MANY BACTERIA AND IN CONTRAST TO MITOCHONDRIA THE 'RAM POSITIVE BACTERIA OFTEN CONTAIN BRANCHED RE SPIRATORY CHAINS /NE IS THE #YT C REDUCTASE OXIDASE BRANCH COMPOSED OF TWO CONSECUTIVE ENZYMES #OM PLEX ))) #YT BC TYPE QUINOL #YT C OXIDOREDUCTASE OR 1C2 AND #OMPLEX )6 #YT C OXIDASE OR #C/ 4HE MAIN #C/ IS A #YT CAA TYPE HEME COPPER OXIDASE 3OX- TYPE BUT THE EXTREMOPHILIC SPECIES SUCH AS THE THERMOPHILIC "ACILLUS THERMODENITRIlCANS AND THE ALKALOPHILIC " HALODURANS CONTAIN A SECOND #C/ A 3OX" TYPE HEME COPPER OXIDASE 4HE SECOND BRANCH BYPASSES 1C2 AND CONSISTS OF QUINOL OXIDASES OF EITHER HEME COPPER OXIDASE OR #YT BDD TYPE 4HE LATTER TYPE OF QUINOL OXIDASES FORM A DISTINCT FAMILY
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"5IF0WFSBMM3FTQJSBUPSZ$IBJO 3EVERAL "ACILLUS SPECIES ARE MODERATELY THERMOPHILIC AND GROW AT TEMPERATURES BETWEEN # AND # " CALDOTENAX AND " CALDOLYTICUS WERE ISOLATED FROM A HOT SPRING IN 9ELLOWSTONE .ATIONAL 0ARK AND CAN GROW AT TEMPERATURES UP TO # "ACILLUS SP 03 WAS ISOLATED FROM A HOT SPRING IN *APAN AND GROW WELL AT n # -ANY THERMOPHILIC "ACILLUS STRAINS FOUND IN SOIL ARE CLASSIlED AS " STEAROTHERMOPHILUS 4HEY USUALLY GROW AT n # " THERMODENITRIlCANS STRAIN + 3TUDHOLME ET AL THAT WAS ISO LATED FROM SOIL IN *APAN AS A TRANSFORMABLE STRAIN WAS AT lRST CLASSIlED AS A STRAIN OF " STEAROTHERMOPHILUS .ARUMI ET AL AND CAN GROW AT n # 4HESE BACTERIA ARE AEROBIC AND IN RICH MEDIA THEIR DOUBLING TIME IS AS SHORT AS n MIN 4HEY ARE RICH IN #YTS AND COLORED BROWN THE #YT CONTENT IN THE MEMBRANE CAN BE HIGHER THAN NMOL MG MEMBRANE PROTEIN ESPECIALLY WHEN THE BACTERIA ARE GROWN AT HIGH TEMPERATURES 4HE THERMOSTABILITY OF THE CELLS ALSO FACILITATES PURIlCATION OF THE INTACT ENZYMES FROM THE RESPIRATORY CHAIN 4HERE IS NO WHOLE GENOME SEQUENCE AVAILABLE FOR ANY THERMOPHILIC "ACILLUS BUT THE BRANCHED RESPIRA TORY CHAIN OF A THERMOPHILIC "ACILLUS AS WE KNOW IT IS OUTLINED IN &IG 4HE QUINONE POOL CONSISTING OF MENAQUINONE -+ IS REDUCED BY SEVERAL DEHYDROGENASES SUCH AS .!$( SUCCINATE MALATE AND GLYCEROLPHOSPHATE DEHYDROGENASES 4HE .!$( DEHYDROGENASE IS OF TYPE )) A SINGLE POLYPEPTIDE WITH &!$ AS THE PROSTHETIC GROUP 8U ET AL AND DOES NOT TRANSLOCATE ( %NERGY CONSERVATION OCCURS UPON MENAQUINOL OXIDATION CATALYZED BY THE BC COMPLEX 1C2 AND #YT CAA #C/ WHICH IS
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&IG 0UTATIVE RESPIRATORY CHAIN OF THERMOPHILIC "ACILLUS + 3OLID ARROWS INDICATE THE MAIN ELECTRON TRANSFER BRANCH OPERATING UNDER HIGHLY AERATED CONDITIONS AND BROKEN ARROWS THE ALTERNATIVE BRANCHES UNDER AIR LIMITED CONDITIONS
ILLUSTRATED BY THE SOLID LINE IN THE lGURE !EROBICALLY GROWN THERMOPHILIC "ACILLUS CELLS SHOWED #YT A B AND C TYPE SPECTRUM SIMILAR TO THAT OF MAMMALIAN MITOCHONDRIA 3ONE ET AL WHICH IS MOSTLY ATTRIBUTABLE TO THE PRESENCE OF 1C2 AND #C/ .OTABLE DIFFERENCES TO THE MITOCHONDRIAL RESPIRATORY CHAIN IS THE ABSENCE OF .!$( DEHYDROGENASE TYPE ) THE PRESENCE OF A QUINOL OXIDASE AND A 3OX" TYPE #C/ AND THAT A #YT C IS FUSED TO SUBUNIT )) OF THE 3OX- TYPE #C/ 3ONE AND 9ANAGITA )SHIZUKA ET AL %SPECIALLY DURING GROWTH OF THERMOPHILES THE OXYGEN CONCENTRATION OF THE CULTURE MEDIUM OFTEN BECOMES VERY LOW IN SPITE OF SHAKING !T HIGH TEM PERATURES THE CONCENTRATION OF DISSOLVED OXYGEN IS LOW IN COMBINATION WITH THAT THE OXYGEN CONSUMPTION OF THE BACTERIA IS VERY RAPID GIVING RISE TO AN AIR LIM ITED CULTURE CONDITION !FTER SEVERAL HOURS OF GROWTH UNDER THESE CONDITIONS THE #YT PATTERN OF THE CELLS CHANGES 4HERE ARE TWO MOLECULAR EVENTS INVOLVED IN THE CHANGE ONE IS CAUSED BY HEME / ACCUMULATION DUE TO / LIMITATION WHICH RESULTS IN THE CONVERSION OF THE CAA #C/ TO CYTOCHROME CAO SEE BELOW AND ANOTHER IS THE APPEARANCE OF QUINOL OXIDASES THE ALTERNATIVE PATHWAYS TO USE / ILLUSTRATED BY THE BROKEN LINES IN &IG #$ZUPDISPNFDBBUZQF$D0 4HE STRUCTURAL GENES ENCODING THE CAA TYPE #C/ WERE CLONED AND SEQUENCED FROM "ACILLUS 03 3ONE ET AL )SHIZUKA ET AL AND FROM " THERMODENITRIlCANS +USANO ET AL 4HE GENES CTA#$%& ARE CLUSTERED WITH & CTA" AND CTA!
JUST LIKE THOSE OF " SUBTILIS 3ARASTE ET AL #TA" IS A HEME / SYNTHASE OR FARNESYL TRANSFERASE 3AIKI ET AL COMPARABLE TO % COLI #OX% 3AIKI ET AL /N THE OTHER HAND #TA! WHICH IS ENCODED FROM THE OTHER STRAND AND SEPARATELY EX PRESSED TURNED OUT TO BE A HEME ! SYNTHASE OR HEME / OXIDASEOXYGENASE WHICH CONVERTS THE METHYL GROUP OF PORPHYRIN INTO A FORMYL GROUP 3AKAMOTO ET AL #TA#$%& ARE STRUCTURAL GENES ENCODING & SUBUNITS )) ) ))) AND )6 RESPECTIVELY OF THE #C/ &IG A 3UBUNIT ) CONTAINS TRANSMEMBRANE HELICES AND BEARS HEME A AND THE HEME A #U" BINUCLEAR CENTER WHERE DIOXYGEN IS REDUCED 3UBUNIT )) CON SISTS OF . TERMINAL HYDROPHOBIC DOMAIN COMPOSED OF TWO TRANSMEMBRANE SEGMENTS AND A # TERMINAL HYDROPHILIC DOMAIN CONTAINING THE #U! CENTER AND A FUSED CYTOCHROME C DOMAIN )SHIZUKA ET AL 4HE SUBUNIT ))) WAS RATHER SHORT AND CONTAINED TRANSMEMBRANE HELICES 4HE ROLE OF SUBUNIT ))) AND )6 3ONE ET AL HAVE NOT BEEN ELUCIDATED BUT SINCE SOME BACTERIAL OXIDASE PREPARATIONS LACK SUBUNITS )6 ANDOR ))) (ALTIA ET AL THEY ARE PROPOSED TO BE NECESSARY ONLY FOR ASSEMBLY OF THE CATALYTIC PORTION COMPOSED OF SUBUNITS ) AND )) 4-0$ 2U.( AND PHENAZINE METHOSULFATE FUNCTION AS ARTIlCIAL ELECTRON DONORS FOR THE #C/ 3ONE AND 9ANAGITA AND #YTS C FROM SEVERAL DIF F FERENT SPECIES SUCH AS 03 #YT C 4 THERMOPHILUS #YT C #ANDIDA KRUSEI #YT C AND 3ACHHAROMYCES CEREVISIAE #YT C ARE OXIDIZED WELL BUT NOT EQUINE #YT C (OWEVER THESE SUBSTRATES SHOW RESPECTIVE +MS OF SEVERAL - INDICATING THAT THE HIGH AFlNITY SITE FOR #YT C IS OCCUPIED BY THE INTRINSIC #YT C DOMAIN
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&IG 'ENE MAP FOR #YT CAA TYPE #C/ AND #YT BC TYPE 1C2 OF " THERMODENITRIlCANS + 'ENE CLUSTERS FOR #YT CAA TYPE #C/ A AND FOR 1C2 B 3YMBOLS USED EMPTY ARROWS ¼ GENES 0 C PUTATIVE PROMOTERS 4 \n PUTATIVE TERMINATORS WITH A PALINDROME SEQUENCE
.ICHOLLS AND 3ONE )T IS THUS LIKELY THAT THE ELECTRONS FROM THE REDUCTANT REACH THE BINUCLEAR SITE THROUGH THE INTRINSIC #YT C AND ARE THEN PASSED ON TO #U! A #YT A A #YT A #U" AS IN THE MITOCHONDRIAL ENZYME (OWEVER RADIATION INACTIVATION EXPERIMENTS INDICATED THAT A MINOR PORTION OF THE DONATED ELECTRONS MAY REDUCE THE BINUCLEAR SITE WITHOUT MEDIATION OF #YT A 3ONE AND +OSAKO 4HE #C/ ENZYME FROM 03 NOT ONLY PUMPS ( 3ONE AND (INKLE 3ONE AND 9ANAGITA BUT ALSO SHOWS MANY PROPERTIES SIMILAR TO THE MI TOCHONDRIAL ENZYME IT PERFORMS RESTING TO PULSED FORM CONVERSION 3ONE ET AL A AND LIGAND BINDING WITH CYANIDE AND AZIDE 3ONE AND .ICHOLLS (OWEVER +I FOR CYANIDE IN THE 03 ENZYME IS - WHICH IS ABOUT FOLD LESS SENSITIVE THAN FOR THE BEEF HEART ENZYME MAINLY DUE TO A FASTER RATE OF DISSOCIATION OF CYANIDE 4HE 03 ENZYME FORMED INTERMEDIATES ! OXYGENATED AND " PEROXY UPON mASH PHOTOLYSIS OF THE #/ REDUCED FORM IN THE PRES ENCE OF / AT LOW TEMPERATURES n # AND n # RESPECTIVELY AS THE BOVINE ENZYME 3ONE ET AL B &LASH PHOTOLYSIS EXPERIMENTS AT THE ROOM TEMPERATURE SHOWED THE OXYGENATED FORM WITH A TIME CONSTANT OF S AS THE BOVINE ENZYME BUT NEITHER THE SECOND PEROXY NOR THE THIRD FERRYL
WAS DETECTED (IROTA ET AL 4HE REASON FOR THIS RESULT WAS DUE TO THE ELECTRON mOW FROM THE IN TRINSIC #YT C HARBORING THE lFTH ELECTRON IN THE FULLY REDUCED ENZYME &LASH INDUCED #/ DISSOCIATION FROM MIXED VALENCE ENZYME SHOWED THAT MORE ELECTRONS WERE BACK TRANSFERRED FROM HEME A TO HEME A THAN IN THE CASE OF THE BOVINE ENZYME SUGGESTING A HIGHER %M| OF THE 03 #YT A 2ESONANCE 2AMAN SPECTROSCOPY SHOWS THAT THE STRUCTURE OF THE 03 ENZYME IN THE VICINITY OF THE CHROMOPHORE IS VERY SIMILAR TO THAT OF THE BOVINE ENZYME INCLUDING THE CMn LINE DUE TO &E HIS TIDINE STRETCHING MODE 4HIS 2AMAN LINE WAS FOUND TOO SUSCEPTIBLE TO SELECTIVE HEAT INACTIVATION WHICH DESTROYS ( PUMP ACTIVITY WITHOUT SEVERE LOSS OF THE OXIDASE ACTIVITY /GURA ET AL 3ONE ET AL !LKALINE TREATMENT WHICH ALSO INDUCE LOSS OF ( PUMPING ACTIVITY IN THE BOVINE ENZYME DID NOT DESTROY THE CMn LINE AND HAD NO EFFECT ON THE 2AMAN LINE OR ( PUMPING IN THE 03 ENZYME 3ONE ET AL )T SHOULD BE NOTED THAT ALKALINIZA TION INDUCED MONOMERIZATION OF THE DIMERIC BOVINE ENZYME WHEREAS THE MONOMERIC BACTERIAL ENZYME 3ONE AND 4AKAGI MAY PUMP ( 4HE #YT CAO TYPE 03 ENZYME WITH HEME / AT THE HIGH SPIN HEME SITE PUMPED ( AS THE CAA TYPE AND
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SHOWED ALMOST THE SAME &E HISTIDINE i&E (IS AND &E #/ STRETCHING FREQUENCIES OF RESONANCE 2AMAN BUT THE FORMYL STRETCHING i#( / BAND OF THE HIGH SPIN HEME WAS ABSENT 3ONE ET AL 4HE +I VALUE FOR CYANIDE OF #YT CAO WAS FOUR TIMES SMALLER THAN THAT OF #YT CAA WHICH IS MAINLY DUE TO THE SLOWER @OFF CONSTANT KKOFFF OF THE FORMER $$ZUPDISPNFC DUZQF2D3 4HE QCR!"# # OPERON &IG B WAS CLONED FROM " THERMODENITRIlCANS AND SEQUENCED 3ONE ET AL 4HE BC COMPLEX EQUIVALENT TO 1C2 IS CATALYZING MENAQUINOL DEPENDENT #YT C REDUC TASE ACTIVITY AND IS COMPOSED OF THREE SUBUNITS ALTHOUGH A FORTH BAND WAS USUALLY FOUND IN THE lNAL PREPARATION +UTOH AND 3ONE 1CR! CONTAINS CYSTEINE AND HISTIDINE LIGANDS FOR A 2IESKE TYPE &E3 CLUSTER 4HE VERY HYDROPHOBIC K$A K$A IN 3$3 0!'% 1CR" SUBUNIT IS HOMOLOGOUS TO #YT B OF THE PLASTID B F COMPLEX AND CORRESPONDS THE . TERMINAL HALF OF MITOCHONDRIAL #YT B .OTABLY HEME B( OF THE " THERMODENITRIlCANS #YT B APPEARS TO BE COVALENTLY BOUND TO PROTEIN +UTOH AND 3ONE 4HE K$A 1CR# SUBUNIT CONTAINS A CLASS ) #YT C 3ONE AND 4OH BUT ITS . TERMINAL SEQUENCE IS HOMOLOGOUS TO SUBUNIT )6 OF THE PLASTID B F COMPLEX 7IDGER ET AL 4HE #YT BC COMPLEX FROM 03 HAS TWO QUINONE BINDING SITES )N PREPARATIONS OF DETERGENT SOLUBILIZED AND PURIlED 1C2 ALMOST ALL THE ENDOGENOUS -+ HAD BEEN LOST AND WAS RATHER DIFlCULT TO RECONSTITUTE DUE TO LOW SOLUBILITY OF -+ (OWEVER PREINCU BATING 1C2 WITH -+ IS MORE FEASIBLE 7HEN -+ +MS FOR THE LOW AND HIGH AFlNITY SITES WERE WAS USED + AND - RESPECTIVELY 5SING THE -+ PRE INCUBATED ENZYME THE +M OF THE LOW AFlNITY SITE FOR DUROQUINOL AND 51 WAS AND - RESPEC TIVELY 4HE ENZYME DONATES ELECTRONS TO #YT C VIA ITS INTRINSIC #YT C WITH RATHER LOW SUBSTRATE SPECIlCITY EQUINE #YT C AND YEAST #YT C CAN BE REDUCED AS WELL AS #YT C FROM "ACILLUS 03 1C2 ACTIVITY WAS INHIBITED BY MYXOTHIAZOL HEPTYL HYDROXYQUINO LINE . OXIDE AND ABIETIC ACID BUT NOT BY ANTIMYCIN ! . AS IN THE CASE OF PLASTID #YT B F COMPLEXES F %4VQFSDPNQMFYPG2D3BOE$D0 4HE TWO MAJOR RESPIRATORY COMPLEXES #YT BC AND #YT CAA SEEM TO FORM A SUPER COMPLEX HAVING
&IG $ITHIONITE REDUCED MINUS AIR OXIDIZED SPECTRUM OF #YT BC CAA TYPE QUINOL OXIDASE SUPERCOMPLEX FROM "ACILLUS 03
MENAQUINOL OXIDASE ACTIVITY 3ONE ET AL 4HE ENZYMES SEE &IG COMIGRATED DURING GEL lLTRATION AFTER SOLUBILIZATION BY A MILD DETERGENT SUCH AS #% 1C2 COPRECIPITATED WITH #C/ WHEN A 03 MEMBRANE FRACTION WAS SOLUBILIZED WITH HEPTYLTHIOGLUCOSIDE AND IMMUNO PRECIPITATED WITH ANTISERUM AGAINST #C/ "OTH THE CHROMATOGRAPHICALLY PURIlED SAMPLE AND THE IMMUNOPRECIPITATE SHOWED 4-0$ OXIDASE QUINOL DEPENDENT #YT C REDUCTASE AND QUINOL OXIDASE ACTIVITIES (OWEVER THE QUINOL OXIDASE ACTIVITY OF EI THER SUPER COMPLEX PREPARATIONS WAS NOT ACCELERATED BY ADDITION OF #YT C FROM THE SAME BACTERIUM 3ONE ET AL )T IS THUS LIKELY THAT THE PRESENCE OF THE SUPER COMPLEX IS PHYSIOLOGICALLY IMPORTANT 4HIS SUPER COMPLEX SEEMS TO BE FORMED IN MOST STRAINS OF THERMOPHILIC "ACILLUS )NTER COMPLEX CROSS LINKING BETWEEN 1C2 AND #C/ ESPECIALLY BETWEEN SUBUNITS ) AND )) OF #C/ AND #YT B OF 1C2 WAS CONlRMED BY BIFUNCTIONAL CROSSLINKERS SUCH AS | DITHIOBISSUCCINIMIDYL PROPIONATE $30 WHICH WAS NOT OBSERVED IN THE PRESENCE OF 4RITON 8 4ANAKA ET AL ! PROLONGED INCUBATION IN THE PRESENCE OF A HIGH CONCENTRATION OF 4RITON 8 IS NECESSARY TO OBTAIN PURE #YT BC COMPLEX ANDD CAA TYPE #C/ SEPARATELY 3ONE AND 9ANAGITA +UTOH AND 3ONE
/PCVIJUP4POF $FDJMJB)ÅHFSIÅMMBOE+VOTIJ4BLBNPUP 4ABLE %FFECTS OF AERATION ON CYTOCHROME CONTENT OF THE THERMOPHILIC "ACILLUS 03 $ATA ARE TAKEN FROM 3ONE ET AL 7AVELENGTH PAIR NM #ONTENT NMOL MG MEMBRANE PROTEIN #YTOCHROME 6IGOROUSLY AERATED /XYGEN LIMITED B n C n A n O
A
#/ DIFFERENCE SPECTRUM
&&GGFDUPG"JS-JNJUBUJPOPOUIF$ZUPDISPNF &YQSFTTJPO1BUUFSO 4HE #YT PATTERN OF THERMOPHILIC "ACILLUS CELLS CUL TURED UNDER AIR LIMITED CONDITIONS IS VERY DIFFERENT FROM THAT OF WELL AERATED CELLS 4HE REDUCED MINUS OXIDIZED AND #/ DIFFERENCE SPECTRA OF CELLS CULTURED UNDER BOTH CONDITIONS ARE COMPARED AND THE AMOUNT OF CYTOCHROMES IS SUMMARIZED IN 4ABLE #ELLS GROWN WITH LOW AERATION SYNTHESIZED HIGHER AMOUNTS OF #YT C AND #YT B AND LOWER AMOUNTS OF #YT A 4HE #/ DIFFERENCE SPECTRA INDICATE THAT THE TERMINAL OXIDASE CONTAINS #YT O UNDER AIR LIMITED CONDITIONS AND A #YT CAA IN WELL AERATED CELLS 3ONE ET AL 3ONE AND &UJIWARA B (EME / HYDROXYFARNESYLATED PROTOHEME )8 IS THE PRECURSOR OF HEME ! HAVING A METHYL GROUP AT THE POSITION NOT FORMYL (EME / FUNCTIONS AS THE PROSTHETIC GROUP OF #YT BO OFF % COLI 0UUSTINEN AND 7IKSTRÚM 7U ET AL 5NDER AIR LIMITED CONDITIONS THE CONVERSION OF HEME / TO HEME ! WHICH IS CATALYZED BY AN OXYGENASEOXIDASE MAY BE HAMPERED BY THE LOW CONCENTRATION OF DISSOLVED / IN THE CULTURE MEDIUM 3ONE AND &UJIWARA A "IOCHEMICAL ANALYSES OF THE #C/ INDICATED THAT THE PROTEIN PART OF THE OXIDASE WAS THE SAME EVEN IF THE ABSOLUTE AMOUNT BECOME LESS UNDER AIR LIMITED CONDI TIONS 4HUS THE #YT CAA TYPE OXIDASE WAS CHANGED TO A CAO CONTAINING THE SAME PROTEIN PARTS 3ONE AND &UJIWARA A 4HIS CHANGE ALSO AFFECTED THE TURNOVER NUMBER OF THE ENZYME SEVERAL KINETIC PA RAMETERS OF THE REACTION WITH CYANIDE AND THE FORMYL STRETCHING MODE IN THE 2ESONANCE 2AMAN SPECTRUM 3ONE ET AL 4HE CYTOCHROME CONTENT CHANGES OBSERVED BY DOWN SHIFT OF AERATION 4ABLE IS DUE TO INCREASED FORMATION OF SEVERAL #YTS OF B ANDD C TYPE 4HE CON CENTRATION OF CAAO TYPE OXIDASE BECAME LOWER AS MENTIONED BUT THE CONCENTRATION OF #YT C IN CREASED FROM ABOUT NMOLMG MEMBRANE PROTEIN
IN WELL AERATED CELLS TO n NMOLMG MEMBRANE PROTEIN IN AIR LIMITED CELLS 3ONE ET AL #YT C IS A MEMBRANE BOUND C TYPE #YT OF K$A &IG 4HE AMOUNT OF B TYPE #YTS WAS ALSO INCREASED 3ONE ET AL WHICH WAS LATER SHOWN TO BE DUE TO APPEARANCE OF #YT BD D QUINOL OXIDASE SIMILAR TO THAT OF % COLI AND A #YT BAO 3OX" TYPE #YT C OXIDASE &IG '$ZUPDISPNFCE 5ZQF2VJOPM0YJEBTF 3EVERAL STRAINS OF " THERMODENITRIlCANS LACKING #YT CAAO WERE SELECTED AFTER RANDOM MUTAGENESIS 3AKAMOTO ET AL 4WO ALTERNATIVE TERMINAL OXIDASES WERE PURIlED FROM MEMBRANES OF ONE SUCH MUTANT + BY ION EXCHANGE AND HYDROXYLAPATITE CHROMATOGRAPHIES #YD!" ENCODING THE TWO SUBUNIT #YT BD WAS ALSO CLONED AND SEQUENCED &IG A "OTH SUBUNITS ) AND )) ARE VERY HYDROPHOBIC PROTEINS AND HAVE EIGHT AND NINE TRANSMEMBRANE HELICES RESPECTIVELY 3AKAMOTO ET AL A #YT BDD FROM " THERMODENITRIlCANS CONTAINED NMOL HEME " AND NMOL HEME $ PER MG PROTEIN AND SHOWED AN ABSORBANCE PEAK AT NM OF REDUCED FORM &IG A AND A PEAK OF THE #/ DIFFERENCE SPECTRUM AT NM WHICH IS ABOUT NM SHORTER WAVELENGTHS THAN WHAT WAS FOUND IN THE % COLI AND ! VINELANDII #YT BD 4HE DIFFERENCE SEEMS TO BE DUE TO STRUCTURAL DIFFERENCES OF THE SURROUNDING POLYPEPTIDE SINCE THE PYRIDINE FERROHEMOCHROME SPECTRUM OF HEME $ IS NORMAL 4HE PURIlED #YT BD FUNCTIONED AS A QUINOL OXIDASE AFTER PRE INCUBATION FOR H WITH 51 OR -+ DUROQUINOL OXIDASE ACTIVITY OF ABOUT Sn WAS DETECTED AT # 3AKAMOTO ET AL A 4HE ACTIVITY WAS EFFECTIVELY INHIBITED BY P BENZOQUINONE DIMETHYL BENZOQUINONE AND :N#L ) OF AND M- RESPECTIVELY BUT ) ONLY SLIGHTLY INHIBITED BY .A#. ) ) OF M-
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&IG 'ENE MAP FOR #YT BD TYPE QUINOL OXIDASE AND #YT D BO TYPE #C/ OF " THERMODENITRIlCANS + 'ENE CLUSTERS FOR #YT BD TYPE QUINOL OXIDASE A AND FOR #YT D BO TYPE #C/ B 3YMBOLS ARE AS FOR &IG
4HE GENE AND SUBUNIT STRUCTURE OF #YT BOA FROM " THERMODENITRIlCANS IS SUMMARIZED IN &IG B #BA" ENCODES A SHORT SUBUNIT )) THE #U U! MOTIF WHILE CBA! ENCODES SUBUNIT ) WHICH CONTAINS THE SIX (IS RESIDUES THAT ARE LIGANDS FOR THE LOW SPIN HEME AND / REDUCING BINUCLEAR CENTER .IKAIDO ET AL #YT BOA FROM " THERMODENITRIlCANS CONTAINED HEME " AS THE LOW SPIN HEME HEME / OR HEME ! AS THE HIGH SPIN HEME AND #U! AND #U" SINCE HEME " HEME /HEME !#U WAS WHEN THE ENZYME WAS PREPARED FROM THE " THERMODENITRIlCANS + STRAIN WHICH LACKS #YT CAAO &IG B 4HE HEME " CONTENT OF ABOUT NMOLMG MEMBRANE PROTEIN IS SLIGHTLY LOWER THAN THAT EXPECTABLE FROM THE -R OF 4HE ENZYME SHOWED A HIGH 4-0$ OXIDASE ACTIVITY Sn AT # WHEN #YT C WAS SUPPLIED AND THE REACTION MEDIUM IS RICH IN SALT &IG 3AKAMOTO ET AL !NALYSIS OF SUBSTRATE SPECIlCITY INDICATED THAT THERE IS A HIGH AFlNITY SITE WHICH IS #YT C SPECIlC AND A LOW AFlNITY SITE WHERE 4-0$ AND MITOCHONDRIAL #YT C CAN REACT REMINISCENT OF IN MITOCHONDRIAL AA TYPE #C/ (OW EVER IN " THERMODENITRIlCANS HIGH CONCENTRATION OF SALT ACTIVATES THE REACTION INSTEAD OF INHIBITING
&IG !BSORPTION SPECTRA OF #YT BD TYPE QUINOL OXIDASE AND #YT D BO TYPE #C/ OF " THERMODENITRIlCANS + 3OLID AND BROKEN LINES ARE FOR REDUCED AND OXIDIZED FORMS RESPECTIVELY OF #YT BD TYPE QUINOL OXIDASE D A 3AKAMOTO ET AL A AND #YT BO TYPE #C/ B 3AKAMOTO ET AL )NSET REDOX DIFFERENCE SPECTRA OF PYRIDINE HEMOCHROME OF THE #C/
/PCVIJUP4POF $FDJMJB)ÅHFSIÅMMBOE+VOTIJ4BLBNPUP VERY SIMILAR TO THE 3OX- TYPE BOVINE ENZYME 4HE MOST PRONOUNCED DIFFERENCE WAS FOUND IN THE &E #/ STRETCHING MODE i&E #/ WHICH APPEARED AT CMn SUGGESTING THAT THE EFFECT OF #U" TO THE &E #/ BINDING WAS MUCH LOWER 4HE %02 DATA OF THE #/ REDUCED ENZYME FORM ALSO SUGGESTED A LOWER INTERACTION BETWEEN HIGH SPIN HEME AND #U" )5IF4NBMM$ZUPDISPNFD
&IG 3ALT DEPENDENCY OF #YT BO ENZYME ACTIVITY #C/ ACTIVITY WAS MEASURED IN THE PRESENCE OF THE INDICATED CONCENTRATIONS OF .A#L U OR +#L D 3AKAMOTO ET AL
IT 4HE ENZYME WAS SUSCEPTIBLE TO CYANIDE + +I OF - AND AZIDE + +I OF M- 4HE AMOUNTS OF #YT BOA FOUND IN STRAIN + WAS VARIABLE FROM CULTURE TO CULTURE AND WAS OFTEN TOO LOW TO ATTEMPT PURIlCATION ! MUTANT +Q HAVING VERY LOW QUINOL OXIDASE ACTIVITY WAS CREATED BY RANDOM MUTAGENESIS AND SELECTION IN THE PRESENCE OF P BENZOQUINONE 3ONE ET AL !N EXPRESSION VECTOR FOR #YT BOA WAS ALSO CONSTRUCTED USING THE NATURAL PROMOTER AND THE STRUCTURAL GENES CBA!" .IKAIDO ET AL 4HE " THERMODENITRIlCANS CELLS CONTAINING THE VECTOR PRODUCED AS NMOL #YT BO PER MG MEMBRANE PROTEIN UNDER AIR RESTRICTED GROWTH CONDITIONS 4HE ENZYME OVER EXPRESSED UNDER THESE CONDITIONS WAS NAMED #YT BO SINCE THE HEME AT THE HIGH SPIN SITE IS HEME / .IKAIDO ET AL JUST AS IN THE CASE OF #YT CAO 4HE SUBUNIT STRUCTURE AND AMINO ACID SEQUENCES INDICATE THAT THIS #C/ IS A 3OX" TYPE HEME #U OXIDASE 3OX" TYPE OXIDASES ARE OFTEN FOUND IN !RCHAEA AND ARE QUINOL OXIDASES CF #HAPTER BY 3AKAMOTO AND 3ONE 6OL ALTHOUGH A FEW 3OX" TYPE #C/ HAVE BEEN FOUND IN EUBACTERIA 4HE ACTIVE SITE ENVIRONMENT OF " THERMODENITRIlCANS #YT BO WAS EXAMINED BY %02 AND RESONANCE 2AMAN SPECTROSCOPIES 5CHIDA ET AL AND FOUND TO BE
"ACILLUS 03 #YT C IS A SMALL MEMBRANE PROTEIN OF ABOUT K$A 3ONE ET AL 4HE $.! FRAG MENT ENCODING #YT C APOPROTEIN COMPOSED OF AMINO ACIDS WAS CLONED &UJIWARA ET AL 4HE HYDROPATHY PLOT INDICATED THAT THE PROTEIN SHOULD NOT BE MEMBRANE BOUND BUT LIPASE TREATMENT GREATLY DIMINISHED THE HYDROPHOBICITY OF THE PROTEIN 4HE MASS NUMBER OF AFTER DEACYLATION SUGGESTED THAT THE . TERMINAL SIGNAL PEPTIDE OF AMINO ACID RESIDUES IS CLEAVED PROBABLY BY THE TYPE )) SIGNAL PEPTIDASE AND SUBSEQUENTLY THE NEW . TERMINAL #YS IS DIACYLGLYCERATED VIA THIOETHER AND THE AMINO GROUP IS BLOCKED BY ACETYLATION AS PREVIOUSLY SHOWN FOR THE #YT C SUBUNIT OF PHOTOSYNTHETIC REACTION CENTER FROM 2HODOPSEUDOMONAS VIRIDIS 7EYER ET AL ! #YT C EXPRESSION VECTOR WAS THEN CONSTRUCTED 4HE TRANSFORMED CELLS OF " THERMODENITRIlCANS EX PRESSED AS MUCH AS NMOL 03 #YT C PER MG OF MEMBRANE PROTEIN 4HE MOLECULAR MASS DETERMINATION AND TREATMENT WITH 2HIZOPUS LIPASE INDICATED THAT THE SAME PROCESSES CLEAVAGE OF SIGNAL PEPTIDASE BLOCK ING OF THE . TERMINAL GROUP AND DIACYL GLYCERYLATION OF THIOL TOOK PLACE IN THE OVER EXPRESSION SYSTEMS .OGUCHI ET AL !S DESCRIBED THE THERMOPHILIC BACILLI CONTAIN TWO ALTERNATIVE BRANCHES IN THE RESPIRA TORY CHAIN &IG #YT C SEEMS TO BE THE NATURAL PHYSIOLOGICAL SUBSTRATE OF THE BOA TYPE OXIDASE SINCE THE +M OF #YT BO FOR THIS #YT C WAS VERY LOW BELOW - AND THE DIACYLGLYCEROL MOIETY OF #YT C SPECIlCALLY ACTIVATES THE OXIDASE 3AKAMOTO ET AL 5SING THE OVER EXPRESSED #YT BO 3OX" TYPE OXI DASE INTERACTION WITH #YT C WAS ANALYZED IN MORE DETAIL .IKAIDO ET AL AND SUBSEQUENTLY ALMOST ALL ,YS RESIDUES IN #YT C WERE CHANGED TO !LA OR 3ER +AGEKAWA ET AL 4ABLE SUMMARIZES THESE RESULTS -OST OF CHANGES DID NOT AFFECT THE ACTIVITY MUCH BUT THE MUTANT #YT C SUCH AS +! +! 2! AND +! HAD LOWER + +M THAN THE WILD TYPE INDICATING THAT REMOVAL OF POSITIVE CHARGE CLOSE TO THE
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A
ND
A
OXIDASE BINDING DOMAIN RESULTED IN INCREMENT OF THE AFlNITY 4HE #! MUTANT THAT LACKS THE HYDROPHOBIC DIACYLGLYCEROL GROUP ALSO LOST ITS SUBSTRATE ACTIVITY 4HE IMPORTANCE OF THE DIACYL GROUP IS ALSO SHOWN BY THE FACT THAT DI DELIPIDATED C ALSO LOST ACTIVITY WHILE MONO DELIPIDATED C RETAINED MOST OF THE ACTIVITY +AGEKAWA ET AL 2ECENTLY SOLUBLE #YT C FROM "ACILLUS PASTEURII WAS PURIlED AND CRYSTALLIZED "ENINI ET AL 4HE SEQUENCE OF " PASTEURII #YT C IS HOMOLOGOUS TO #YT C OF 03 BUT IS CLEAVED BY UNPHYSIOLOGICAL PROTEOLYSIS AND THUS LOST THE LIPID ANCHOR (OWEVER THE CRYSTAL STRUCTURE OF #YT C SHOWED THAT THE FRONT FACE OF THE HEME CREVICE IS MOSTLY COVERED WITH HYDROPHOBIC AMINO ACID RESIDUES SUGGESTING HYDROPHOBIC INTERACTION BETWEEN THE CYTOCHROME C AND SUBUNIT )) OF THE #YT BO TYPE OXIDASE
**OEJWJEVBM3PMFTPGUIF5ISFF5FSNJOBM0YJ EBTFT 4HE MOST IMPORTANT POINTS OF THIS ISSUE ARE THE + +M FOR / AND THE ( TRANSLOCATING EFlCIENCY OF THE ENZYMES 4ABLE SUMMARIZES SEVERAL DATA ON THESE POINTS 4HE +M VALUES FOR / CAN BE DETERMINED BY TRACING THE TIME COURSES OF / CONSUMPTION WITH AN OXYGEN ELECTRODE IF THE + +M IS ABOVE - &OR LOWER VALUES LIKE THE +M OF THE BD TYPE OXIDASE FROM D % COLI THAT WAS AROUND N- RHEGHEMOGLOBIN CAN BE USED AS OXYGEN SEN SOR $MELLO ET AL )N THE CASE OF THE "ACILLUS 03 OR " THERMODENITRIlCANS ENZYMES THE + +M OF #YT CAA IS HIGHER THAN THAT OF #YT BO AND THAT OF #YT BD IS VERY LOW 4HE SECOND KEY VALUE ( En OR ( / RATIO IS USUALLY MEASURED USING PROTEOLIPOSOMES RE CONSTITUTED FROM PURIlED ENZYME AND PHOSPHOLIPIDS 4HERE IS HOWEVER A POSSIBILITY THAT THE ENZYME MAY LOOSE ( PUMPING ACTIVITY WHILE KEEPING ITS OXIDASE ACTIVITY INTACT 3ONE AND .ICHOLLS 3ONE ET AL OR THAT THE RECONSTITUTION WAS NOT APPROPRIATE !NOTHER POSSIBLE METHOD IS TO MEASURE THE ( / RATIO OF RESTING CELLS ALTHOUGH USING THIS APPROACH THERE IS SOME AMBIGUITY AS TO WHICH RESPIRATORY COMPLEXES PARTICIPATES AND WHAT SUBSTRATE IS USED )T IS USU ALLY ASSUMED THAT .!$( ANDOR .!$0( ARE USED TO REDUCE / DURING THE RAPID OXYGEN PULSE GIVEN TO THE ANAEROBIC CELL SUSPENSION 3ONE )N "ACIL LUS SPECIES THE FORMER PROBLEM IS SIMPLIlED SINCE PROTON PUMPING .$( ) IS ABSENT AND 1C2 TRANSLOCATE ( USING THE QUINONE CYCLE AS IN THE MITOCHONDRIAL 1C2 4RUMPOWER 4HE ( / RATIOS OF RESTING CELLS OF " THERMODENITRIlCANS + MAINLY USING BRANCH + MAINLY USING BRANCH WITHOUT HAV ING #YT CAA AND +Q USING #YT BOA WITHOUT HAVING #YT CAA OR #YT BD WERE ABOUT 3ONE ET D AL 4HUS THE EXPECTED ( En RATIOS FOR THREE TERMINAL OXIDASES WERE ALMOST IN ACCORDANCE
4ABLE #OMPARISON OF THREE TERMINAL OXIDASES IN " THERMODENITRIlCANS &EATURE #YT CAA #YT BO #YT BD %LECTRON DONOR 1C2 #YT BC #YT C -+ +M FOR / - ( / OF WHOLE CELLSA ( / RECONSTITUTED B B C #YANIDE SENSITIVITY ) - D A #ELLS CONTAIN MAINLY THE INDICATED TERMINAL OXIDASE BUT THE NUMBERS LISTED REPRESENT THE ( / STOICHIOMETRY OF THE WHOLE RESPIRATORY CHAIN B $ATA WERE TAKEN FROM 3ONE AND 9ANAGITA FOR #YT CAA AND FROM .IKAIDO ET AL FOR #YT BO C .OT DETERMINED 4HE VALUE SHOWN IS THE ONE EXPECTED FROM CHEMICAL PROTON D /BTAINED FROM A TITRATION CURVE OF CELL RESPIRATION 3ONE ET AL
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&IG 3UGGESTED RESPIRATORY CHAIN OF " SUBTILIS
WITH THE VALUES OBTAINED USING THE RESPECTIVE PURIlED ENZYME INLAYED IN LIPOSOMES )T IS THUS LIKELY THAT THREE TERMINAL OXIDASES HAVE DIFFERENT ROLES 7HEN THE / CONCENTRATION IS SUFlCIENT THE MOST EFlCIENT ENERGY CONSERVING OXIDASE #YT CAA IS EXPRESSED BUT WHEN / CONCENTRATION IS VERY LOW THE MORE WASTE FUL QUINOL OXIDASE #YT BD IS WORKING WHEREAS THE D 3OX" TYPE #YT BO IS INTERMEDIATE )T SEEMS LIKELY THAT ONE OF THE MAIN ROLES OF #YT BD IS TO REMOVE OXYGEN D WHICH MAY CAUSE INJURY TO CELLS
CORRESPOND TO THE ENZYME ISOLATED FROM STRAIN 7 %XPRESSION OF 9JL$ WAS REPRESSED UNDER ANAEROBIC GROWTH CONDITIONS -ARINO ET AL .O GENES ENCODING A #OMPLEX ).$( ) ARE PRESENT IN THE " SUBTILIS GENOME ! GENE THAT IS DENOTED NDH IN THE DATABASES DUE TO PRIMARY SEQUENCE SIMILARITY WITH THE #OMPLEX ) .$.UO, SUBUNIT ENCODES AN ANTIPORTER LIKE POLYPEPTIDE OF UNKNOWN FUNCTION -ATHIESEN AND (ÊGERHÊLL #4VDDJOBUF%FIZESPHFOBTF
***#BDJMMVTTVCUJMJT !MONG THE MESOPHILIC 'RAM POSITIVE BACTERIA THE RESPIRATORY CHAIN OF " SUBTILIS IS THE BY FAR MOST EX TENSIVELY STUDIED 4HE COMPLETE GENOME OFF " SUBTILIS STRAIN HAS BEEN SEQUENCED +UNST ET AL AND MANY OF THE COMPONENTS THAT CONSTITUTE THE " SUBTILIS RESPIRATORY CHAIN 4ABLE HAS ALSO BEEN STUDIED BIOCHEMICALLY IN GREAT DETAIL ,IKE THE THERMOPHILE "ACILLUS " SUBTILIS CONTAINS A BRANCHED AEROBIC RE SPIRATORY CHAIN WITH A #YT C REDUCTASE OXIDASE BRANCH AND QUINOL OXIDASE BRANCHES &IG "/"%)%FIZESPHFOBTF .!$( DEHYDROGENASE TYPE )) WAS ISOLATED FROM " SUBTILIS STRAIN 7 AND CHARACTERIZED BY "ERGSMA ET AL 4HE ENZYME IS A SINGLE MEMBRANE ASSOCI ATED POLYPEPTIDE CONTAINING &!$ AS THE PROSTHETIC GROUP 4HE GENOME OF " SUBTILIS CONTAINS THREE PUTATIVE .$( )) ENCODING GENES YJL$ YUT** AND YUM" )T IS PRESENTLY UNKNOWN WHICH ONE THAT MAY
4HE SUCCINATEMENAQUINONE OXIDOREDUCTASE 312 IN " SUBTILIS CONSISTS OF THREE POLYPEPTIDES ENCODED BY THE SDH#!" OPERON 4HOSE ARE A mAVOPROTEIN RESIDUES ENCODED BY SDH! CONTAINING COVALENTLY BOUND &!$ &0 AN IRON SULFUR PROTEIN RESIDUES ENCODED BY SDH" CONTAINING THREE IRON SULFUR CLUSTERS )0 AND A DIHEME #YT B MEMBRANE ANCHOR ENCODED BY SDH# # RESIDUES 3TRUCTURALLY THE ENZYME IS VERY SIMILAR TO THAT OF THE 7OLINELLA SUCCINOGENES FUMARATE REDUCTASE THAT ALSO POSSESSES A ONE POLYPEP TIDE DIHEME MEMBRANE ANCHOR FOR WHICH THE THREE DIMENSIONAL STRUCTURE HAS BEEN SOLVED ,ANCASTER ET AL )T SHOULD BE EMPHASIZED THOUGH THAT OTHER MEMBERS OF THIS ENZYME FAMILY THAT HAVE TWO POLYPEPTIDE ANCHORS CONTAINING ONE HEME OR LACKING HEME ALSO HAVE A CONSERVED STRUCTURE (ÊGERHÊLL AND (EDERSTEDT )VERSON ET AL ,ANCASTER ET AL 4HE TWO HEMES IN " SUBTILIS 312 HAVE DIFFERENT OPTICAL AND %02 SPECTRAL PROPERTIES AND A HIGH M6 AND LOW n M6 REDOX MIDPOINT POTENTIAL (ÊGERHÊLL ET AL 4HE TWO HEMES HAVE BIS HISTIDINE AXIAL LIGANDS 4HE HIGH POTENTIAL
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HEME HEME B0 IS LOCATED TOWARDS THE NEGATIVE SIDE OF THE MEMBRANE NEAR IRON SULFUR CLUSTER 3 AND IS LIGATED BY ( AND ( AND THE LOW POTENTIAL HEME HEME B$ IS LOCATED TOWARDS THE POSITIVE SIDE OF THE MEMBRANE LIGATED BY ( AND ( (ÊGERHÊLL ET AL (EME IS REQUIRED FOR PROPER ASSEMBLY OF THE 312 ENZYME IN " SUBTILIS (EDERSTEDT AND 2UTBERG 4HE ENZYME IS A SUCCINATE DEHYDROGENASE OF #LASS (ÊGERHÊLL CATALYZING OXIDATION OF SUCCINATE % %M| M6 AND REDUCTION OF -+ %M| n M6 A SEEMINGLY ENERGETICALLY UNFAVOR % ABLE REACTION )T WAS OBSERVED THAT SUCCINATE OXIDATION ACTIVITIES WERE SEVERELY REDUCED WHEN THE BACTERIA WERE DISRUPTED OR TREATED WITH UNCOUPLER ,EMMA ET AL 3CHIRAWSKI AND 5NDEN )T HAS THUS BEEN POSTULATED THAT THE #LASS ENZYMES CATALYZING -+ REDUCTION BY SUCCINATE ARE ENERGY COUPLED AND RELY ON THE TRANSMEMBRANE ELECTROCHEMICAL POTENTIAL FOR FUNCTION !LL #LASS 312S INVESTIGATED SO FAR CONTAIN TWO HEMES IN THE MEMBRANE ANCHOR WHEREAS ENZYMES OF #LASS UBIQUINONE REDUCING 312S OR #LASS FUMARATE REDUCTASES CAN HAVE DIFFERENT COMPOSITIONS (ÊGERHÊLL /THER FEATURES THAT IDENTIFY A #LASS 312 IS AN %M| OF THE BINUCLEAR IRON SULFUR CLUSTER 3 HIGHER THAN THAT OF THE SUCCINATEFU MARATE COUPLE M6 IN " SUBTILIS AND A LOWER % %M| OF THE TRINUCLEAR IRON SULFUR CLUSTER 3 n M6 IN " SUBTILIS 4HE -+ LIKE INHIBITOR (1./ WAS SHOWN TO BIND AT THE DISTAL QUINONE BINDING SITE 1$ NEAR HEME B$ IN " SUBTILIS 312 3MIRNOVA ET AL 4HE (9 AND (9 MUTANTS USED IN IDENTIlCATION OF THE HEME B$ AXIAL LIGANDS DO NOT ASSEMBLE A 312 ENZYME WITH &0 AND )0 BUT SUBSEQUENT MUTATIONS AT THE SAME POSITIONS (, AND (- GAVE RISE TO ASSEMBLED 312 4HE LATTER MUTANTS CONTAINED HEME B$ WITH ALTERED PROPERTIES ANDOR EXHIBITED NO OR VERY POOR QUINONE REDUCTASE ACTIVITY -ATTSSON ET AL 4HIS STRONGLY SUGGESTS THAT -+ REDUCTION OCCURS AT 1$ )N % COLI FUMARATE REDUCTASE A #LASS ENZYME THAT CARRY OUT THE REVERSE BUT ENERGETICALLY FAVORABLE REACTION COMPARED TO THE " SUBTILIS ENZYME (1./ BINDS AT THE PROXIMAL QUINONE BINDING SITE 10 CLOSE TO IRON SULFUR CLUSTER &2 (ÊGERHÊLL ET AL 4AKEN TOGETHER THE DATA lTS A MODEL WHERE IN " SUBTILIS 312 ELECTRONS ARE TRANSFERRED FROM SUCCINATE VIA &!$ A 3 A 3 A 3 A HEME B0 A HEME B$ TO -+ AT THE DISTAL QUINONE BINDING SITE USING THE 0-& TO DRIVE THE -+ REDUCTASE REACTION (EDERSTEDT )F THIS HOLDS TRUE WE EXPECT THAT FUMARATE REDUCTION BY A #LASS 312 SHOULD CREATE A MEMBRANE POTENTIAL 3CHNORPFEIL ET AL
INDEED DEMONSTRATED THAT FUMARATE REDUCTION BY .!$( WAS COUPLED TO MEMBRANE POTENTIAL GENERA TION IN " SUBTILIS )N CONTRAST TO SUCCINATE RESPIRATION THIS ACTIVITY WAS NOT INHIBITED BY UNCOUPLERS )N THE STRUCTURALLY VERY SIMILAR FUMARATE REDUCTASE FROM 7 SUCCINOGENES MENAQUINOL OXIDATION BY FUMARATE APPEARS TO BE AN ELECTRONEUTRAL PROCESS +RÚGER ET AL ,ANCASTER &INALLY IT SHOULD BE EMPHA SIZED THAT THE REQUIREMENT FOR MEMBRANE ENERGIZATION FOR RESPIRATORY ACTIVITY IN " SUBTILIS IS NOT A SPECIlC FEATURE FOR SUCCINATE OXIDATION USING THE LOW POTENTIAL MENAQUINONE BUT WAS ALSO OBSERVED IN A MUTANT STRAIN LACKING 312 AND USING OTHER SUBSTRATES THAN SUCCINATE !ZARKINA AND +ONSTANTINOV $2D3 4HE QCR!"# # OPERON OF " SUBTILIS IS VERY SIMILAR TO D AND THAT OF " THERMODENITRIlCANS ALREADY DESCRIBED ENCODES A 1C2 CLOSELY RELATED TO BUT DISTINCT FROM BC AND B F COMPLEXES 1CR! IS SIMILAR TO 2IESKE TYPE IRON SULFUR PROTEINS 1CR" RESEMBLES THE CYTOCHROME B OF B F COMPLEXES AND 1CR# APPEARS TO BE A FUSION OF A SUBUNIT )6 OF B F COMPLEXES AND A #YT F C DO MAIN 9U ET AL 4HE 1CR" AND 1CR# SUBUNITS GIVE RISE TO BANDS AT AND K$A RESPECTIVELY ON 3$3 0!'% 9U AND ,E"RUN )NTERESTINGLY NOT ONLY THE K$A BAND BUT ALSO THE K$A BAND RETAINED HEME AFTER DENATURATION )T WAS SUGGESTED THAT HEME B( IS COVALENTLY LINKED TO THE POLYPEPTIDE VIA #YS WHICH IS CONSERVED IN SEVERAL "ACILLUS SPECIES AND CYANOBACTERIAPLASTIDS +UTOH AND 3ONE 9U ET AL )N " SUBTILIS 1C2 EXPRESSION IS INDUCED AT THE END OF THE EXPONENTIAL GROWTH PHASE AND REDUCED AS THE BACTERIA MOVE FROM THE TRANSITION STATE INTO SPORULATION 9U ET AL %4NBMM$ZUD " SUBTILIS CONTAINS TWO SMALL C TYPE CYTOCHROMES WITH HOMOLOGOUS HEME DOMAINS BUT DIFFERENT MODE OF MEMBRANE ANCHORING #CC! OR CYTOCHROME C CONTAINS AN . TERMINAL TRANSMEMBRANE POLYPEPTIDE SEGMENT VON 7ACHENFELDT AND (EDERSTEDT WHEREAS #CC" OR CYTOCHROME C IS A LIPOPROTEIN "ENGTSSON ET AL B SIMILAR TO THE PREVIOUSLY DESCRIBED "ACILLUS 03 #YT C 4HE CCC" GENE IS COTRANSCRIBED WITH THE YVJ! GENE BUT 9VJ! IS NOT REQUIRED FOR #YT C SYNTHESIS AND ITS FUNCTION REMAINS UNKNOWN 4HE SMALL #YTS C SHOULD BE PART OF THE 1C2 #C/ BRANCH OF THE RESPIRATORY CHAIN BUT
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INDIVIDUAL SPECIlC FUNCTIONS HAVE NOT BEEN IDENTIlED -UTANTS DELETED FOR CCC! OR CCC" SHOW NO APPARENT PHENOTYPIC DIFFERENCES VON 7ACHENFELDT AND (ED ERSTEDT "ENGTSSON ET AL A &5FSNJOBM0YJEBTFT 4HE GENOME OF " SUBTILIS CONTAINS GENES FOR FOUR POTENTIAL OXIDASES 4ABLE &IG TWO HEME COPPER OXIDASES VAN DER /OST ET AL OF WHICH CAA IS A #C/ AND AA IS A QUINOL OXIDASE ,AURAEUS ET AL AND ONE POSSIBLY TWO #YT BD TYPE OXI D
DASES 4HE CAA TYPE #C/ IS ENCODED BY CTA#$%& 3ARASTE ET AL #TA# THAT ENCODES SUBUNIT )) OF THE #C/ WAS SHOWN TO BE A LIPOPROTEIN BUT THE LIPID MODIlCATION WAS SEEMINGLY NOT REQUIRED FOR FORMATION OF A FUNCTIONAL ENZYME "ENGTSSON ET AL B 4HE QUINOL OXIDASE AA IS ENCODED BY QOX !"#$ 3ANTANA ET AL 4WO ADDITIONAL GENES CTA! AND CTA" ARE REQUIRED FOR PRODUCTION OF THE TWO HEME COPPER OXIDASES SINCE THEIR GENE PRODUCTS ARE INVOLVED IN HEME ! BIOSYNTHESIS 3VENSSON ET AL 3VENSSON AND (EDERSTEDT 3TURR ET AL #YT BD D IS ENCODED BY THE CYD!"#$ OPERON WHERE CYD! AND " CONSTITUTE THE STRUCTURAL GENES ENCODING THE OXIDASE AND #YD# AND $ RESEMBLE A MEMBRANE BOUND !"# TRANSPORTER AND ARE REQUIRED FOR EXPRESSION OF A FUNCTIONAL #YT BD 7INSTEDT ET AL 4HE CYD D OPERON IS MAINLY EXPRESSED UNDER LOW OXYGEN GROWTH CONDITIONS 4HE OTHER PUTATIVE D IS PREDICTED FROM $.! SEQUENCE CYTOCHROME BD ANALYSES 4HE GENES YTH! AND YTH" ARE CLOSELY RELATED TO CBD! AND CBD" FROM " THERMODENITRIlCANS ENCOD ING A #YT BD 3AKAMOTO ET AL A BUT THERE IS NO D DIRECT EXPERIMENTAL EVIDENCE FOR THE PRESENCE OF THIS OXIDASE IN " SUBTILIS %ACH OF THE OXIDASES CAN BE DELETED INDIVIDUALLY WITHOUT ANY NOTICEABLE EFFECT ON GROWTH (OWEVER IT HAS BEEN DEMONSTRATED THAT ONE QUINOL OXIDASE EITHER AA OR #YT BD IS REQUIRED FOR D AEROBIC GROWTH AND THAT ONE HEME COPPER OXIDASE EITHER CAA OR AA IS NEEDED FOR EFlCIENT SPORULATION 7INSTEDT AND VON 7ACHENFELDT )N ADDITION THERE IS SPECTROSCOPIC DATA INDICATING THE PRESENCE OF A BB| TYPE OXIDASE IN " SUBTILIS BUT THE GENES ENCODING SUCH AN OXIDASE HAVE NOT BEEN IDENTIlED !ZARKINA ET AL 4HE 1C2 #C/ BRANCH #YT B C COMPLEX AND #YT CAA IS ONLY EXPRESSED IN VERY LOW AMOUNT IN VEGETATIVE " SUBTILIS CELLS SINCE SUCH CELLS LACKED ABSORBANCE FEATURES AROUND NM VON 7ACHENFELDT AND (EDERSTEDT INDICATING A VERY LOW C TYPE
#YT CONTENT 4HE ( / RATIO OF " SUBTILIS HAS BEEN DETERMINED TO BE AROUND *ONES WHICH IS IN AGREEMENT WITH THE VALUE EXPECTED FOR THE #YT AA TYPE QUINOL OXIDASE BRANCH )T IS THUS LIKELY THAT THE MAIN TERMINAL OXIDASE IN " SUBTILIS IS #YT AA ALTHOUGH THE A COMBINATION OF A #YT B C CAA BRANCH ( / OF AND A #YT BD QUINOL OXIDASE ( D / OF COULD ACCOUNT FOR THE OBSERVED ( / RATIO "OTH #YT B C AND #YT CAA WITH A CLEAR ABSORBANCE SHOULDER AT AROUND NM WAS OBSERVED IN MEMBRANES FROM " SUBTILIS GROWN IN SUCCINATE MEDIUM AND PURIlED #YT CAA WAS PREPARED FROM SUCH CELLS ,AURAEUS ET AL (ENNING ET AL 4HE ENZYME CONTAINS A MIXED VALENCE BINUCLEAR #U! COPPER CENTER VON 7ACHENFELDT AND (EDERSTEDT )N ADDITION THE PRESENCE OF HEME #U TERMINAL OXIDASES HAS BEEN SHOWN IN SEVERAL OTHER MESOPHILIC "ACILLUS SPECIES !LMOST EQUAL AMOUNTS OF #YT CAA AND #YT AA WERE FOUND TO BE PRESENT IN " CEREUS 'ARCIA (ORSMAN ET AL AND " BREVIS 9AGINUMA ET AL )NTERESTINGLY THE ( / RATIO OF THESE BACTERIA IS CLEARLY HIGHER THAN *7"MLBMJQIJMJD#BDJMMVT 3EVERAL "ACILLUS SPECIES ARE KNOWN TO GROW AT P( AS HIGH AS 3OME OF THEM ARE OBLIGATE ALKALIPHILES LIKE "ACILLUS 9. " ALCALOPHILUS AND " lRMUS 2!" WHILE SEVERAL SPECIES ARE FACULTATIVE AND ALSO ABLE TO GROW AT NEUTRAL P( %XAMPLES OF THE LATTER ARE "ACILLUS 9. " HALODURANS # AND " lRMUS /& "&OFSHZ$PVQMJOHJOUIF"MLBMJQIJMFT )T HAS BEEN DEMONSTRATED THAT A .A CYCLE PLAYS A CENTRAL ROLE FOR P( HOMEOSTASIS IN THE ALKALIPHILES +RULWICH ET AL THAT OFTEN MAINTAINS THE INTERNAL P( UP TO TWO P( UNITS MORE ACIDIC THAN THE EXTERIOR P( 4HIS REVERSE DIRECTION 6P( POSES A FUNDAMENTAL PROBLEM FOR A TRADITIONAL -ITCHELLIAN ENERGY COUPLING 9ET ALKALIPHILIC "ACILLUS THRIVE ON NON FERMENTABLE SUBSTRATES CONSERVING REDOX ENERGY IN THE FORM OF 0-& AND THE !40 SYNTHASE IS ( DE PENDENT ALTHOUGH MOST OF SECONDARY TRANSLOCATORS AND THE mAGELLAR MOTOR ARE .A DRIVEN )T MIGHT THUS BE EXPECTED THAT THE RESPIRATORY CHAIN COMPONENTS OF THESE BACTERIA ARE SPECIALLY ADAPTED FOR FUNCTION UNDER THESE CONDITIONS (ICKS AND +RULWICH ! SYSTEMATIC INVESTIGATION OF THE COMPONENTS OF THE
$IBQUFS (SBN1PTJUJWF#BDUFSJBM3FTQJSBUJPO 4ABLE %M| OF C TYPE CYTOCHROMES OF ALKALIPHILES IN COMPARISON WITH NEUTROPHILIC "ACILLUS SPECIES 4HE DATA WERE TAKEN FROM THE FOLLOWING REFERENCES #YT C OF THE THERMOPHILIC "ACILLUS 03 +UTOH AND 3ONE #YT C FROM " lRMUS 2!" $AVIDSON ET AL #YT C FROM "ACILLUS 9. 9UMOTO ET AL #YT C FROM " SUBTILIS VON 7ACHENFELDT AND (EDER STEDT #YT C IN CYT CAA FROM "ACILLUS 9. 9UMOTO ET AL #YT C IN #YT CAA OF THE THERMOPHILIC "ACILLUS 03 0OOLE ET AL | -ID POINT POTENTIAL %M| M6 !LKALIPHILE .EUTROPHILE 1C2 CYT C A /& 03 3MALL #YT C 2!" C 03 C 9. C "SU C #C/ #YT C OF CAA 9. 03 A %M| OF THE 2IESKE &E 3 CENTER IS SHOWN 2IEDLE ET AL
0-& HAS BEEN CARRIED OUT BY +RULWICHS GROUP WHEN THE P( OF THE CULTURE MEDIUM OF " lRMUS /& WAS CHANGED SUMMARIZED IN 4ABLE FOR REVIEWS SEE (ICKS AND +RULWICH +RULWICH ET AL &OR EXAMPLE 0-& ACROSS " lRMUS MEMBRANE ARE n M6 AT P( AND n M6 AT P( SINCE P(IN ARE AND AND 6^ ARE n AND n M6 RESPECTIVELY /N THE CONTRARY THE CELLS TO CULTURED AT P( FORM 6^ OF n M6 WITHOUT 6P( 4HE MEASURED 0-& WAS FAR SMALLER THAN M6 WHICH IS COMMONLY FOUND IN CELLS THAT SYNTHESIZE ONE !40 PER THREE ( )T IS THUS POSSIBLE EITHER THAT THE ( !40 RATIO MAY BE DIFFERENT OR CHANGEABLE IN ALKALIPHILES OR THAT THERE MAY BE SOME SPACE WHICH IS NOT IN EQUILIBRIUM WITH THE BULK PHASE THROUGH WHICH ( IS TRANSLOCATED BETWEEN THE REDOX COMPLEX AND ( !40 SYNTHASE 4HEY TEND TO POSTULATE SUPER COM PLEX FORMATION BETWEEN #C/ AND ( !40 SYNTHASE +RULWICH ET AL #3FTQJSBUPSZ$IBJO$PNQPOFOUTJOUIF "MLBMJQIJMFT 2ECENTLY THE WHOLE GENOME SEQUENCE OF THE ALKA LIPHILIC " HALODURANS WAS REPORTED 4AKAMI ET AL 5NFORTUNATELY VERY LITTLE IF ANY BIOCHEMICAL WORK HAS BEEN CARRIED OUT ON THE RESPIRATORY CHAIN ENZYMES OF " HALODURANS )N " lRMUS THE MAIN RESPI RATORY CHAIN COMPONENTS WERE REPORTED TO BE .!$( DEHYDROGENASE A -+ A 1C2 A #YT CAA A / WITH AN ALTERNATIVE QUINOL OXIDASE BRANCH USING A BD D TYPE QUINOL OXIDASE 'ILMOUR AND +RULWICH 4HE LATTER OXIDASE IS MAINLY EXPRESSED IN STATIONARY GROWTH PHASE )N " lRMUS NEITHER THE PRESENCE OF A
3OX" TYPE #C/ NOR AN AA TYPE QUINOL OXIDASE HAVE BEEN REPORTED )N THE GENOME OF " HALODURANS THERE ARE lVE GENE CLUSTERS ENCODING PUTATIVE DIFFERENT TERMINAL OXIDASES A #YT CAAnTYPE #C/ A QUINOL OXIDASE #YT AA A 3OX" TYPE #C/ AND TWO #YT BD )T REMAINS TO BE CONlRMED WHETHER " lRMUS /& LACKS THE GENES FOR A #YT AA TYPE QUINOL OXIDASE AND A 3OX" TYPE #C/ OR IF IT ONLY DID NOT EXPRESS THEM IN DETECTABLE AMOUNTS /"%)%FIZESPHFOBTF (ICKS AND +RULWICH POSTULATE THAT " lRMUS CONTAIN AN .!$( DEHYDROGENASE TYPE ) SINCE DE AMINO .!$( WAS OXIDIZED BY THE MEMBRANE FRACTION OF " lRMUS 4HE GENOME OF " HALODURANS 4ABLE DID NOT CONTAIN .$( ) ENCODING GENES (OWEVER SINCE THE SUBSTRATE SPECIlCITY OF .$( )) DEPEND ON THE ORGANISM AND THAT .$( )) FROM 4 THERMOPHILUS WAS SHOWN TO BE ABLE TO OXIDIZE DEAMINO .!$( 9AGI ET AL IT REMAINS TO BE ESTABLISHED IF " lRMUS CONTAINS A PROTON TRANSLOCATING .$( ) 8U ET AL HAVE ISOLATED AND CHARACTER IZED .$( )) AND ITS GENE FROM "ACILLUS 9. 4HE ENZYME CONTAINING ONE &!$ HAS MOLECULAR MASS OF K$A AND ITS SEQUENCE HAS HIGH SIMILARITY TO THIOREDOXIN REDUCTASE FROM % COLI 423 " HALODURANS GENOME ALSO TELLS THAT ITS 312 SHOULD BE VERY SIMILAR TO THAT OF " SUBTILIS 4HE ENZYME WAS PURIlED FROM " lRMUS /& WHICH WAS COMPOSED OF THREE SUBUNITS 'ILMOUR AND +RULWICH 2D3 ! 1C2 HAS NOT BEEN ISOLATED OR DIRECTLY DEMONSTRATED IN ANY ALKALIPHILE "ACILLUS SPECIES (OWEVER A 2IESKE TYPE &E 3 CENTER WAS DETECTED IN " lRMUS /& MEM BRANES 2IEDLE ET AL AND A BAND AT K$A WAS DETECTED WHEN A 3$3 0!'% OF THE SAME MEMBRANES WAS STAINED FOR HEME (ICKS AND +RULWICH ! QCR!"# # OPERON IS PRESENT IN THE " HALODURANS GENOME AND ITS DEDUCED AMINO ACID SEQUENCE IS VERY SIMILAR TO THAT OF " THERMODENITRIlCANS $4NBMM$ZUPDISPNFD 4WO MEMBRANE BOUND C TYPE #YTS HAVE BEEN ISOLATED FROM "ACILLUS 9. 9UMOTO ET AL /NE
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THE #YT C IS A MULTI SUBUNIT PROTEIN AND ITS FUNC TION HAS NOT BEEN ELUCIDATED 4HE OTHER #YT C IS A SMALL K$A POLYPEPTIDE WITH P) WHICH ) WAS SHOWN TO BE AN EFFECTIVE ELECTRON DONOR TO THE CAO OXIDASE IN THE PRESENCE OF POLY , LYSINE 4HE " HALODURANS GENOME CONTAINS ONE GENE FOR A SMALL #YT C POTENTIALLY ENCODING A AMINO ACID POLYPEPTIDE WITH ONE HEME # MOTIF 4HE PRIMARY SEQUENCE IS MORE SIMILAR TO " SUBTILIS #CC! THAN TO #CC" AND THE ESSENTIAL #YS RESIDUE IN THE . TERMINAL REGION IS ABSENT 4HUS THIS #YT C IS MOST LIKELY NOT A LIPOPRO TEIN BUT MAY BE MEMBRANE BOUND BY AN . TERMINAL HYDROPHOBIC SIGNAL SEQUENCE AS IN " SUBTILIS #YT C VON 7ACHENFELDT AND (EDERSTEDT 4HE C TYPE #YTS OF ALKALIPHILES ARE VERY ACIDIC PROBABLY BECAUSE THEY ARE LOCATED ON THE OUTER SURFACE OF THE CYTOPLASMIC MEMBRANE !NOTHER PECULIARITY OF THE C TYPE #YTS IN ALKALIPHILES IS THAT THEIR REDOX MIDPOINT POTENTIALS ARE MUCH LOWER THAN THOSE OF NEUTROPHILE AND THERMOPHILE "ACILLUS 4ABLE ! POSSIBLE REASON FOR THIS COULD BE THAT THE LOW %M| OF THE C TYPE #YTS MAY FACILITATE ELECTRON TRANSFER TO THE ACTIVE CENTERS OF THE OXIDASES LOCATED INSIDE OF THE MEMBRANE /TH ERWISE THE LARGE NEGATIVE INSIDE MEMBRANE POTENTIAL FOUND IN ALKALIPHILIC "ACILLUS 4ABLE MIGHT RETARD THE REACTION 9UMOTO ET AL %5FSNJOBM0YJEBTFT /NLY TWO OXIDASES FROM ALKALIPHILIC "ACILLUS HAVE BEEN CHARACTERIZED BIOCHEMICALLY NAMELY $ZUDBBUZQF$D0 ! #YT CAA #C/ PURIlED FROM " lRMUS /& WAS COMPOSED OF THREE SUBUNITS AND K$A AND IN ADDITION A POSSIBLE CANDIDATE FOR THE SMALL SUB UNIT )6 WAS FOUND 4HE ENZYME SHOWED VERY SIMILAR PROPERTIES TO THE #C/ FROM THE THERMOPHILIC "ACILLUS 03 1UIRK ET AL 4HE GENE ORGANIZATION IS ALSO VERY SIMILAR WITH CTA! AND CTA" PRECEDING THE FOUR STRUCTURAL GENES CTA#$%& 4HE DEDUCED AMINO ACID SEQUENCE IS ALSO VERY SIMILAR TO THAT OF "ACILLUS 03 EXCEPT FOR A CHARACTERISTIC FEATURE OF MEMBRANE SPANNING ENZYMES OF ALKALIPHILIC BACTERIA ! NUMBER OF OTHERWISE CONSERVED BASIC RESIDUES PREDICTED TO BE FACING THE OUTSIDE OF THE MEMBRANE ARE IN ALKALIPHILES SUBSTITUTED BY NEUTRAL OR ACIDIC RESIDUES &OR EXAMPLE THE #YT C BINDING DOMAIN OF SUBUNIT )) FROM " lRMUS HAS A PREDICTED P) OF WHICH IS MUCH LOWER THAN ) THE OR ABOVE FOUND FOR SUBUNIT )) IN " SUBTILIS AND
"ACILLUS 03 (ICKS AND +RULWICH ! #YT CAO TYPE #C/ WAS PURIlED FROM "ACILLUS 9. GROWN UNDER AIR LIMITED CONDITIONS AT P( 1URESHI 4HE #YT CONSISTED OF THREE POLYPEP TIDES AND K$A AND THE HEME COMPOSITION OF THE ENZYME WAS FOR HEME ! HEME # AND HEME / 4HE ENZYME CONTAINED ABOUT #U &E AND -G PER MOLE OF HEME ! 4HE PRESENCE OF #U! WAS SHOWN BY %02 AND 2ESONANCE 2AMAN SPECTRA INDICATED THAT THE ENVIRONMENT AROUND HEME / WAS SIMILAR TO THAT OF HEME A OF THE MITOCHONDRIAL AND "ACILLUS 03 ENZYMES 9UMOTO ET AL 4HESE MOLECULAR FEATURES INDICATE THAT THIS IS A TYPICAL "ACILLUS #C/ OF #YT CAA TYPE EXCEPT FOR THAT THE HEME AT THE HIGH SPIN SITE WAS SUBSTITUTED BY HEME / AS ALSO SEEN IN THE "ACILLUS 03 ENZYME 3ONE AND &UJIWARA A $ZUCEUZQF2VJOPM0YJEBTF ! #YT BDD QUINOL OXIDASE IS FOUND IN " lRMUS /& GROWN AT HIGH P( WHEN REACHING THE STATIONARY PHASE (ICKS ET AL 4HIS OXIDASE WAS PURIlED FROM A STRAIN OF /& WITH A DISRUPTED CTA GENE CLUSTER IN WHICH THE AMOUNT OF #YT BDD INCREASED SEVERAL TIMES ALTHOUGH THE MUTANT CELLS CAN GROW ONLY WITH FERMENT ABLE SUBSTRATE OR AT NEUTRAL P( WITH A NON FERMENTABLE # SOURCE 'ILMOUR AND +RULWICH 4HE ISOLATED ENZYME CONSISTED OF TWO POLYPEPTIDE SUBUNITS OF AND K$A AND SHOWED AN ABSORPTION PEAK AT NM UPON REDUCTION 4HESE PROPERTIES ARE VERY SIMILAR TO THOSE OF THE " THERMODENITRIlCANS #YT BD 3AKAMOTO ET AL A 7 $PSZOFCBDUFSJVNHMVUBNJDVN BOE0UIFS )JHI( $(SBN1PTJUJWF#BDUFSJB # GLUTAMICUM IS AN AEROBIC 'RAM POSITIVE HIGH ' # BACTERIUM WHICH IS OF INDUSTRIAL IMPORTANCE IN PRODUC ING AMINO ACIDS USED AS NUTRITIOUS ADDITIVES TO FOOD AND FEED 4HE ORGANISMS PREVIOUSLY NAMED "REVIBAC TERIUM LACTOFERMENTUM AND "R mAVUM ARE FOUND TO BE CLOSELY RELATED TO # GLUTAMICUM AND ARE THUS NOW CLASSIlED AS SUBSPECIES )N EARLY STUDIES #YTS A B AND C HAVE BEEN IDENTIlED SPECTROSCOPICALLY +AWAHARA ET AL 4HE #ORYNEBACTERIUM CELLS CONTAIN DI OR TETRAHYDROGENATED MENAQUINONE -+ ( OR -+ ( AS REPORTED FOR # DIPHTHERIAE 3CHOLES AND +ING AND "R LIPOLYTICUM 9AMADA ET AL ! REMARKABLE BIOCHEMICAL FEATURE WAS THAT THE
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&IG 3UGGESTED RESPIRATORY CHAIN OF # GLUTAMICUM
ORGANISM HAS ONLY ONE POLYPEPTIDE WITH COVALENTLY BOUND HEME # WHOSE APPARENT MOLECULAR WEIGHT IS ABOUT K$A AS JUDGED BY 3$3 0!'% OF THE TOTAL PROTEINS AND HEME STAINING OF THE GEL )N A TYPICAL #YT BC C AA RESPIRATORY PATHWAY THERE ARE TWO C TYPE #YTS A PERIPHERAL SMALL #YT C AND THE #YT C SUBUNIT OF THE 1C2 WHILE IN A QUINOL OXIDASE BRANCH THERE ARE NO C TYPE #YTS 4HEREFORE THE PRESENCE OF A SOLE C TYPE #YT HAD BEEN MYSTERIOUS &IG "5IF$ZUPDISPNFCDDUZQF2D3 4HIS SINGLE C TYPE #YT WAS SOLUBILIZED WITH DETERGENTS AND PURIlED TO HOMOGENEITY FROM # GLUTAMICUM AND FOUND TO CONTAIN TWO MOLES OF HEME # PER MOLE POLY PEPTIDE 3ONE ET AL 4HE . TERMINAL AMINO ACID RESIDUE WAS NOT DETECTED WITH %DMAN DEGRADATION SUGGESTING THAT ITS AMINO GROUP WAS MODIlED WHILE TWO INTERNAL PEPTIDE SEQUENCES COULD BE OBTAINED )N PARALLEL THE STRUCTURAL GENES FOR THE 1C2 QCR#!" WERE CLONED AND THE AMINO ACID SEQUENCE DEDUCED FROM THE QCR# # GENE MATCHED THE PARTIAL PEPTIDE SEQUENCES FROM THE PURIlED C TYPE #YT &IG A )NDEED 1CR# CONTAINS TWO HEME # BINDING MOTIFS #88#(8N- 3ONE ET AL .IEBISCH AND "OTT )N ADDITION 1CR# HAS A HYDROPHOBIC SEGMENT AT THE # TERMINUS WHICH COULD FORM A TRANSMEMBRANE _ HELIX 4AKEN TOGETHER THESE lNDINGS CLEARLY INDI CATE THAT THIS #YT IS A HYDROPHOBIC DIHEME PROTEIN COMPOSED OF TWO #YT C DOMAINS FUSED IN TANDEM AND THUS THE PROTEIN WAS NAMED #YT CC 4HE MOLECULAR WEIGHT OF #YT CC MEASURED WITH MATRIX ASSISTED LASER DESORPTION IONIZATION TIME OF mIGHT -!,$) 4/& MASS SPECTROMETRY WAS SMALLER THAN THAT OF THE TOTAL WEIGHT OF TWO HEME # MOLECULES PLUS A 1CR# POLYPEPTIDE DEDUCED FROM THE NUCLEOTIDE SEQUENCE SUGGESTING A POSTTRANS
LATIONAL MODIlCATION 3AKAMOTO ET AL UNPUBLISHED 4HE PRIMARY SEQUENCE OF #YT CC IS NOT HOMOLOGOUS TO THE #YT C SUBUNIT OF REGULAR #YT BC COMPLEXES )NSTEAD THE . TERMINAL DOMAIN IS MOST SIMILAR TO "ACILLUS SMALL #YT C SUCH AS #YT C FROM THERMO PHILIC "ACILLUS 03 AND #YT C FROM " SUBTILIS #CC! 4HE # TERMINAL DOMAIN RESEMBLES TO #YT C FROM CYANOBACTERIA 3ONE ET AL 4HE 1CR! COMPRISING THE 2IESKE &E3 PROTEIN AND 1CR" FORMING THE CYTOCHROME B IN # GLUTAMI CUM ARE HOMOLOGOUS TO THE CORRESPONDING SUBUNITS OF REGULAR #YT BC COMPLEX IN AEROBIC ORGANISMS (OWEVER THESE TWO PROTEINS FROM # GLUTAMICUM TOGETHER WITH THE COUNTERPARTS FROM OTHER ACTINOBAC TERIA ARE MOST SIMILAR TO THOSE FROM RADIO RESISTANT 'RAM NEGATIVE BACTERIA 4HERMUS THERMOPHILUS AND $EINOCOCCUS RADIODURANS AND COMPOSE A NEW GROUP )T IS ALSO NOTEWORTHY THAT THE #YT B AND 2IESKE &E3 PROTEIN COMPOSE RELATIVELY CONSERVATIVE CORE OF 1C2 WHEREAS A DIFFERENT #YT C IS ADOPTED AS THE SUBUNIT CONTAINING HEME # IN AN ANCESTRAL ORGANISM OF EACH PHYLOGENETIC GROUP OR SOMETIMES TWO SUCH AS IN THE CASE OF # GLUTAMICUM 4HE 1C2 SUBUNITS EASILY DISSOCIATE IN THE PRESENCE OF VARIOUS DETERGENTS AND THUS IT IS DIFlCULT TO PURIFY THE INTACT PROTEIN COMPLEX 3ONE ET AL /N THE OTHER HAND A HETEROGENEOUS SAMPLE OF 1C2 PARTIALLY PURIlED IN A MILD DETERGENT ALSO CONTAINS #YT AA SEE ALSO THE FOLLOWING SECTION 4HIS OBSERVATION SUGGESTS THAT THE TWO ENZYMES COMPOSE A LOOSELY ASSOCIATED SUPER COMPLEX /NE OF THE POLYPEPTIDES IN THIS LATTER PREPARATION WAS IDENTIlED AS 1CR! THE 2IESKE &E3 PROTEIN BY DETERMINING THE . TERMINAL AMINO ACID SEQUENCE WHICH LACKS THE INITIAL -ET BUT STARTS WITH THE SECOND RESIDUE ...$+19x 3AKAMOTO ET AL UNPUBLISHED 6ERY RECENTLY .IEBISCH AND "OTT SHOWED THAT THE TWO ENZYMES INDEED FORM
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A SUPER COMPLEX USING AFlNITY CHROMATOGRAPHY OF STREP TAGGED #YT B 1CR" AND STREP TAGGED SUBUNIT ) #TA$ %ITHER OF THE TWO SUBUNITS EXPRESSED IN THE CELLS WERE ALWAYS COPURIlED WITH ALL THE SUBUNITS OF #YT BCC COMPLEX AND #YT AA INCLUDING THE FOURTH SUBUNIT #TA& OF THE LATTER ENZYME ! UNIQUE FEATURE OF THE 2IESKE &E3 PROTEIN IN THE HIGH ' # 'RAM POSITIVES IS THAT IT CONTAINS THREE . TERMINAL TRANS MEMBRANE _ HELICES WHEREAS IN ALL OTHER ORGANISMS ONLY ONE TRANSMEMBRANE SEGMENT IS PRESENT 3INCE NO SMALL #YTS C IS PRESENT AND THE ONLY C TYPE #YT IS THE DIHEME # #YT SUBUNIT OF THE 1C2 IT IS LIKELY THAT ONE OF THE TWO C TYPE #YT DOMAINS OF 1C2 REPLACES THE SMALL #YT C IN MEDIATING ELECTRON TRANSFER )N OTHER WORDS THE SMALL #YT C HAS BEEN GE NETICALLY FUSED TO THE ANCESTRAL MONO HEME # SUBUNIT OF THE 1C2 RESULTING IN A #YT BCC COMPLEX &IG A #ONSEQUENTLY IT IS LIKELY THAT THE #YT BCC COMPLEX MUST DONATE ELECTRONS DIRECTLY TO THE TERMINAL OXIDASE
&IG AND THUS THE PRESENCE OF A SUPER COMPLEX IS NOT SURPRISING #$ZUPDISPNFBBUZQF$D0 #YT AA WAS PURIlED FROM THE # GLUTAMICUM MEM BRANES AS A THREE SUBUNIT #YT C OXIDASE 3AKAMOTO ET AL )N ADDITION ALL THE THREE STRUCTURAL GENES HAVE BEEN CLONED &IG A B 4HE MOLECULAR WEIGHTS OF SUBUNITS ) AND ))) DETERMINED WITH -!,$) 4/& MASS SPECTROMETRY WERE IDENTICAL WITHIN THE EXPERI MENTAL ERROR TO THOSE DEDUCED FROM THE NUCLEOTIDE SEQUENCE WHEREAS THAT MEASURED FOR SUBUNIT )) WAS LOWER THAN THAT DEDUCED FROM THE PRIMARY SEQUENCE %DMAN DEGRADATION COMBINED WITH DATA FROM MASS SPECTROMETRY INDICATED THAT THE . TERMINAL RESI DUES OF SUBUNIT )) ARE CLEAVED OFF AND #YS IS DIACYL GLYCERATED 3AKAMOTO ET AL 4HE ! TYPE HEME IN THIS ENZYME IS HEME !S WHICH HAS A GERANYLGERANYL
&IG 'ENE MAPS FOR THE RESPIRATORY ENZYMES OF # GLUTAMICUM 'ENE CLUSTERS FOR ALL THE THREE SUBUNITS OF #YT BC TYPE 1C2 AND D SUBUNITS )) ))) AND )6 ORF OF #YT CAA TYPE #C/ A FOR SUBUNIT ) OF THE #C/ B AND FOR BOTH OF THE TWO SUBUNITS OF #YT BD TYPE QUINOL OXIDASE
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CLUSTER MAY PLAY A ROLE IN THE SPECIlC INTERACTION OF THE OXIDASE WITH THE #YT C SUBUNIT OF THE NEW TYPE OF BCC COMPLEX !S DESCRIBED ABOVE NO C TYPE #YTS OR INTRINSIC SUBSTRATES FOR THE #YT AA OXIDASE HAVE BEEN FOUND IN THE CELLS BESIDES THE #YT BCC COMPLEX /XIDASE ACTIVITIES MEASURED USING #YTS C FROM YEAST AND HORSE HEARTS #YT C FROM THERMOPHILIC "ACIL LUS OR OTHER 'RAM POSITIVE BACTERIA AND THE ARTIlCIAL
PHENYLENE DIAMINE SUBSTRATE . . .| .| TETRAMETHYL P 4-0$ ARE ALL VERY LOW 3AKAMOTO ET AL 4HE EXTRA CHARGED INSERTED IN SUBUNIT )) MIGHT CONTRIBUTE TO THE SUBSTRATE SPECIlCITY OF THE OXIDASE 4HE GENES EN CODING SUBUNITS )) CTA# AND ))) # CTA% OF THE OXIDASE ARE LOCATED DIRECTLY UPSTREAM OF THE QCR#!" OPERON WHILE THAT FOR SUBUNIT ) CTA$ IS LOCATED SEPARATELY 4HE CLOSE LOCATION OF THE GENES FOR #YT BCC COMPLEX AND SUBUNIT )) OF #YT AA IS A FURTHER INDICATION OF THE CLOSE RELATIONSHIP IN THE ELECTRON TRANSFER BETWEEN THE #YT C REDUCTASE AND THE OXIDASE $$ZUPDISPNFCEUZQF2VJOPM0YJEBTF
&IG &IVE EXAMPLES OF #YT C MEMBRANE INTERACTION A 3OLUBLE #YT C AS IN THE MITOCHONDRIAL RESPIRATORY CHAIN B #YT C FUSED TO #C/ C #YT C HAVING A LIPID ANCHOR AS IN THE "ACILLUS SPECIES D #YT C HAVING AN . TERMINAL PEPTIDE ANCHOR AS IN " SUBTILIS AND E #YT C FUSED TO 1C2 AS IN 'RAM POSITIVE BACTERIA WITH HIGH ' #
GROUP SIDE CHAIN INSTEAD OF A FARNESYL (EME !S WAS lRST IDENTIlED IN !RCHAEA ,àBBEN AND -ORAND 4HE ENZYME IS SIMILAR TO OTHER 3OX- TYPE #YT C OXIDASES HOWEVER AN EXTRA CHARGED AMINO ACID CLUSTER IS INSERTED BETWEEN THE ` AND ` STRANDS IN THE SUBSTRATE BINDING DOMAIN OF SUBUNIT )) 4HE ` ` LOOP OF THIS OXIDASE IS ABOUT RESIDUES LONGER THAN THAT OF THE MAJOR 3OX- TYPE #C/S AND RICH IN BOTH ACIDIC AND BASIC RESIDUES 4HIS EXTRA CHARGED
! SECOND TERMINAL OXIDASE THE QUINOL OXIDASE #YT BDD HAS BEEN PURIlED FROM # GLUTAMICUM CELLS AND WAS SHOWN TO HAVE HIGH MENAQUINOL OXIDASE ACTIVITY +USUMOTO ET AL 4HE GENES CYD!" ENCODING SUBUNITS ) AND )) HAVE ALSO BEEN CLONED &IG C 4HE #YTS BDD CAN BE DIVIDED INTO TWO SUBFAMILIES 3AKA MOTO ET AL A 4HOSE FROM MANY PROTEOBACTERIA INCLUDING % COLI HAVE A LONG HYDROPHILIC LOOP CALLED @1 LOOP WHICH IS SUGGESTED TO BE IMPORTANT FOR QUINOL BINDING AND OXIDATION BETWEEN TH AND TH TRANS MEMBRANE SEGMENT WHILE THOSE FROM 'RAM POSITIVES CYANOBACTERIA ARCHAEA AND SOME PROTEOBACTERIA HAVE A SHORTER 1 LOOP !LTHOUGH THE SPECTRAL PROPERTIES AND THE SIZE OF SUBUNIT ) OF # GLUTAMICUM #YT BD ARE SIMILAR TO THOSE OF % COLI AND ! VINELANDII #YT BD THE ENZYMATIC PROPERTIES AND DEDUCED AMINO ACID SEQUENCE CLEARLY INDICATE THAT THE #YT IS FUNCTIONALLY AND STRUCTURALLY CLOSER TO " THERMODENITRIlCANS #YT BDD AND BELONGS TO THE LATTER SUBFAMILY %3FTQJSBUPSZ$IBJO$PNQPOFOUTBOE1SP UPO1VNQJOH 4HE WHOLE GENOME SEQUENCE OF # GLUTAMICUM BECAME AVAILABLE IN THE PUBLIC $.! DATABASE OF $$"*%-",'EN"ANK IN 4HE SEQUENCE CONlRMS THAT THE RESPIRATORY CHAIN CONTAINS ONLY TWO ELECTRON TRANSFER BRANCHES THE BCC AA ROUTE AND THE #YT BD TYPE QUINOL OXIDASE ROUTE DOWNSTREAM OF THE D
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QUINONE POOL AS FAR AS THE CONVENTIONAL HOMOLOGY SEARCHES CAN DETECT ! CYANIDE INSENSITIVE OXIDASE ACTIVITY HAS BEEN REPORTED -ATSUSHITA ET AL +USUMOTO ET AL BUT ITS MOLECULAR ENTITY HAS NOT BEEN IDENTIlED 5PSTREAM OF THE QUINONE POOL A PROTON PUMPING .$( )COMPLEX ) IS ABSENT IN # GLUTAMICUM WHILE .$( )) IS PRESENT 3UCCINATE DEHYDROGENASE IS PRESENT ALBEIT WITH AN UNUSUAL PUTATIVE 3DH# MEMBRANE ANCHOR POLYPEPTIDE WITH VERY LITTLE PRIMARY SEQUENCE SIMILARITY TO KNOWN 3DH# ANDOR 3DH$ POLYPEPTIDES FROM ORGANELLES BACTERIA OR !RCHEA -ALATE MENAQUINONE OXIDOREDUCTASE OR MEMBRANE BOUND MALATE DEHYDROGENASE HAS BEEN REPORTED TO PLAY AN IMPORTANT ROLE IN THE CITRIC ACID CYCLE ALTHOUGH THERE IS ALSO A CONVENTIONAL SOLUBLE SUCCINATE DEHYDROGENASE WHICH TRANSFERS ELECTRONS TO .!$ -OLENAAR ET AL (OWEVER THERE SEEM TO BE NO PROTON PUMPING ENZYMES UPSTREAM OF THE QUINONE POOL 4HERE ARE TWO ELECTRON TRANSFER BRANCHES A BCC AA ROUTE OR MORE GENERALLY A BC C AA ROUTE AND BD ROUTE DOWN STREAM OF THE QUINONE POOL WHICH MUST EXHIBIT DIFFERENT OF ENERGY CONSERVATION EFlCIENCY #YT BC AND #YT AA HAVE BEEN SHOWN TO PUMP PROTONS IN SEVERAL ORGANISMS WHILE #YT BDD DOES NOT PUMP BUT MERELY TRANSLOCATE CHEMICAL PROTONS 3ONE !LTOGETHER THE ENZYMES IN THE BC C AA ROUTE ARE EX PECTED TO TRANSLOCATE ABOUT THREE PROTONS PER ELECTRON WHILE THE #YT BDD ROUTE TRANSLOCATES ONLY ONE PROTON PER ELECTRON 4HE EFlCIENCY OF ENERGY METABOLISM MUST THUS DEPEND ON WHICH RESPIRATORY PATHWAY IS MAINLY OPERATING 4HUS GENETIC MANIPULATIONS OF RESPIRATORY CHAIN COMPONENTS MAY ENHANCE THE CELL GROWTH 6ERY RECENTLY IT WAS FOUND THAT DESTRUCTION OF THE CYD!" GENES INCREASED THE CELL YIELD IN STATIONARY GROWTH PHASE WHEREAS THE GROWTH RATE WAS DECREASED DURING THE EXPONENTIAL GROWTH PHASE 3AKAMOTO ET AL UNPUBLISHED 4HIS OBSERVATION IS IN AGREEMENT WITH THE ABOVE CONSIDERATIONS ABOUT CELLULAR ENERGY METABOLISM
# GLUTAMICUM 5PSTREAM OF THE QUINONE POOL THERE ARE MANY DIFFERENCES BETWEEN # GLUTAMICUM AND - TUBERCULOSIS 4HE LATTER BACTERIA CONTAIN GENES ENCOD ING A PROTON PUMPING .$( ) IN ADDITION TO .$( )) 4ABLE ! SUCCINATE DEHYDROGENASE PREDICTED TO BE A DIHEME #LASS 312 IS PRESENT AND A FUMARATE REDUCTASE THAT EXHIBIT HIGH SEQUENCE SIMILARITY TO THE HEME LESS % COLI FUMARATE REDUCTASE -YC LEPRAE ANOTHER HUMAN PATHOGEN LACKS CYD!" GENES #OLE ET AL WHEREAS -YC SMEGMATIS A FAST GROWING NONPATHOGENIC SAPROPHYTE HAVE TWO GENE SETS FOR #YT BDD OR ITS HOMOLOGUE CYD!" AND CBD!" YTH!" +ANA ET AL $ELETION OF CYD!" GENES CAUSED LOSS OF THE ABSORPTION PEAK AT NM THAT IS DUE TO #YT D 4HIS CYD!" LESS MUTANT GROWS AS WELL AS WILD TYPE CELLS IN THE PRESENCE OF / HIGHER THAN WHILE GROWTH IS SUPPRESSED AT / CONCENTRATIONS LOWER THAN +ANA ET AL D 4HE AUTHORS SUGGESTED THAT #YT BD PLAYS A ROLE UNDER AIR LIMITED GROWTH CONDITIONS WHERE AN OXIDASE WITH HIGH AFlNITY TO / IS NEEDED AS IS THE CASE WITH % COLI #YT BD 3INCE -YC LEPRAE IS AN INTRACELLULAR PARASITE THAT LIVES UNDER LOW / CONDITIONS THE PRESENCE OF #YT BD MIGHT BE IMPORTANT FOR ITS PATHOGENESIS D 4HE WHOLE GENOME OF 3TREPTOMYCES COELICOLOR BELONGING TO THE GROUP OF lLAMENTOUS ACTINOMYCETES HAS ALSO BEEN SEQUENCED "ENTLEY ET AL 4HE GENE SETS ENCODING RESPIRATORY CHAIN ENZYME COM PLEXES ARE SIMILAR TO THOSE FOUND IN -YC TUBERCU LOSIS 4ABLE EXCEPT THAT THE % COLI LIKE FUMARATE REDUCTASE IS ABSENT 'ENES FOR #YT BCC WAS ALSO ISOLATED FROM 2HODOCOCCUS RHODOCHROUS BELONGING TO .OCARDIACEAE OF THE lLAMENTOUS ACTINOMYCETES 3ONE ET AL SUGGESTING THAT THE DIHEME C #YT SUBUNIT IS A COMMON FEATURE OF THE WHOLE HIGH ' # ACTINOBACTERIA
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4HE WHOLE GENOME SEQUENCE OF -YCOBACTERIUM TUBERCULOSIS A MAJOR HUMAN PATHOGEN #OLE ET AL INDICATES THAT IT CONTAINS TWO ELECTRON TRANSFER PATHWAYS BCC AA ROUTE AND BD ROUTE SIMILAR TO WHAT D WAS DESCRIBED FOR # GLUTAMICUM 4HE STRUCTURAL GENES FOR #YT BCC QCR#!" AA CTA#$% AND BD CYD!" ARE HIGHLY HOMOLOGOUS TO THE COUNTERPARTS IN
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4ABLE SEE ALSO 3ECTION ))) !S MENTIONED TWO HEMES SEEM TO BE REQUIRED TO CARRY OUT SUCCINATE OXIDATION USING A LOW POTENTIAL QUINONE IN ALL SPECIES &ROM THE LOW ' # GROUP " SUBTILIS " HALODURANS 0AENIBACILLUS MACERANS AND 3 AUREUS HAVE ONLY ONE #LASS 312 AND A DIHEME MEMBRANE ANCHOR VERY SIMILAR TO THAT OF " SUBTILIS (EDERSTEDT )N THE (IGH ' # GROUP THE PRIMARY SEQUENCE SIMILAR ITY TO OTHER 312S IS LOWER AND A SECOND ENZYME A FUMARATE REDUCTASE CAN BE PRESENT
PER ELECTRON TRANSFER AS DISCUSSED IN THE PREVIOUS SECTIONS 4HE MULTIPLE BRANCHES ARE NOT JUST REDUNDANT BUT SELECTIVELY EXPRESSED AND OPERATING DEPENDING ON THE ENVIRONMENTAL CONDITIONS ESPECIALLY DIOXYGEN CONCENTRATION /N THE OTHER HAND HIGH ' # ACTINOBACTERIA SEEM TO HAVE A #YT BCC CAA BRANCH AND #YT BD D BRANCH AS THE FUNDAMENTAL COMBINATION OF ENZYMES IN THE RESPIRATORY CHAIN EXCEPT FOR -YC LEPRAE THAT LACK GENES FOR #YT BD IN THE GENOME 4ABLE D
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!EROBIC "ACILLUS SPECIES BELONGING TO THE LOW ' # GROUP TYPICALLY HAVE MORE THAN THREE OXIDASES 4HEY COMMONLY CONTAIN #YT CAA TYPE 3OX- TYPE #YT C OXIDASE AND #YT BD TYPE D QUINOL OXIDASE )N ADDITION TO THESE TWO OXIDASES THE THERMOPHILIC " THERMODENITRIlCANS 3AKAMOTO ET AL .I KAIDO ET AL AND THE ALKALIPHILIC " HALODURANS 4AKAMI ET AL HAVE 3OX" TYPE #YT C OXIDASE WHILE THE MESOPHILIC " SUBTILIS DOES NOT +UNST ET AL 3OX" TYPE OXIDASES AND ITS GENES HAVE MAINLY IF NOT EXCLUSIVELY BEEN IDENTIlED IN ARCHAEA AND EUBACTERIA LIVING IN MORE OR LESS EXTREME ENVI RONMENTS INCLUDING 'RAM NEGATIVE BACTERIA SUCH AS 4HERMUS THERMOPHILUS $ISTRIBUTION OF 3OX" TYPE OXIDASE IS THUS NOT PARALLEL TO THE PHYLOGENY OF THE LIVING ORGANISMS 4HIS IS @EPIDEMIOLOGICAL EVIDENCE FOR THE HYPOTHESIS THAT THIS TYPE OF OXIDASE MAY BE SOMEHOW SUITABLE TO EXTREMOPHILES AND THE GENES HAVE BEEN LATERALLY TRANSFERRED DURING THE EVOLUTION 4HE COMBINATION OF THE #YT BC CAA OXIDASE BRANCH AND THE ALTERNATIVE 3OX" TYPE #C/ BRANCH FOUND IN THE THERMOPHILIC AND ALKALOPHILIC "ACILLUS SPECIES IS THE SAME AS THAT FOUND IN 4 THERMOPHILUS WHILE NO SIMILAR COMBINATION IS FOUND IN THE HIGH ' # 'RAM POSITIVES 3OX- TYPE #YT AA TYPE QUINOL OXIDASE IS THE MAIN TERMINAL OXIDASE OF " SUBTILIS AND A HOMOLO GOUS GENE IS ALSO FOUND IN THE " HALODURANS GENOME 4ABLE 4HE GENOMES OF THESE TWO "ACILLUS SPECIES ALSO CONTAIN TWO GENE SETS FOR #YT BD !LTOGETHER " HALODURANS HAS lVE TERMINAL OXIDASES WHILE " SUBTILIS SEEMS TO HAVE FOUR 3 AUREUS CONTAINS TWO QUINOL OXIDASES A 3OX- TYPE HEME COPPER OXIDASE AND #YT BDD +URODA ET AL "ABA ET AL 4HE DIFFERENT OXIDASES HAVE VARIOUS AFlNITIES TO DIOXY GEN AND THE MULTIPLE ELECTRON TRANSFERRING BRANCHES SHOW DIFFERENT EFlCIENCIES OF CHEMIOSMOTIC ENERGY TRANSDUCTION OR VARIOUS RATIOS OF PROTON TRANSPORTED
#YT BC TYPE 1C2 IS NOT ONLY PRESENT IN THE RESPIRA TORY CHAIN OF AEROBIC ORGANISMS #YT BC BUT ALSO IN THE ELECTRON TRANSFER CHAIN OF PHOTOSYNTHETIC ORGAN ISMS #YT B F 3EQUENCE ANALYSIS OF #YT F BB IN A WIDE RANGE OF ORGANISMS INDICATES THAT THEY CAN BE DIVIDED INTO lVE GROUPS &IG 3ONE ET AL &IRST #YTS B FROM PROTEOBACTERIA AND MITOCHONDRIA COMPOSE A DISTINCT GROUP 3ECONDLY #YTS B OF #YT B FF COMPLEX FROM CYANOBACTERIA AND CHLOROPLAST COMPOSE ANOTHER DISCRETE GROUP 3EQUENCE COMPAR ISON SHOW THAT #YT B LACKS A PORTION CORRESPONDING TO THE # TERMINAL PART OF #YT B CONTAINING THREE TRANSMEMBRANE HELICES )NSTEAD #YT B F COMPLEX HAVE AN ADDITIONAL SUBUNIT SUBUNIT )6 ENCODED JUST DOWNSTREAM OF THE GENE FOR #YT B AND ITS SEQUENCE IS SIMILAR TO THE # TERMINAL PART OF #YT B )N OTHER WORDS #YT B IS SPLIT INTO #YT B AND SUBUNIT )6 IN THIS GROUP 4HIRDLY #YTS B FROM THE LOW ' # lRMICUTES COMPOSES ANOTHER GROUP -OST OF THESE #YT BC COM PLEXES OPERATE IN THE RESPIRATORY CHAIN HOWEVER THE #YT B SEQUENCE IS MORE SIMILAR TO THAT OF #YT B THAN TO MITOCHONDRIAL OR PROTEOBACTERIAL #YT B 4HE MAJOR DIFFERENCE OF #YT BC IN THIS GROUP FROM #YT B F IS THAT F SUBUNIT )6 IS NOT PRESENT IN AN ISOLATED POLYPEPTIDE BUT GENETICALLY FUSED TO THE . TERMINUS OF THE #YT C SUBUNIT (ELIOBACILLUS MOBILIS IS A PHOTOSYNTHETIC LOW ' # lRMICUTE )TS #YT BC COMPLEX ENCODED FROM A LARGE GENE CLUSTER FOR PHOTOSYNTHETIC APPARATUS IS MORE SIMILAR TO A #YT B F COMPLEX AND HAS SUBUNIT )6 F ENCODED BY A SEPARATE GENE 8IONG ET AL 4HE FORTH GROUP #YTS B FROM THE HIGH ' # ACTINOBACTERIA IS SIMILAR TO THOSE FROM RADIATION RESISTANT BACTERIA 4 THERMOPHILUS AND $EINOCOCCUS RADIODURANS 4HIS GROUP IS DISTINCT BUT RELATIVELY SIMILAR TO #YT B OF THE lRST GROUP !RCHAEAL #YTS B COMPOSE A lFTH DISTINCT GROUP 4HE PHYLOGENETIC TREE DRAWN FOR THE 2IESKE &E3 PROTEIN IS QUITE SIMILAR TO THAT FOR #YT B &IG 3ONE
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&IG 0OSSIBLE EVOLUTION OF THE GENE CLUSTER FOR THE 1C2 #YT BC COMPLEX
ET AL SUGGESTING THAT GENES FOR THESE TWO PROTEINS HAVE DESCENDED AND DIVERGED TOGETHER IN THE GENOME DURING THE MOLECULAR EVOLUTION OF #YT BC COMPLEX ! REMARKABLE FEATURE OF THE &E3 PROTEIN FROM THE HIGH ' # ACTINOBACTERIA IS THAT IT HAS THREE TRANSMEMBRANE HELICES AT THE . TERMINAL IN CONTRAST TO THOSE OF OTHER ORIGINS WHICH HAVE ONLY ONE 4AKEN TOGETHER THE TOTAL SIZE OF &E3 PROTEIN AND THAT OF #YT B OF THE HIGH ' # GROUP IS MUCH LARGER THAN IN THE COUNTERPARTS FROM OTHER EUBACTERIA !LTHOUGH THERE ARE DIVERGENT SUBGROUPS OF #YT B
AND &E3 ALL THE MEMBERS OF THE EACH PROTEIN GROUP ARE SO SIMILAR THAT THEY CAN BE RELATED IN A SINGLE PHY LOGENETIC TREE )N CONTRAST THE C TYPE #YT SUBUNIT OF BC COMPLEX IS HIGHLY DIVERGENT )T IS LIKELY THAT DIFFERENT PERIPHERAL SMALL SIZED #YT C WERE ADOPTED AS THE @C SUBUNIT OF 1C2 INDEPENDENTLY IN EACH PHYLOGENETIC GROUP OF ORGANISMS )N THE HIGH ' # 'RAM POSITIVES TWO #YT C WERE ADOPTED TO 1C2 'ENES FOR SUCH A #YT CC 1C2 SUBUNIT ARE FOUND IN THE GENOMES OF ¡ PROTEOBACTERIA (ELICOBACTER PYLORI 4OMB ET AL AND #AMPYLOBACTER JEJUNI 0ARKHILL ET AL
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! #YT CC GENE IS ALSO FOUND IN A LARGE GENE CLUSTER FOR PHOTOSYNTHESIS IN THE LOW ' # lRMICUTE (ELIOBACILLUS MOBILIS 8IONG ET AL BUT NONE HAS BEEN DETECTED IN THE RESPIRATORY CHAIN OF THE LOW ' # GROUP )T SHOULD ALSO BE NOTICED THAT NO SEQUENCE SIMILARITY IS SEEN BETWEEN THE THREE GROUPS OF #YTS CC THE AEROBIC HIGH ' # ACTINOBACTERIA THE ¡ PROTEOBACTERIA AND THE PHOTOSYNTHETIC LOW ' # lRMICUTE SUGGESTING THAT THESE DIHEME C PROTEINS HAVE EMERGED INDEPENDENTLY DURING MOLECULAR EVO LUTION OF 1C2 @-OLECULAR ADOPTION OF #YT C HAS ALSO TAKEN PLACE IN TERMINAL OXIDASES RESULTING IN THE #YT CAA TYPE OXIDASE IN THE LOW ' # lRMICUTES THE RADIATION RESISTANT 'RAM NEGATIVES IN &LAVOBACTERIA "ACTER OIDES SPECIES AND IN SOME PROTEOBACTERIA 3EQUENCE COMPARISON OF THE #YT C DOMAINS SUGGESTS THAT THE GENETIC FUSION OF #YT C HAS OCCURRED INDEPENDENTLY IN THESE GROUPS
OF "ACILLUS SUBTILIS BY MEMBRANE ENERGIZATION * "ACTERIOL n !ZARKINA . 3ILETSKY 3 "ORISOV 6 VON 7ACHENFELDT # (EDER STEDT , AND +ONSTANTINOV !! ! CYTOCHROME BB| TYPE QUINOL OXIDASE IN "ACILLUS SUBTILIS STRAIN * "IOL #HEM n "ABA 4 4AKEUCHI & +URODA - 9UZAWA ( !OKI + /GUCHI ! .AGAI 9 )WAMA . !SANO + .AIMI 4 +URODA ( #UI , 9AMAMOTO + (IRAMATSU + 'ENOME AND VIRULENCE DETERMINANTS OF HIGH VIRULENCE COMMUNITY ACQUIRED -23! ,ANCET n "ARSKY %, .AZQARENKO !6 3AMUILOV 6$ AND +HAKIMOV 3! )NHIBITION OF RESPIRATION IN "ACILLUS SUBTILIS CELLS BY ACIDIlCATION OF THE CYTOPLASM "IOL -EMBR -OSCOW n "ENGTSSON * 2IVOLTA # (EDERSTEDT , AND +ARAMATA $ A "ACILLUS SUBTILIS CONTAINS TWO SMALL C TYPE CYTOCHROMES WITH HOMOLOGOUS HEME DOMAINS BUT DIFFERENT TYPES OF MEMBRANE ANCHORS * "IOL #HEM n "ENGTSSON * 4JALSMA ( 2IVOLTA # AND (EDERSTEDT , B 3UBUNIT )) OF "ACILLUS SUBTILIS CYTOCHROME C OXIDASE IS A LIPO PROTEIN * "ACTERIOL n "ENINI 3 'ONZALEZ ! 2YPNIEWSKI 72 7ILSON + 6AN "EEUMEN * AND #IURLI 3 #RYSTAL STRUCTURE OF OXIDIZED "ACILLUS PASTEURII CYTOCHROME C AT ¯ RESOLUTION "IOCHEMISTRY n "ENTLEY 3$ #HATER +& #ERDENO 4ARRAGA !- #HALLIS ', 4HOM SON .2 *AMES +$ (ARRIS $% 1UAIL -! +IESER ( (ARPER $ "ATEMAN ! "ROWN 3 #HANDRA ' #HEN #7 #OLLINS - #RONIN ! &RASER ! 'OBLE ! (IDALGO * (ORNSBY 4 (OWARTH 3 (UANG #( +IESER 4 ,ARKE , -URPHY , /LIVER + /.EIL 3 2ABBINOWITSCH % 2AJANDREAM -! 2UTHERFORD + 2UTTER 3 3EEGER + 3AUNDERS $ 3HARP 3 3QUARES 2 3QUARES 3 4AYLOR + 7ARREN 4 7IETZORREK ! 7OODWARD * "ARRELL "' 0ARKHILL * AND (OPWOOD $! #OMPLETE GENOME SEQUENCE OF THE R .ATURE MODEL ACTINOMYCETE 3TREPTOMYCES COELICOLOR ! n "ERGSMA * VAN $ONGEN -"- AND +ONINGS 7. 0URIlCATION AND CHARACTERIZATION OF .!$( DEHYDROGENASE FROM "ACILLUS SUBTILIS %UR * "IOCHEM n #OLE 34 "ROSCH 2 0ARKHILL * 'ARNIER 4 #HURCHER # (ARRIS $ 'ORDON 36 %IGLMEIER + 'AS 3 "ARRY #% RD 4EKAIA & "ADCOCK + "ASHAM $ "ROWN $ #HILLINGWORTH 4 #ONNOR 2 $AVIES 2 $EVLIN + &ELTWELL 4 'ENTLES 3 (AMLIN . (OLROYD 3 (ORNSBY 4 *AGELS + +ROGH ! -C,EAN 3 -OULE 3 -URPHY , /LIVER + /SBORNE * 1UAIL -! 2AJANDREAM - ! 2OGERS * 2UTTER 3 3EEGER + 3KELTON * 3QUARES 2 3QUARES 3 3ULSTON *% 4AYLOR + 7HITEHEAD 3 AND "ARRELL "' $ECIPHERING THE BIOLOGY OF -YCOBACTERIUM TUBERCULOSIS FROM THE COMPLETE GENOME SEQUENCE .ATURE n #OLE 34 %IGLMEIER + 0ARKHILL * *AMES +$ 4HOMSON .2 7HEELER 02 (ONORE . 'ARNIER 4 #HURCHER # (ARRIS $ -UNGALL + "ASHAM $ "ROWN $ #HILLINGWORTH 4 #ONNOR 2 $AVIES 2- $EVLIN + $UTHOY 3 &ELTWELL 4 &RASER ! (AMLIN . (OLROYD 3 (ORNSBY 4 *AGELS + ,ACROIX # -ACLEAN * -OULE 3 -URPHY , /LIVER + 1UAIL -! 2AJANDREAM -! 2UTHERFORD +- 2UTTER 3 3EEGER + 3IMON 3 3IMMONDS - 3KELTON * 3QUARES 2 3QUARES 3 3TEVENS + 4AYLOR + 7HITEHEAD 3 7OODWARD *2 AND "ARRELL "' -ASSIVE GENE DECAY IN THE LEPROSY BACILLUS .ATURE n $AVIDSON -7 'RAY +! +NAFF $" AND +RULWICH 4!
7**$PODMVEJOH3FNBSLT 4HE MOLECULAR STUDIES ON THE RESPIRATORY ENZYME COMPLEXES OF 'RAM POSITIVE BACTERIA HAVE FOCUSED ON A HANDFUL OF MODEL SPECIES AS DESCRIBED IN THIS CHAP TER )N ADDITION THE RECENT WHOLE GENOME SEQUENCING PROJECTS HAVE PROVIDED COMPREHENSIVE INFORMATION ON THE COMPOSITION OF THE RESPIRATORY CHAIN IN PARTICULAR 'RAM POSITIVE BACTERIA 4HE BIOCHEMICAL STUDIES AND PRIMARY SEQUENCE COMPARISONS HAVE DEMONSTRATED A HIGH DIVERGENCE OF THE ENZYME COMPLEXES AND HOW THEY ARE COMBINED INTO RESPIRATORY CHAIN OF 'RAM POSITIVE PROKARYOTES 4HESE RESULTS CLEARLY INDICATE THAT EACH OF THE TWO GROUPS THE LOW AND THE HIGH ' # CONTAINING 'RAM POSITIVES CONSTITUTES A DISTINCT CLASS BOTH IN TERMS OF SIMILARITIES IN AMINO ACID SEQUENCES AND THE COMBINATIONS OF THE RESPIRATORY ENZYME COMPLEXES BEING PRESENT 4HERE ARE FEW TIES ENABLING US TO COMBINE THE TWO GROUPS INTO AN UR GROUP AT A HIGHER LEVEL &URTHER STUDIES ON THE RES PIRATION OF THESE LARGE DIVERGED GROUPS WILL PROVIDE INSIGHTS INTO THE MOLECULAR MECHANISM OF THE ENERGY TRANSDUCTION AND POSSIBILITY TO MANIPULATE THE ENERGY METABOLISM OF SUCH USEFUL ORGANISMS 3FGFSFODFT !ZARKINA . AND +ONSTANTINOV !! 3TIMULATION OF MENA QUINONE DEPENDENT ELECTRON TRANSFER IN THE RESPIRATORY CHAIN
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0URIlCATION AND CHARACTERIZATION OF TWO SOLUBLE CYTOCHROMES FROM THE ALKALOPHILE "ACILLUS lRMUS 2!" "IOCHIM "IOPHYS !CTA n $MELLO 2 (ILL 3 AND 0OOLE 2+ 4HE CYTOCHROME BD QUINOL OXIDASE IN %SCHERICHIA COLI HAS AN EXTREMELY HIGH OXYGEN AFlNITY AND TWO OXYGEN BINDING HAEMS )MPLICATIONS FOR REGULATION OF ACTIVITY IN VITRO BY OXYGEN INHIBITION -ICRO BIOLOGY n &UJIWARA 9 /KA - (AMAMOTO 4 AND 3ONE . #YTOCHROME C OF THE THERMOPHILIC BACTERIUM 03 $.! SEQUENCE AND ANALYSIS OF THE MATURE CYTOCHROME "IOCHIM "IOPHYS !CTA n 'ARCIA (ORSMAN *! "ARQUERA " AND %SCAMILLA *% 4WO DIFFERENT AA TYPE CYTOCHROMES CAN BE PURIlED FROM THE BACTE RIUM "ACILLUS CEREUS %UR * "IOCHEM n 'ILMOUR 2 AND +RULWICH 4! 0URIlCATION AND CHARAC TERIZATION OF THE SUCCINATE DEHYDROGENASES COMPLEX AND #/ REACTIVE B TYPE CYTOCHROMES FROM THE FACULTATIVE ALKALIPHILE "ACILLUS lRMUS /& "IOCHIM "IOPHYS !CTA n 'ILMOUR 2 AND +RULWICH 4! #ONSTRUCTION AND CHARAC TERIZATION OF A MUTANT OF ALKALIPHILIC "ACILLUS lRMUS /& WITH A DISRUPTED CTA OPERON AND PURIlCATION OF A NOVEL CYTOCHROME BD "ACTERIOL n (ÊGERHÊLL # 3UCCINATEQUINONE OXIDOREDUCTASES VARIA TIONS ON A CONSERVED THEME "IOCHIM "IOPHYS !CTA n (ÊGERHÊLL # AND (EDERSTEDT , ! STRUCTURAL MODEL FOR THE MEMBRANE INTEGRAL DOMAIN OF SUCCINATEQUINONE OXIDO REDUCTASES &%"3 ,ETT n (ÊGERHÊLL # !ASA 2 VON 7ACHENFELDT # AND (EDERSTEDT , 4WO HEMES IN "ACILLUS SUBTILIS 3UCCINATE -ENAQUINONE OXI DASE "IOCHEMISTRY n (ÊGERHÊLL # &RIDEN ( !ASA 2 AND (EDERSTEDT , 4RANS MEMBRANE TOPOLOGY AND AXIAL LIGANDS TO HEMES IN CYTOCHROME B SUBUNIT OF "ACILLUS SUBTILIS SUCCINATEMENAQUINONE REDUCTASE "IOCHEMISTRY n (ÊGERHÊLL # -AGNITSKY 3 3LED 6 3CHRÚDER ) 'UNSALUS 20 #ECCHINI ' AND /HNISHI 4 !N %SCHERICHIA COLI MUTANT QUINOLFUMARATE REDUCTASE CONTAINS AN %02 DETECTABLE SEMIQUINONE STABILIZED AT THE PROXIMAL QUINONE BINDING SITE * "IOL #HEM n (ALTIA 4 3ARASTE - AND 7IKSTRÚM - 3UBUNIT ))) OF CY TOCHROME C OXIDASE IS NOT INVOLVED IN PROTON TRANSLOCATION ! SITE DIRECTED MUTAGENESIS STUDY %-"/ * n (EDERSTEDT , 3UCCINATEQUINONE OXIDOREDUCTASE IN THE BACTERIA 0ARACOCCUS DENITRIlCANS AND "ACILLUS SUBTILIS "IOCHIM "IOPHYS !CTA n (EDERSTEDT , AND 2UTBERG , "IOSYNTHESIS AND MEMBRANE BINDING OF SUCCINATE DEHYDROGENASE IN "ACILLUS SUBTILIS * "ACTERIOL n (ENNING 7 6O , !LBANESE * AND (ILL "# (IGH YIELD PURIlCATION OF CYTOCHROME AA AND CYTOCHROME CAA OXIDASES FROM "ACILLUS SUBTILIS PLASMA MEMBRANES "IOCHEM * n (ICKS $" AND +RULWICH 4! 4HE RESPIRATORY CHAIN OF ALKALIPHILIC BACILLI "IOCHIM "IOPHYS !CTA n (ICKS $" 0LASS 2* AND 1UIRK 0' %VIDENCE FOR MULTIPLE TERMINAL OXIDASES INCLUDING CYTOCHROME D IN FACULTATIVELY ALKALIPHILIC "ACILLUS lRMUS /& * "ACTERIOL n (IROTA 3 3VENSSON " !DELROTH 0 3ONE . .ILSSON 4 -ALMSTRÚM "' AND "REZEZINSKI 0 ! mASH PHOTOLYSIS STUDY OF THE
REACTIONS OF A CAA TYPE CYTOCHROME C OXIDASE WITH DIOXYGEN AND CARBON MONOXIDE * "IOENERG "IOMEMBR n )SHIZUKA - -ACHIDA + 3HIMADA 3 -OGI ! 4SUCHIYA 4 /HMORI 4 3OUMA 9 'ONDA - AND 3ONE . .UCLEOTIDE SEQUENCE OF THE GENE CODING FOR FOUR SUBUNITS OF CYTOCHROME C OXIDASE FROM THE THERMOPHILIC BACTERIUM 03 * "IOCHEM n )VERSON 4- ,UNA #HAVEZ # #ECCHINI ' AND 2EES $# 3TRUCTURE OF THE %SCHERICHIA COLI FUMARATE REDUCTASE RESPIRATORY COMPLEX 3CIENCE n *ONES #7 "RICE *- $OWNS !* AND $ROZD *7 "ACTERIAL RESPIRATION LINKED PROTON TRANSLOCATION AND ITS RELATIONSHIP TO RESPIRATORY CHAIN COMPOSITION %UR * "IOCHEM n +AGEKAWA 3 -IZUKAMI - .OGUCHI 3 3AKAMOTO * AND 3ONE . )MPORTANCE OF HYDROPHOBIC INTERACTION BETWEEN A 3OX" TYPE CYTOCHROME C OXIDASE WITH ITS NATURAL SUBSTRATE CYTOCHROME C AND ITS MUTANTS * "IOCHEM n +ANA "$ 7EINSTEIN %! !VARBOCK $ $AWES 33 2UBIN ( -IZRAHI 6 #HARACTERIZATION OF THE CYD!" ENCODED CYTOCHROME BDD OXIDASE FROM -YCOBACTERIUM SMEGMATIS * "ACTERIOL n +AWAHARA 9 4ANAKA 4 )KEDA 3 AND 3ONE . #OUPLING SITE OF THE RESPIRATORY CHAIN OF "REVIBACTERIUM LACTOFERMENTUM !GRIC "IOL #HEM n +OYANAGI 3 .AGATA + 4AMURA 4 4SUKITA 3 AND 3ONE . 0URIlCATION AND CHARACTERIZATION OF CYTOCHROME C FROM (ELICOBACTER PYLORI * "IOCHEM n +RÚGER ! "IEL 3 3IMON * 'ROSS 2 5NDEN ' AND ,ANCASTER #2$ &UMARATE RESPIRATION OF 7OLINELLA SUCCINOGENES %NZYMOLOGY ENERGETICS AND COUPLING MECHANISM "IOCHIM "IOPHYS !CTA n +RULWICH 4! )TO - 'ILMOUR 2 (ICKS $" AND 'UFFANTI !! %NERGETICS OF ALKALIPHILIC "ACILLUS SPECIES 0HYSIOLOGY AND MOLECULES !DV -ICROBIAL 0HYSIOL n +UNST & /GASAWARA . -OSZER ) !LBERTINI !- !LLONI ' !ZEVEDO 6 "ERTERO -' "ESSIERES 0 "OLOTIN ! "ORCHERT 3 "ORRISS 2 "OURSIER , "RANS ! "RAUN - "RIGNELL 3# "RON 3 "ROUILLET 3 "RUSCHI #6 #ALDWELL " #APUANO 6 #ARTER .- #HOI 3+ #ODANI ** #ONNERTON )& #UMMINGS .* $ANIEL 2! $ENIZOT & $EVINE +- $URTERHOFT ! %HRLICH 3$ %MMERSON 04 %NTIAN +$ %RRINGTON * &ABRET # &ERRARI % &OULGER $ &RITZ # &UJITA - &UJITA 9 &UMA 3 'ALIZZI ! 'ALLERON . 'HIM 3 9 'LASER 0 'OGGEAU ! 'OLIGHTLY %* 'RANDI ' 'UISEPPI ' 'UY "* (AGA + (AIECH * (ARWOOD #2 (ENAUT ! (ILBERT ( (OLSAPPEL 3 (OSONO 3 (ULLO - & )TAYA - *ONES , *ORIS " +ARAMATA $ +ASAHARA 9 +LABBER "LANCHARD - +LEIN # +OBAYASHI 9 +OET TER 0 +ONINGSTEIN ' +ROUGH 3 +UMANO - +URITA + ,APIDUS ! ,ARDINOIS 3 ,AUBER * ,AZAREVIC 6 ,EE 3 - ,EVINE ! ,IU ( -ASUDA 3 -AUEL # -EDIGUE # -EDINA . -ELLADO 20 -IZUNO - -OESTL $ .AKAI 3 .OBACK - .OONE $ /2EILLY - /GAWA + /GIWARA ! /UDEGA " 0ARK 3 ( 0ARROT 6 0OHL 4- 0ORTETELLE $ 0ORWOLLIK 3 0RESCOTT !- 0RESECAN % 0UJIC 0 0URNELLE " 2APOPORT ' 2EY - 2EYNOLDS 3 2IEGER - 2IVOLTA # 2OCHA % 2OCHE " 2OSE - 3ADAIE 9 3ATO 4 3CANLAN % 3CHLEICH 3 3CHROETER 2 3COFFONE & 3EKIGUCHI * 3EKOWSKA ! 3EROR 3* 3HIN "3 3OLDO " 3OROKIN ! 4ACCONI % 4AKAGI 4 4AKAHASHI ( 4AKEMARU + 4AKEUCHI - 4AMAKOSHI ! 4ANAKA 4 4ERPSTRA 0 4OGNONI ! 4OSATO 6 5CHIYAMA 3 6ANDENBOL - 6ANNIER & 6ASSAROTTI ! 6IARI ! 7ANBUTT 2 7EDLER % 7EDLER ( 7EITZENEGGER 4 7INTERS 0 7IPAT ! 9AMAMOTO ( 9AMANE + 9ASUMOTO + 9ATA + 9OSHIDA + 9OSHIKAWA ( & :UMSTEIN % 9OSHIKAWA ( AND $ANCHIN ! 4HE COMPLETE GENOME
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4VNNBSZ 4HE MICROAEROPHILIC HUMAN GASTRO INTESTINAL PATHOGENS #AMPYLOBACTER JEJUNI AND (ELICOBACTER PYLORI ARE CLOSELY RELATED PHYLOGENTICALLY YET DISTINCT IN SOME MAJOR ASPECTS OF THEIR PHYSIOLOGY ESPECIALLY WITH REGARD TO ELECTRON TRANSPORT # JEJUNI IS A MORE VERSATILE AND METABOLICALLY ACTIVE PATHOGEN WITH A COMPLETE CITRIC ACID CYCLE AND A COMPLEX AND HIGHLY BRANCHED RESPIRATORY CHAIN ALLOWING THE USE OF A VARIETY OF ELECTRON DONORS SUCH AS FORMATE HYDROGEN $ LACTATE SUCCINATE MALATE .!$0 ( AND ALTERNATIVE ELECTRON ACCEPTORS TO OXY GEN INCLUDING FUMARATE NITRATE NITRITE . OR 3 OXIDES AND HYDROGEN PEROXIDE ( PYLORI IS A MORE SPECIALIZED PATHOGEN LARGELY RESTRICTED TO THE HUMAN STOMACH WITH AN INCOMPLETE CITRIC ACID CYCLE AND A SIMPLER RESPIRA TORY CHAIN !LTERNATIVE ELECTRON ACCEPTORS TO OXYGEN INCLUDE FUMARATE HYDROGEN PEROXIDE AND POSSIBLY . OR 3 OXIDES "OTH ORGANISMS CONTAIN A NOVEL TYPE OF COMPLEX ) WHICH LACKS THE TWO SUBUNITS .1/ .UO% AND .1/ .UO& RESPONSIBLE FOR .!$( BINDING AND INITIAL ELECTRON TRANSFER REACTIONS IN OTHER BACTERIA 4HE NATURE OF THE ELECTRON DONOR TO COMPLEX ) HAS YET TO BE IDENTIlED )N BOTH BACTERIA REDUCING EQUIVALENTS ARE TRANSFERRED TO THE SOLE QUINONE MENAQUINONE -ENAQUINOL REDUCES THE CYTOCHROME BC COMPLEX WHICH IN TURN REDUCES PERIPLASMIC CYTOCHROME C 5NUSUALLY FOR A BACTERIUM ( PYLORI CONTAINS ONLY A SINGLE TERMINAL OXIDASE A CB TYPE CYTOCHROME C OXIDASE (OWEVER # JEJUNI ALSO POSSESSES A BD LIKE QUINOL OXIDASE IN ADDI D
TION TO THE CB TYPE OXIDASE ALLOWING AN ADDITIONAL BUT LESS COUPLED PATHWAY OF ELECTRON TRANSFER TO OXYGEN **OUSPEVDUJPO -ANY SPECIES IN THE RELATED GENERA (ELICOBACTER AND #AMPYLOBACTERR ARE EXTREMELY IMPORTANT GASTRO INTESTINAL PATHOGENS WHICH HAVE COME TO PROMINENCE IN RECENT YEARS AS THE CAUSATIVE AGENTS OF MAJOR ANIMAL AND HUMAN DISEASES 4HEY ARE 'RAM NEGATIVE SPIRAL SHAPED BACTERIA WHICH ARE FASTIDIOUS MICROAEROPHILIC AND WHICH GENERALLY ALSO REQUIRE ELEVATED LEVELS OF CARBON DIOXIDE FOR GROWTH $ISCOVERED IN STUD IES ON THE HUMAN HELICOBACTER SPECIES ( PYLORI HAVE EXPANDED RAPIDLY AND THERE ARE NOW MANY ASPECTS OF THE BIOLOGY OF THIS BACTERIUM THAT ARE UNDERSTOOD IN GREATER DETAIL THAN IN #AMPYLOBACTERR SPECIES EVEN THOUGH MEMBERS OF THE LATTER GENUS HAVE BEEN KNOWN FOR MANY DECADES 4HIS IS PARTICULARLY TRUE SINCE THE GENOME SEQUENCE OF ( PYLORI WAS PUBLISHED 4OMB ET AL 4HE SEQUENCE OF ANOTHER STRAIN OF ( PYLORI * HAS ALSO BEEN DETERMINED !LM ET AL MAKING DIRECT COMPARISONS OF GENOME STRUCTURE AND THE IDENTIlCATION OF STRAIN SPECIlC GENES POSSIBLE (OWEVER STUDIES ON THE BIOLOGY OF CAMPYLOBACTERS HAVE BEEN GIVEN NEW IMPETUS WITH THE PUBLICATION OF THE GENOME SEQUENCE OF # JEJUNI 0ARKHILL ET AL $ESPITE THE IMPORTANCE OF THESE BACTERIA IN HUMAN AND ANIMAL DISEASE STUDIES OF THEIR METABOLISM AND PHYSIOLOGY HAVE LAGGED BEHIND WORK ON PROPERTIES !BBREVIATIONS ##0 n CYTOCHROME C PEROXIDASE $-3/ n DIMETH YLSULPHOXIDE -+ n MENAQUINONE .$( n .!$( DEHYDROGENASE .1/ n .!$( QUINOLOXIDASE 2.2 n RIBONUCLEOTIDE REDUCTASE 4-!/ n TRIMETHYLAMINE . OXIDE
RELATED TO VIRULENCE .EVERTHELESS SEVERAL REVIEWS HAVE BEEN PUBLISHED WHICH HAVE COLLATED THE AVAIL ABLE BIOCHEMICAL INFORMATION FROM THE LITERATURE AND PREDICTED PHYSIOLOGICAL PROPERTIES INCLUDING ELECTRON TRANSPORT PATHWAYS FROM GENOME SEQUENCE INFORMA TION +ELLY $OIG ET AL -ARAIS ET AL +ELLY ET AL )N THIS CHAPTER WE DESCRIBE THE CURRENT STATE OF KNOWLEDGE ABOUT THE COMPOSITION AND FUNCTION OF THE RESPIRATORY ELECTRON TRANSPORT PATHWAYS IN THESE BACTERIA **$IBSBDUFSJTUJDTBOE1BUIPHFOJDJUZPG $BNQZMPCBDUFSKFKVOJJ BOE )FMJDPCBDUFS QZMPSJ #AMPYLOBACTER JEJUNI IS THE LEADING CAUSE OF ACUTE BACTERIAL GASTROENTERITIS IN BOTH THE WESTERN WORLD AND IN DEVELOPING COUNTRIES &RIEDMAN ET AL WHERE IT IS PREDOMINANTLY ACQUIRED BY INGESTING CONTAMINATED FOOD MILK OR WATER # JEJUNI IS A COMMENSAL INHABITANT OF THE GASTROINTESTINAL TRACT OF POULTRY AND OTHER BIRDS &RIEDMAN ET AL AND AS A CONSEQUENCE POULTRY SERVES AS THE PRIMARY SOURCE OF CONTAMINATION !CUTE SYMPTOMS OF # JEJUNII INFECTION IN HUMANS INCLUDE DIARRHEA FEVER AND ABDOMINAL PAIN BUT THE COMPLICATIONS CAN INCLUDE REACTIVE ARTHRITIS AND NEUROLOGICAL DISTURBANCES SUCH AS THE -ILLER &ISHER AND THE 'UILLAINE "ARRÏ SYNDROMES 3KIRROW AND "LASER 4HE PATHOGENIC MECHANISMS OF # JEJUNI ARE MEDIATED BY A NUMBER OF VIRULENCE FACTORS ,EACH INCLUDING MOTILITY ADHESION AND THE
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER ABILITY TO INVADE HOST CELLS AS WELL AS THE PRODUCTION OF SEVERAL TOXINS AT PRESENT NOT WELL CHARACTERIZED ,EACH WHICH ARE LIKELY TO BE RESPONSIBLE FOR MANY OF THE ACUTE MANIFESTATIONS OF INFECTION ( PYLORI IS CLOSELY RELATED TO # JEJUNI (OWEVER IT IS NOT A FOOD BORNE PATHOGEN AND THERE IS LITTLE EVIDENCE THAT IT CAN SURVIVE OR GROW IN THE ENVIRON MENT OUTSIDE OF A SUITABLE HOST )T COLONIZES THE GASTRIC OR DUODENAL MUCOSA RATHER THAN THE INTESTINAL TRACT ALTHOUGH OTHER HELICOBACTERS DO COLONIZE THE LOWER ') TRACT OF ANIMALS AND IS THOUGHT TO BE TRANSMITTED DIRECTLY FROM PERSON TO PERSON VIA THE ORAL ORAL OR FECAL ORAL ROUTE -ITCHELL ( PYLORI IS ONE OF THE MOST COMMON INFECTIONS IN -AN AND IS NOW RECOGNIZED AS THE MAJOR ETIOLOGICAL FACTOR IN CHRONIC ACTIVE TYPE " GASTRITIS GASTRIC AND DUODENAL ULCER ATION AND AS A RISK FACTOR FOR GASTRIC CANCER $IXON -AJOR VIRULENCE FACTORS INCLUDE MOTILITY THE PRODUCTION OF A POTENT UREASE WHICH IS INVOLVED IN ACID RESISTANCE A VACUOLATING CYTOTOXIN 6AC! AND G PATHOGENICITY ISLAND #ENSINI THE PRODUCTS OF THE CAG ET AL 4HE MICROAEROPHILIC NATURE OF # JEJUNI AND ( PYLORI MEANS THAT THE MAJORITY OF STRAINS HAVE TO BE CULTURED IN AN ATMOSPHERE CONTAINING n VV OXYGEN AND n VV CARBON DIOXIDE 4HEY ARE NUTRITIONALLY FASTIDIOUS BACTERIA ROUTINELY CULTURED IN COMPLEX MEDIA WITH ADDITIONAL GROWTH SUPPLE MENTS "LOOD DERIVATIVES ARE OFTEN ADDED BUT THE GROWTH OF ( PYLORI CAN BE IMPROVED WITH CHARCOAL STARCH BOVINE SERUM ALBUMIN "3! CATALASE OR ` CYCLODEXTRIN (AZELL ET AL /LIVIERI ET AL 4HE FUNCTION OF THESE SUPPLEMENTS MAY BE TO REDUCE OXIDATIVE DAMAGE OR TO ADSORB POTENTIALLY TOXIC LONG CHAIN FATTY ACIDS 4HE ROLE OF MEDIA SUPPLEMENTS IN PREVENTING OXIDATIVE DAMAGE IS WELL ESTABLISHED FOR #AMPYLOBACTERR "OLTON ET AL !LTHOUGH DElNED MEDIA HAVE BEEN DEVELOPED FOR BOTH # JEJUNI ,EACH ET AL AND ( PYLORI .EDENSKOV 2EYNOLDS AND 0ENN WHICH CONTAIN SEVERAL ESSENTIAL AMINO ACIDS AND VITAMINS SOME KIND OF SUPPLEMENT IS OFTEN STILL REQUIRED !FTER SEVERAL DAYS OF GROWTH IN VITRO BOTH H # JEJUNI AND ( PYLORI BATCH CULTURES UNDERGO A MORPHOLOGI CAL CHANGE FROM SPIRAL BACILLARY TO COCCOID FORMS 4HIS STATIONARY PHASE MORPHOLOGICAL CONVERSION IS CHARACTERIZED BY THE LOSS OF CULTURABILITY 4HERE IS CONTROVERSY OVER WHETHER THESE COCCOID FORMS ARE @ALIVE AND WHETHER THEY COULD BE A MEANS OF TRANS MISSION -ITCHELL 4HERE IS EVIDENCE THAT THE FORMATION OF COCCOID CELLS OF # JEJUNI IS NOT AN ACTIVE
PROCESS AND REPRESENTS A DEGENERATE FORM RESULTING FROM OXIDATIVE DAMAGE (ARVEY AND ,EACH &OR ( PYLORI +USTERS ET AL SHOWED THAT INHIBITION OF PROTEIN OR 2.! SYNTHESIS DID NOT AFFECT CONVERSION TO THE COCCOID FORM AND MOREOVER THAT COCCOID CELLS DID NOT EXHIBIT A MEASURABLE CYTOPLASMIC MEMBRANE POTENTIAL 4HESE DATA STRONGLY INDICATE THAT THE COC COID CELLS ARE DEAD AND ARE THE END RESULT OF A PASSIVE CONVERSION FROM THE SPIRAL FORM ***.JDSPBFSPQIJMZ 0HYSIOLOGICAL EXPLANATIONS FOR THE MICROAEROPHILIC NATURE OF # JEJUNI AND ( PYLORI HAVE LONG BEEN SOUGHT AND IT IS LIKELY THAT ARE SEVERAL CONTRIBUTORS TO THIS PHENOTYPE 4HE POSSESSION OF OXYGEN SENSITIVE ENZYMES IS ONE POSSIBILITY "OTH BACTERIA POSSESS PYRUVATE AND OXOGLUTARATE OXIDOREDUCTASES WHICH ARE NORMALLY ASSOCIATED WITH OBLIGATELY ANAEROBIC BACTERIA +ELLY AND (UGHES (UGHES ET AL FOUND THAT THE ( PYLORI ENZYMES WERE VERY OXYGEN SENSITIVE AND THE PRESENCE OF OXYGEN AND THE OMISSION OF DITHIOTHREITOL FROM PURIlCATION BUFFERS RESULTED IN THEIR RAPID INACTIVATION )T SEEMS LIKELY THAT THIS PROPERTY COULD BE A MAJOR CONTRIBUTOR TO THE MICROAEROPHILIC GROWTH PHENOTYPE AS BOTH ENZYMES ARE ESSENTIAL FOR VIABILITY (UGHES ET AL BUT THERE MAY BE SOME FORM OF PROTECTION TO PREVENT INACTIVATION BY OXYGEN IN VIVO 4HE STEPWISE ONE ELECTRON REDUCTION OF / RESULTS IN THE FORMATION OF THE SUPEROXIDE RADICAL /n AND HYDROGEN PEROXIDE (/ #AMPYLOBACTER JEJUNI AND ( PYLORI POSSESS WELL KNOWN MAJOR DEFENSE MECHANISMS AGAINST SUCH OXIDATIVE STRESS INCLUDING PROTEINS SUCH AS SUPEROXIDE DISMUTASE CATALASE AND FERRITIN YET BOTH ORGANISMS ARE STILL OXYGEN SENSITIVE 4HE EARLY WORK OF (OFFMAN ET AL A B SUG GESTED THAT CAMPYLOBACTERS MAY BE MORE SENSITIVE TO EXOGENOUS SUPEROXIDE AND PEROXIDES WHICH CAN BE EASILY GENERATED SPONTANEOUSLY IN GROWTH MEDIA BY EG EXPOSURE TO LIGHT THAN ARE AEROTOLERANT BACTERIA DESPITE THEIR POSSESSION OF THESE PROTECTIVE ENZYMES 4HIS IS CONSISTENT WITH OBSERVATIONS THAT ESTIMATES OF OXYGEN TOLERANCE CAN VARY WIDELY DEPENDING UPON THE MEDIA USED (ODGE AND +RIEG 4HE IMPORTANCE OF ALKYLHYDROPEROXIDE REDUCTASE !HP# IN AEROTOLERANCE AND OXIDATIVE STRESS RESISTANCE HAS BEEN DEMONSTRATED IN # JEJUNI "AILLON ET AL BY THE ANALYSIS OF AN AHP# MUTANT WHICH WAS CONSIDERABLY MORE SENSITIVE TO OXYGEN AND ORGANIC
HYDROPEROXIDES BUT NOT HYDROGEN PEROXIDE THAN ITS ISOGENIC PARENT 4HE AHP# # AND CATALASE KAT! GENES IN # JEJUNI ARE REGULATED BY IRON AND ALSO BY 0ER2 A &UR LIKE PROTEIN VAN 6LIET ET AL ( PYLORI ALSO POSSESSES !HP# WHICH HAS BEEN SHOWN TO BE AN ESSENTIAL PEROXIREDOXIN AND WHICH PLAYS A SIMILAR ROLE "AKER ET AL 4HERE ARE SOME ADDITIONAL PROTEINS PREDICTED BY THE # JEJUNI GENOME SEQUENCE 0ARKHILL ET AL WHICH ARE POTENTIALLY INVOLVED IN OXYGEN METABOLISM BUT WHICH ARE NOT SHARED WITH ( PYLORI 4HESE INCLUDE A BACTERIAL GLOBIN HOMOLOGUE AND THE PROTEINS RUBERYTHRIN AND HAEMERYTHRIN WHICH COULD BE OXYGEN BINDING PROTEINS 4HE ROLES OF THESE PROTEINS ARE CURRENTLY UNKNOWN
-RQDWKDQ'0\HUVDQG'DYLG-.HOO\ OTHER PRIMARY SUBSTRATE DEHYDROGENASES &IG B )N TERMS OF OXYGEN DEPENDENT ELECTRON TRANSPORT A CB TYPE CYTOCHROME C OXIDASE IS PRESENT 0ARKHILL ET AL BUT # JEJUNI ALSO HAS AN ALTERNATIVE QUINOL OXIDASE #YD!" HOMOLOGUE AND TWO SEPARATE GENES ENCODING CYTOCHROME C PEROXIDASES WHICH MAY BE IMPORTANT IN DETOXIFYING HYDROGEN PEROXIDE IN THE PERIPLASM -OREOVER THE GENOME SEQUENCE INDICATES THE PRESENCE OF SEVERAL ALTERNATIVE ELECTRON TRANSPORT PATHWAYS TO ELECTRON ACCEPTORS SUCH AS FUMARATE NITRATE NITRITE 4-!/ AND $-3/ &IG B 4HE RESPIRATORY CHAIN IN # JEJUNI APPEARS TO BE HIGHLY BRANCHED AND IS SIGNIlCANTLY MORE COMPLEX THAN THAT IN ( PYLORI #&MFDUSPOEPOPST
*7&MFDUSPO5SBOTQPSU$IBJOT )ZESPHFO "0WFSWJFX 4HE MOST IMPORTANT CHARACTERISTICS OF BACTERIAL RE SPIRATORY CHAINS ARE I BRANCHING AT BOTH @DEHYDRO GENASE AND @REDUCTASE ENDS II THE USE OF OXYGEN AS WELL AS ALTERNATIVE ELECTRON ACCEPTORS III THE PRESENCE OF NUMEROUS TYPES OF CYTOCHROMES AND OFTEN MORE THAN ONE TYPE OF QUINONE IV @CROSS TALK BETWEEN PATHWAYS OPTIMIZING THE POSSIBILITY OF EACH REDUCTANT BEING PAIRED WITH A WIDE CHOICE OF OXIDANTS AND V CONCOMITANT PROTON TRANSLOCATION AND ENERGY TRANSDUCTION 3IMPLE LINEAR PATHWAYS INVOLVING A SMALL NUMBER OF DEHYDROGENASES A QUINONE AND A SINGLE TERMINAL OXIDASE OR REDUCTASE ARE UNCOMMON IN BACTERIA .EVERTHELESS GENOME AND BIOCHEMICAL ANALYSES -ARCELLI ET AL +ELLY #HEN ET AL $OIG ET AL DO SUGGEST A SURPRISINGLY SIMPLE ORGANIZATION &IG A FOR THE RESPIRATORY AP PARATUS OF ( PYLORI IN WHICH DEHYDROGENASES PASS REDUCING EQUIVALENTS TO MENAQUINONE ONLY THEN THROUGH A CYTOCHROME BC COMPLEX AND A SOLUBLE CYTOCHROME C TO A SINGLE TERMINAL OXIDASE )N CONTRAST EVEN EARLY WORK ON THE RESPIRATORY PHYSIOLOGY OF CAMPYLOBACTERS LED TO THE IDENTIlCATION OF A RICH COMPLEMENT OF CYTOCHROMES ,ASCELLES AND #ALDER AND THE WORK OF (OFFMAN AND 'OODMAN AND #ARLONE AND ,ASCELLES INDICATED THAT THE RESPIRATORY CHAIN OF # JEJUNI WAS COMPLEX 4HEIR WORK CAN NOW BE INTERPRETED IN TERMS OF THE AVAILABLE GENOME SEQUENCE OF STRAIN 0ARKHILL ET AL %LECTRON INPUT TO THE QUINONE POOL IS VIA A COMPLEX ) TYPE MEMBRANE BOUND QUINONE REDUCTASE A HYDROGENASE AND A VARIETY OF
5PTAKE HYDROGENASE MEDIATES THE TRANSFER OF ELECTRONS FROM HYDROGEN TO THE MENAQUINONE POOL 4HE ENZYME IS MEMBRANE BOUND AND CONSISTS OF THREE POLYPEP TIDES (YD! (YD" AND (YD# "OTH ( PYLORI AND # JEJUNI HAVE BEEN SHOWN TO POSSESS HYDROGENASE ACTIVITY IN MEMBRANE FRACTIONS #ARLONE AND ,AS CELLES (OFFMAN AND 'OODMAN -AIER ET AL 5SING ANTISERA RAISED AGAINST THE "RADYRHI ZOBIUM JAPONICUM UPTAKE HYDROGENASE -AIER ET AL IDENTIlED ( PYLORI HYDROGENASE POLYPEPTIDES OF AND K$A 4HESE ARE THE PRODUCTS OF THE HYD" (0*(0 AND HYD! (0*(0 GENES RESPECTIVELY WHICH ARE PART OF A HYD!"#$ OPERON ALSO ENCODING A CYTOCHROME SUBUNIT (YD# AND A MATURATION PROTEIN (YD$ 'ILBERT ET AL CLONED A HYP" HOMOLOGUE WHICH IN "RADYRHIZOBIUM JAPONICUM ENCODES A NICKEL BINDING PROTEIN NECES SARY FOR HYDROGENASE BIOSYNTHESIS 4HE HYP" GENE (0*(0 IS CO TRANSCRIBED WITH TWO FURTHER BIOGENESIS PROTEINS ENCODED BY THE HYP$# # GENES !DDITIONAL PROTEINS NEEDED FOR HYDROGENASE ASSEMBLY ARE ENCODED BY HYP! (0*(0 AND HYP%& (0 (0*(0 *(0 HOMOLOGUES /L SON ET AL HAVE SHOWN THAT (YP! AND (YP" ARE REQUIRED FOR THE FULL ACTIVITY OF BOTH THE HYDROGENASE AND THE UREASE OF ( PYLORI (OWEVER UREASE ACTIVITY WAS NORMAL IN HYP$ OR HYP& & MUTANTS 4HESE RESULTS INDICATE SOME SHARING OF THE ACCESSORY PROTEINS NECESSARY FOR THE CORRECT INCORPORATION OF NICKEL INTO BOTH OF THESE ENZYMES (YDROGENASE HAS BEEN SHOWN TO HAVE AN IMPORTANT ROLE IN SUSTAINING GROWTH OF ( PYLORI IN VIVO AS HYDROGENASE DElCIENT MUTANTS
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER
&IG #OMPARISON OF PREDICTED ELECTRON TRANSPORT CHAINS IN A ( PYLORI AND B # JEJUNI )N A INTEGRAL MEMBRANE OXIDOREDUCTASES INCLUDE AN .$( LIKE COMPLEX (0 THE ELECTRON DONOR TO WHICH IS UNKNOWN AND HYDROGENASE 0ERIPHERALLY ASSOCIATED OXIDOREDUCTASES INCLUDE AMONG SEVERAL OTHERS MALATEQUINONE OXIDOREDUCTASE AND A $ LACTATE DEHYDROGENASE 2EDUCING EQUIVALENTS ARE TRANSFERRED TO THE SOLE QUINONE MENAQUINONE -+ IN THE LIPID BILAYER OF THE INNER MEMBRANE -ENAQUINOL REDUCES THE TRIMERIC CYTOCHROME BC COMPLEX WHICH IN TURN REDUCES PERIPLASMIC CYTOCHROME C #YTOCHROME C IS REOXIDIZED BY THE SOLE TERMINAL OXIDASE A CB TYPE CYTOCHROME C OXIDASE #YTOCHROME C MAY ALSO BE REOXIDIZED BY HYDROGEN PEROXIDE IN THE PERIPLASM THROUGH THE ACTIVITY OF A SINGLE CYTOCHROME C PEROXIDASE &UMARATE REDUCTASE &RD!"# CATALYSES ELECTRON TRANSFER FROM MENAQUINOL TO FUMARATE AS TERMINAL AC CEPTOR (0 MAY ENCODE AN 3 OR . OXIDE OXIDOREDUCTASE )N # JEJUNI B A SIMILAR RANGE OF PRIMARY DEHYDROGENASES IS PRESENT WITH THE NOTABLE ADDITION OF FORMATE AND SUCCINATE DEHYDROGENASES ! CB TYPE CYTOCHROME C OXIDASE IS PRESENT IN ADDITION TO A #YD!" LIKE QUINOL OXIDASE NOT PRESENT IN ( PYLORI 4HERE ARE ALSO TWO SEPARATE PEROXIDASES IN # JEJUNI 3EVERAL ALTERNATIVE REDUCTASES ARE PREDICTED IN # JEJUNI 4HESE INCLUDE NITRATE NITRITE AND A 4-!/$-3/ REDUCTASE 4OR! )N BOTH A AND B SOLID LINES INDICATE EXPERIMENTALLY ESTABLISHED OR HIGHLY LIKELY ROUTES OF ELECTRON TRANSPORT WHILE DOTTED LINES INDICATE UNCERTAINTY AS TO THE EXACT ROUTE POSSIBLY WITH THE PARTICIPATION OF UNIDENTIlED ADDITIONAL REDOX PROTEINS
WERE UNABLE TO COLONIZE THE STOMACHS OF MICE /LSON AND -AIER AND EVIDENCE WAS OBTAINED THAT THE CONCENTRATION OF HYDROGEN DERIVED FROM COLON FERMENTATION REACTIONS IN STOMACH TISSUES WAS WELL ABOVE THE +- VALUE OF THE ENZYME /LSON AND -AIER # JEJUNI CONTAINS A SIMILAR SET OF STRUCTURAL GENES FOR A .I&E TYPE HYDROGENASE AS WELL AS ACCES SORY GENES FOR NICKEL INCORPORATION BUT NO FUNCTIONAL STUDIES HAVE YET BEEN DONE ON THESE GENES 'PSNBUF &ORMATE DEPENDENT OXYGEN RESPIRATION IN # JEJUNI HAS BEEN DEMONSTRATED AND CHARACTERIZED BY (OFF F MAN AND 'OODMAN AND #ARLONE AND ,ASCELLES 4HE RESPIRATORY ACTIVITIES DETERMINED WITH MEMBRANE VESICLES WERE TO TIMES GREATER WITH FORMATE OR HYDROGEN AS SUBSTRATES WHEN COMPARED TO THE RATES ACHIEVED WITH SUCCINATE LACTATE MALATE OR .!$( SUGGESTING THAT THE FORMER ARE EXCELLENT ELECTRON DONORS (OFFMAN AND 'OODMAN )N % COLI FORMATE DEHYDROGENASE . FORMATE 1UINONE /XIDOREDUCTASE . CONSISTS OF THREE SUBUNITS A LARGE SELENOMOLYBDOPROTEIN CONTAINING THE CATALYTIC SITE &DN' A SMALLER IRON SULFUR PROTEIN &DN( AND A B TYPE CYTOCHROME &DN) 'ENNIS AND 3TEWART 4HE THREE SUBUNITS _ ` AND a ARE AND K$A RESPECTIVELY %NOCH AND ,ESTER "ERG ET AL &ORMATE DEHYDROGENASE . CA TALYSES TRANS MEMBRANE PROTON TRANSLOCATION FROM THE CYTOPLASM TO THE PERIPLASM *ONES 4HE GENOME SEQUENCE OF # JEJUNI REVEALS AN OPERON ENCODING PUTATIVE FORMATE DEHYDROGENASE SUBUNITS FDH! $ #JC #JC # JEJUNI FDH! I #JC ENCODES A LARGE K$A SELENOCYSTEINE CONTAINING MOLYBDO PROTEIN EQUIVALENT TO THE % COLI K$A &DN' _ SUBUNIT FDH" #JC ENCODES A K$A IRON SULFUR SUBUNIT EQUIVALENT TO THE % COLI K$A &DN( ` SUBUNIT FDH# #JC ENCODES A # K$A CYTOCHROME B SUBUNIT EQUIVALENT TO THE % COLI K$A &DN) a SUBUNIT )N ADDITION TO THESE THREE SUBUNITS BOTH % COLI AND # JEJUNI ENCODE AN &DH$ PROTEIN K$A REQUIRED FOR ACTIVITY OF THE FORMATE DEHYDROGENASE ENZYME COMPLEX "ERG ET AL )N CONTRAST TO # JEJUNI ( PYLORI DOES NOT CONTAIN ANY ORTHOLOGOUS GENES ENCODING FORMATE DEHYDRO GENASE SUBUNITS 4OMB ET AL !LM ET AL AND FORMATE RESPIRATION HAS NOT BEEN DEMONSTRATED 4HIS DIFFERENCE MAY WELL BE RELATED TO THE DIFFERENT NICHES THAT THE BACTERIA OCCUPY IN VIVO FORMATE MAY BE PRODUCED BY THE RICH INTESTINAL mORA AND THUS BE
-RQDWKDQ'0\HUVDQG'DYLG-.HOO\ AVAILABLE TO # JEJUNI BUT NOT TO ( PYLORI LIVING IN THE STOMACH -BDUBUF (OFFMAN AND 'OODMAN DEMONSTRATED THE ACTIVITY OF A LACTATE DEHYDROGENASE IN OXYGEN LINKED RESPIRATION OF # JEJUNI ! ( / RATIO OF WAS FOUND FOR LACTATE IN WHOLE CELL PROTON PULSE ASSAYS (OFFMAN AND 'OODMAN !NALYSIS OF THE GENOME SEQUENCE HAS YET TO PRODUCE A CANDIDATE LOCUS FOR A MEMBRANE ASSOCIATED LACTATE DEHYDRO GENASE ENZYME IN # JEJUNI ALTHOUGH AN ANNOTATED , LACTATE DEHYDROGENASE #J IS PRESENT WHICH IS PROBABLY A FERMENTATIVE ENZYME (ELICOBACTER PYLORI ALSO SHOWS LACTATE DEHYDROGENASE ACTIVITY AND A PUTATIVE mAVOPROTEIN $ LACTATE DEHYDROGENASE $LD (0*(0 COULD ACCOUNT FOR THE $ LACTATE DEPENDENT RESPIRATION OBSERVED IN THIS BACTERIUM #HANG ET AL .BMBUF -ALATE CAN REDUCE C TYPE CYTOCHROMES IN ( PY LORI AND A MEMBRANE BOUND .!$( INDEPENDENT DYE LINKED ACTIVITY SUGGESTED THE PRESENCE OF A mAVOPROTEIN TYPE MALATE OXIDOREDUCTASE +ELLY +ATHER ET AL CHARACTERIZED THIS ACTIVITY FULLY AND SHOWED THAT IT WAS DUE TO A MALATEQUINONE OXIDOREDUCTASE -QO A K$A &!$ DEPENDANT MEMBRANE ASSOCIATED ENZYME THAT DONATES ELECTRONS TO QUINONE AND WHICH COULD ALSO PARTICIPATE IN THE CITRIC ACID CYCLE +ATHER ET AL 4HE ENZYME WAS UNEQUIVOCALLY SHOWN TO BE ENCODED BY (0 IN ( PYLORI BUT THE DEDUCED PROTEIN HAS FAIRLY LOW SEQUENCE SIMILARITY WITH OTHER -QO ENZYMES +ATHER ET AL !NALYSIS OF THE # JEJUNI GENOME REVEALS A PUTATIVE OXIDOREDUCTASE #JC WITH IDENTITY TO % COLI MALATEQUINONE OXIDOREDUCTASE AND IDENTITY TO THE CLOSELY RELATED -QO OF ( PYLORI INDICATING THAT BOTH BACTERIA USE A SIMILAR ENZYME FOR MALATE OXIDATION 4HIS IS CONSISTENT WITH THE OBSERVATIONS OF (OFFMAN AND 'OODMAN WHO DEMONSTRATED THAT MALATE STIMULATED RESPIRATION IN # JEJUNI PRODUCING A ( / RATIO OF /"% 1 ) )T IS WIDELY HELD THAT THE FUNCTION OF .!$( IN BIO ENERGETICS IS TO ACT AS THE MAJOR ELECTRON DONOR FOR OXIDATIVE PHOSPHORYLATION VIA INTERACTION WITH A PRO
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER TON TRANSLOCATING QUINONE OXIDOREDUCTASE #OMPLEX ) OR .$( WHILE THE ROLE OF .!$0( IS AS A SOURCE OF ELECTRONS FOR BIOSYNTHETIC REACTIONS .$( IS A LARGE ENZYME WITH A MULTITUDE OF REDOX CENTERS AND IS MOST COMMONLY MADE UP OF DIFFERENT SUBUNITS &RIEDRICH 9AGI ET AL %LECTRONS ARE PASSED FROM .!$( VIA &-. AND &E 3 CENTERS IN .$( TO UBIQUINONEMENAQUINONE IN THE RESPIRA TORY CHAIN &OUR PROTONS ARE TRANSLOCATED ACROSS THE MEMBRANE FOR EVERY TWO ELECTRONS TRANSFERRED BY .$( ACCORDING TO THE OVERALL EQUATION .!$( 1 ( A .!$ 1( (
3EVERAL STUDIES HAVE SHOWN THAT MEMBRANE PREPARA TIONS OF ( PYLORI EXHIBIT LOW OR INSIGNIlCANT RATES OF .!$( OXIDATION BUT THAT THE RATES WITH .!$0( AS ELECTRON DONOR ARE MUCH HIGHER #HANG ET AL (UGHES ET AL #HEN ET AL 4HIS UNUSUAL SITUATION SUGGESTS THAT .!$0( IS THE PHYSIOLOGICAL ELECTRON DONOR TO THE RESPIRATORY CHAIN IN ( PYLORI RATHER THAN .!$( AS IN THE MAJORITY OF BACTERIA 4HE GENOME SEQUENCE OF ( PYLORI AND # JEJUNI SHOWS THAT THESE BACTERIA CONTAIN A CLUSTER OF GENES ENCOD ING A POTENTIAL .!$( QUINONE OXIDOREDUCTASE OF THE .$( TYPE (OWEVER AN EXAMINATION OF THE DEDUCED PROTEINS ENCODED BY THIS GENE CLUSTER LED &INEL TO CONCLUDE THAT THE COMPLEX MAY NOT ACTUALLY OXIDIZE .!$( BECAUSE OF THE LACK OF THE .1/ .UO% AND .1/ .UO& SUBUNITS 4HESE SUBUNITS ARE THOUGHT TO BE ESSENTIAL COMPONENTS FOR THE FUNCTION OF THE .$( COMPLEX .UO& BINDS .!$( AND ALSO POSSESSES A BOUND &-. AND AN &E 3 CENTER .UO% IN CONJUCTION WITH .UO' " AND ) HAVE CYSTEINE RESIDUES THAT PROBABLY COORDINATE &E 3 CLUSTERS 3MITH ET AL )N THE PLACE OF THE EXPECTED NUO% % AND NUO& GENES & # JEJUNI AND ( PYLORI HAVE /2&S OF UNKNOWN FUNCTION #JC FROM # JEJUNI ENCODES A PROTEIN AA IN LENGTH K$A WHICH HAS IDENTITY TO THE ( PYLORI EQUIVALENT (0 WHICH ENCODES A PROTEIN AA IN LENGTH K$A )N ADDITION #JC FROM # JEJUNI ENCODES A PROTEIN AA IN LENGTH K$A WITH IDENTITY TO THE ( PYLORI EQUIVALENT (0 WHICH ENCODES A PROTEIN AA IN LENGTH K$A 4OMB ET AL 0ARKHILL ET AL 3IGNIlCANTLY THESE PROTEINS DO NOT CONTAIN ANY OBVIOUS .!$0( BINDING MOTIF AND THEY ARE APPARENTLY UNIQUE TO THESE TWO BACTERIA !N OBVIOUS POSSIBILITY IS THAT ELECTRONS FROM .!$0( ARE TRANSFERRED TO #OMPLEX ) VIA AN INTER MEDIATE THAT INTERACTS WITH THE .1/ AND .1/
REPLACEMENTS &INEL 3MITH ET AL !L TERNATIVELY COUPLING OF .!$0( WITH THE RESPIRATORY CHAIN MAY NOT OCCUR VIA THE .$( HOMOLOGUE AT ALL BUT THROUGH AN ALTERNATIVE QUINONE REDUCTASE WHICH MAY NOT BE PROTON TRANSLOCATING )T SHOULD BE NOTED THAT THERE IS NO OBVIOUS HOMOLOGUE OF AN .$( TYPE PROTEIN WHICH MAY FULlLL THIS ROLE IN EITHER ( PYLORI OR # JEJUNI &INEL SUGGESTED THAT (0 COULD ACT AS A DOCKING SITE FOR A PROTEIN THAT DELIVERS ELECTRONS DIRECTLY TO THE &E3 CLUSTER OF THE .1/ HOMOLOGUE !LTHOUGH THE IDENTITY OF SUCH A PROTEIN IS UNKNOWN ONE POSSIBILITY IS mAVODOXIN OR FERRE DOXIN REDUCED BY THE ACTIVITIES OF THE PYRUVATE AND OXOGLUTARATE OXIDOREDUCTASES WHICH ARE PRESENT IN PLACE OF THE USUAL .!$( PRODUCING MULTI ENZYME DEHYDROGENASE COMPLEXES FOUND IN CONVENTIONAL AEROBES (UGHES ET AL &URTHER EVIDENCE THAT THE .$( HOMOLOGUE IS UNUSUAL AND IS NOT A CONVENTIONAL .!$0 ( QUINONE OXIDOREDUCTASE HAS COME FROM THE OBSERVATION THAT .!$( OXIDATION IN ( PYLORI IS INSENSITIVE TO THE CLASSICAL #OMPLEX ) INHIBITOR ROTENONE #HEN ET AL 7HETHER THIS TYPE OF ENZYME IS PROTON TRANSLOCATING IS AN IMPORTANT UNRESOLVED QUESTION FOR UNDERSTANDING THE BIOENER GETICS OF THESE BACTERIA 4VDDJOBUF )N % COLI THE INTERCONVERSION OF FUMARATE AND SUC CINATE CAN BE CARRIED OUT BY TWO RELATED ENZYMES SUCCINATE DEHYDROGENASE WHICH IS EXPRESSED UNDER AEROBIC CONDITIONS OR FUMARATE REDUCTASE WHICH IS INDUCED UNDER ANAEROBIOSIS 3PIRO AND 'UEST 3UCCINATE DEHYDROGENASE SUCCINATEQUINONE OXIDO REDUCTASE CATALYSES ELECTRON TRANSFER FROM SUCCINATE TO UBIQUINONE WHERE SUCCINATE IS OXIDIZED TO FUMARATE AS PART OF THE TRICARBOXYLIC ACID CYCLE ( PYLORI HAS AN INCOMPLETE CITRIC ACID CYCLE AND LACKS SUCCINYL #O! SYNTHETASE AND SUCCINATE DEHYDROGENASE 4OMB ET AL !LM ET AL 0ITSON ET AL WHILE THE PRESENCE OF A FUMARATE REDUCTASE HAS BEEN CONlRMED BY BIOCHEMICAL ANALYSIS -ENDZ AND (AZELL "IRKHOLZ ET AL 0ITSON ET AL AND SEQUENCE DATA 'E ET AL 4OMB ET AL !LM ET AL (OWEVER ( PYLORI CAN RESPIRE SUCCINATE SUCCINATE RESPIRATION RESULTS IN THE PRODUCTION OF ACETATE SUGGESTING REVERSAL OF FUMARATE REDUCTASE AND PARTICIPATION OF ENZYMES TO CONVERT FUMARATE TO PYRUVATE AND THEN ACETATE +ELLY # JEJUNI APPEARS TO HAVE A COMPLETE CITRIC ACID CYCLE WITH GENES ENCODING CLEARLY IDENTIlABLE HOMO
LOGUES OF ALL OF THE CONVENTIONAL ENZYMES 0ARKHILL ET AL INCLUDING SEPARATE SDH AND FRDD OPERONS )N % COLI THE FOUR SUBUNITS OF SUCCINATE DEHYDROGENASE ARE ENCODED BY THE SDH#$!" OPERON 3DH! K$A CONTAINS THE ACTIVE SITE AND COVALENTLY BOUND &!$ 3DH" K$A CONTAINS THREE IRON SULFUR CLUSTERS AND IS RESPONSIBLE FOR THE TRANSFER OF ELECTRONS FROM 3DH#$ TO 3DH! 3UBUNITS 3DH# AND $ AND K$A RESPECTIVELY COMBINE TO SHARE A (EME B AND ARE RESPONSIBLE FOR THE ELECTRON TRANSFER FROM QUINONE TO THE 3DH" MEMBRANE ANCHOR 'ENNIS AND D 3TEWART 4HE # JEJUNI GENOME REVEALS AN SD H!"# OPERON 0ARKHILL ET AL # SDH! #J ENCODES A K$A PROTEIN WITH IDENTITY TO % COLI 3DH! AND CONTAINS AN &!$ BINDING DOMAIN SDH" #J ENCODES A +$A PUTATIVE IRON SULFUR PROTEIN SDH# #J ENCODES A K$A PUTATIVE SUCCINATE DEHYDROGENASE SUBUNIT # WHICH IS A PROBABLE FUNCTIONAL EQUIVALENT TO % COLI SUCCINATE DEHYDROGENASE SUBUNITS # AND $ 0UIFS0SHBOJD$PNQPVOETBT&MFDUSPO %POPST #HANG ET AL WERE UNABLE TO DETECT OXYGEN UPTAKE WHEN ACETATE GLYCEROL , LACTATE OXALOACETATE OXOBUTYRATE AND SEVERAL AMINO ACIDS INCLUDING ASPARTATE AND GLUTAMATE WERE ADDED TO ( PYLORI CELLS (OMOLOGUES OF PROLINE DEHYDROGENASE 0UT! (0*(0 GLYCOLATE OXIDASE 'LC$ (0 *(0 AND A $ AMINO ACID DEHYDROGENASE $AD! (0*(0 PROVIDE ADDITIONAL POSSIBILITIES FOR SUBSTRATE DERIVED ELECTRONS TO BE DONATED TO THE MEMBRANE BOUND ELECTRON TRANSPORT CHAIN AND .A GATA ET AL HAVE DEMONSTRATED THAT , SERINE $ ALANINE AND , PROLINE CAN INDEED ACT AS ELECTRON DONORS FOR WHOLE CELL OXYGEN LINKED RESPIRATION IN ( PYLORI $5IF2VJOPOF1PPM % COLI UTILIZES THE HIGHER POTENTIAL UBIQUINONE % %M M6 DURING AEROBIC RESPIRATION BUT UNDER ANAEROBIC CONDITIONS IT SWITCHES TO USING THE LOWER MIDPOINT POTENTIAL MENAQUINONE % %M n M6 )NGELDEW AND 0OOLE (ELICOBACTER PYLORI AND # JEJUNI DO NOT CONTAIN UBIQUINONE MENAQUINONE IS THE SOLE ISOPRENOID QUINONE PRESENT #ARLONE AND !NET #OLLINS ET AL -ARCELLI ET AL -ARCELLI ET AL DEMONSTRATED THAT -+ IS THE DOMINANT FORM OF MENAQUINONE WITH TRACES ^
-RQDWKDQ'0\HUVDQG'DYLG-.HOO\ OF -+ IN SEVERAL STRAINS OF ( PYLORI TESTED .O ALTERATION IN QUINONE TYPE WAS DETECTED DURING GROWTH WITH VARIOUS / LEVELS BETWEEN n VV AND LEVELS OF MENAQUINONE WERE ALSO FOUND TO BE HIGHEST AT OP TIMUM GROWTH CONCENTRATIONS OF OXYGEN OF TO VV )N # JEJUNI THE MENAQUINONE ALSO CONTAINS SIX ISOPRENE UNITS )N ADDITION TO -+ #AMPYLOBACTER SSP HAVE BEEN FOUND TO CONTAIN A NOVEL METHYL SUB STITUTED -+ THAT HAS NOT BEEN REPORTED IN ( PYLORI #ARLONE AND !NET #OLLINS ET AL %5IF$ZUPDISPNF EF $PNQMFY 4HE CYTOCHROME BC COMPLEX IS AN OLIGOMERIC MEM BRANE PROTEIN COMPLEX CONTAINING THREE SUBUNITS AND FOUR REDOX CENTERS WHICH COUPLES ELECTRON TRANSFER FROM QUINONE TO PROTON TRANSLOCATION ACROSS THE MEMBRANE LEADING TO THE FORMATION OF AN ELECTRO CHEMICAL PROTON GRADIENT 4RUMPOWER 4HE BC COMPLEX IS FOUND IN MANY 'RAM NEGATIVE AND 'RAM POSITIVE BACTERIA AEROBIC ANAEROBIC AND PHO TOSYNTHETIC BACTERIA AND IN MITOCHONDRIA OF LOWER AND HIGHER EUKARYOTES ALTHOUGH IT IS NOT PRESENT IN % COLI )NGELDEW AND 0OOLE )T IS USUALLY FOUND IN RESPIRATORY CHAINS THAT CONTAIN UBIQUINONE AN IMPORTANT CONSEQUENCE OF THE USE OF MENAQUINONE IN ( PYLORI AND # JEJUNI IS THAT THE BC COMPLEX MUST BE ABLE TO OXIDIZE THE LOWER POTENTIAL MENAQUINOL RATHER THAN UBIQUINOL %VIDENCE FOR THE OPERATION OF THE CYTOCHROME BC COMPLEX IN ( PYLORI HAS COME FROM RESPIRATORY IN HIBITION STUDIES AND GENOME SEQUENCE INFORMATION ,ACTATE RESPIRATION IN INTACT CELLS IS INHIBITED BY THE SPECIlC CYTOCHROME BC COMPLEX INHIBITORS ANTIMY CIN ! AND MYXOTHIAZOL !LDERSON ET AL )N ADDITION SUCCINATE CYTOCHROME C REDUCTASE ACTIVITY WAS INHIBITED BY THESE REAGENTS IN SONICATES #HEN ET AL )N ( PYLORI GENES ENCODING THE THREE SUBUNITS OF THE COMPLEX ARE PRESENT IN AN OPERON #YTOCHROMES B AND C ARE PRESENT AS SEPARATE POLY PEPTIDE SUBUNITS ENCODED BY (0*(0 AND (0*(0 RESPECTIVELY ALONG WITH AN &E 3 PROTEIN THE 2IESKE PROTEIN CONTAINING A &E 3 CLUSTER ENCODED BY (0*(0 .O STUDIES TO DATE HAVE BEEN PUBLISHED ON THE PROPERTIES OR OPERATION OF THE # JEJUNI CYTOCHROME I BC COMPLEX ALTHOUGH THE CYTOCHROME B AND CYTOCHROME C REDOX CENTERS OF THIS COMPLEX WERE DETECTED IN THE EARLY WORK OF #ARLONE AND ,ASCELLES 'ENES ENCODING THE THREE SUBUNITS OF THE BC COMPLEX CAN BE FOUND IN # JEJUNI #JC C
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER & FUZQF$ZUPDISPNFTJO +S\ORUL BOE & MHMXQL #YTOCHROMES C ARE PROTEINS THAT CONTAIN COVALENTLY BOUND HEME AND FUNCTION IN A RANGE OF REDOX REAC TIONS IN BACTERIA C TYPE CYTOCHROMES OCCUR EITHER AS SOLUBLE PERIPLASMIC PROTEINS OR ANCHORED TO THE CYTOPLASMIC MEMBRANE VIA . TERMINAL HYDROPHO BIC EXTENSIONS 4HÚNY -EYER "IOGENESIS OF C TYPE CYTOCHROMES IS COMPLEX THE BIOSYNTHESIS OF HEME EXPORT OF THE APOPROTEIN TO THE PERIPLASM AND COVALENT LINKAGE OF THE HEME MOIETY TO THE APO PROTEIN ARE ALL ESSENTIAL STEPS IN THIS PROCESS 4HÚNY -EYER 4HERE ARE INTERESTING DIFFER ENCES IN THE BIOGENESIS OF CYTOCHROMES C BETWEEN ( PYLORI AND # JEJUNI WHICH ARE APPARENT FROM AN ANALYSIS OF THE RESPECTIVE GENOME SEQUENCES # JE JUNI CONTAINS HOMOLOGUES OF THE CLASSICAL CCM GENES THAT ARE PRESENT IN MANY 'RAM NEGATIVE BACTERIA AND WHICH CONSTITUTE THE COMPLEX @3YSTEM ) PATHWAY ( PYLORI HOWEVER EMPLOYS THE MUCH SIMPLER @3YSTEM )) PATHWAY WHICH IS CHARACTERISTIC OF 'RAM POSITIVE BACTERIA +RANZ ET AL 3ONICATES OF ( PYLORI WERE SHOWN TO HAVE ASCORBIC ACID OXIDIZING ACTIVITY DUM AND !NDERSON WHICH WAS PROPOSED TO BE RESPONSIBLE FOR THE DESTRUC TION OF GASTRIC VITAMIN # SEEN IN ( PYLORI INFECTED PATIENTS ! WATER SOLUBLE COMPONENT OF THE ( PYLORI SONICATE WAS SHOWN TO BE RESPONSIBLE FOR THE ASCORBATE OXIDIZING ACTIVITY AND THIS WAS TENTATIVELY ASSIGNED TO A LOW MOLECULAR MASS K$A CYTOCHROME C DUM AND !NDERSON 3PECTROSCOPIC STUDIES ON ( PYLORI -ARCELLI ET AL .AGATA ET AL CONlRMED THE PRESENCE OF C TYPE CYTOCHROMES IN THE ORGANISM 4HE PURIlCATION AND . TERMINAL SEQUENCE OF A PERIPLASMIC SOLUBLE C TYPE CYTOCHROME HAS BEEN REPORTED %VANS AND %VANS +OYANAGI ET AL AND CHARACTERIZED SPECTROSCOPICALLY AS CYTO CHROME C #YTOCHROME C HAS BEEN IDENTIlED AS A POTENTIAL ELECTRON DONOR TO THE CB TYPE CYTOCHROME C OXIDASE 4SUKITA ET AL +OYANAGI ET AL 4HE REDOX POTENTIAL OF THE PROTEIN WAS REPORTED TO BE M6 +OYANAGI ET AL 4HE GENOME SE QUENCES OF ( PYLORI AND * CONTAIN ONLY ONE ANNOTATED C TYPE CYTOCHROME GENE CYC! (0 *(0 WHICH ENCODES THE PROTEIN IDENTIlED IN THESE BIOCHEMICAL STUDIES )NTERESTINGLY THE CYC! GENE IS TRANSCRIBED DIVERGENTLY FROM A HOMOLOGUE . GENE ENCODING AN IMPORTANT ENZYME OF THE HEM. IN THE ANAEROBIC BIOSYNTHESIS OF HEME 3EARCHING THE WHOLE GENOME SEQUENCE FOR GENES
THAT ENCODE PROTEINS CONTAINING THE #XX#( MOTIF HAS IDENTIlED A SECOND PUTATIVE LOW MOLECULAR MASS CYTOCHROME C +ELLY ET AL 4HE GENE ENCODING THIS PUTATIVE CYTOCHROME (0 DESIGNATED CYC" IS FOUND AS THE DISTAL GENE OF AN EIGHT GENE OPERON 3IGNIlCANTLY HOWEVER IN ADDITION TO THE PUTATIVE CYTOCHROME GENE THERE ARE TWO GENES WITHIN THIS OPERON THAT ARE INVOLVED IN HEME BIOSYNTHESIS HEM! AND HEM# "OTH #YC! AND #YC" CONTAIN A SINGLE #XX#( MOTIF INDICATING THAT THEY ARE MONOHEME C TYPE CYTOCHROMES 4HE ROLE OF #YC" IN ELECTRON TRANSPORT IS UNKNOWN !NALYSIS OF THE # JEJUNI GENOME REVEALS A PUTATIVE PERIPLASMIC MONO HEME CYTOCHROME C #J AS THE MOST LIKELY CANDIDATE TO BE AN ELECTRON DONOR TO THE CYTOCHROME C OXIDASE WITH IDENTITY WITH THE ( PYLORI CYTOCHROME C AND IDENTITY TO THE CYTOCHROME C OF $ESULFOVIBRIO VULGARIS +OYANAGI ET AL 0ARKHILL ET AL 5NLIKE ( PYLORI # JEJUNI CONTAINS MANY PROTEINS WITH A POTENTIAL HEME # BINDING MOTIF #88#( ONLY SOME OF WHICH HAVE BEEN ANNOTATED AS CYTOCHROMES IN THE GENOME DATABASE 0ARKHILL ET AL -ANY OF THESE ARE ASSOCIATED WITH TERMINAL REDUCTASES FOR ALTERNATIVE ELECTRON ACCEPTORS AND ARE DISCUSSED IN 3ECTION ( BUT THE OTHERS HAVE AS YET UNKNOWN FUNCTIONS '5FSNJOBM0YJEBTFTGPS0YZHFO%FQFOEFOU 3FTQJSBUJPO -OST BACTERIA POSSESS AT LEAST TWO TERMINAL OXIDASES OFTEN A QUINOL OXIDASE AND A CYTOCHROME C OXIDASE 0OOLE AND #OOK WITH DIFFERENT CATALYTIC PROP ERTIES AND TRANSCRIPTIONAL CONTROLS 'ENOME ANALYSIS OF # JEJUNI AND ( PYLORI INDICATES THE ABSENCE OF ANY HOMOLOGUES FOR AN AA TYPE CYTOCHROME C OXI DASE /NLY ONE TERMINAL OXIDASE A CB CBB TYPE CYTOCHROME C OXIDASE HAS BEEN FOUND TO TERMINATE THE RESPIRATORY CHAIN OF (ELICOBACTER PYLORI -ARCELLI ET AL .AGATA ET AL )T IS ENCODED BY 0 THE CCO./10 OPERON AND THE SUBUNIT STRUCTURE IS ESSENTIALLY THE SAME AS IN ALL OTHER ORGANISMS FROM WHICH THIS ENZYME HAS BEEN IDENTIlED EXCEPT THAT THE #CO. SUBUNIT FROM ( PYLORI IS TRUNCATED AT THE . TERMINUS WHEN COMPARED TO #CO. SUBUNITS FROM OTHER BACTERIA 4HE AMINO ACID SEQUENCES OF THE 0 GENES REVEAL CONSERVED MOTIFS FOR CCO/ AND CCO0 THE BINDING OF HEME # #88#( #CO/ IS CAPABLE OF BINDING A SINGLE HEME # AND #CO0 IS CAPABLE OF BINDING TWO HEMES # 4HE ( PYLORI OXIDASE HAS BEEN REPORTED TO HAVE A +- FOR OXYGEN OF - .AGATA
ET AL OR - 4SUKITA ET AL BUT THE VALUES WERE DETERMINED USING A RELATIVELY INSENSITIVE MEMBRANE COVERED / ELECTRODE WHICH PROBABLY UNDERESTIMATES THE TRUE AFlNITY 4HE OXIDASE HAS BEEN PURIlED 4SUKITA ET AL AND CONTAINS THREE HEMES # AND TWO PROTOHEMES ONE HIGH SPIN ONE LOW SPIN AS PREDICTED (OWEVER SURPRISINGLY #CO. AND #C// APPEAR TO FORM A PROTEIN COMPLEX EVEN IN THE PRESENCE OF 3$3 4HERE IS EVIDENCE THAT THE ENZYME CAN PUMP PROTONS ALTHOUGH THE ( PUMPING ACTIVITY BY RECONSTITUTED PROTEOLIPOSOMES WAS LOW 4SUKITA ET AL #AMPYLOBACTER JEJUNI HAS TWO TERMINAL OXIDASES AS CLEARLY REVEALED BY GENOME ANALYSIS 0ARKHILL ET AL 4HIS CONlRMS THE EARLIER SPECTROSCOPIC WORK OF #ARLONE AND ,ASCELLES AND (OFFMAN AND 'OODMAN /NE OXIDASE IS A CB TYPE CY TOCHROME C OXIDASE VERY SIMILAR TO THAT DESCRIBED . HAS ABOVE IN ( PYLORI 3UBUNIT ) CCO.#JC A MOLECULAR WEIGHT OF K$A AND CONTAINS THE HEME COPPER OXIDASE CATALYTIC CENTER AND A COPPER " BINDING REGION SIGNATURE 3UBUNIT )) CCO/#JC HAS A MOLECULAR WEIGHT OF K$A AND CONTAINS A CYTOCHROME C FAMILY HEME BINDING SITE SIGNATURE 0 3UBUNIT ))) CCO0#JC HAS A MOLECULAR WEIGHT OF K$A AND CONTAINS TWO HEME # BINDING MOTIFS 3UBUNIT )6 CCO1#JC HAS A MOLECULAR WEIGHT OF K$A WITH IDENTITY TO THE CORRESPOND ING ( PYLORI SUBUNIT .O FUNCTIONAL STUDIES HAVE YET BEEN REPORTED ON THIS OXIDASE #YTOCHROME BDD ENCODED BY THE CYD!" OPERON IS WELL CHARACTERIZED IN % COLI 'REEN ET AL WHERE IT IS EXPRESSED UNDER MICROAEROBIC CONDITIONS &U ET AL AS A HETERODIMER LOCATED IN THE CYTO PLASMIC MEMBRANE -ILLER ET AL 4HE ENZYME OXIDIZES UBIQUINOL IN THE LIPID BILAYER AND REDUCES OXYGEN TO WATER CONTRIBUTING TO THE GENERATION OF A PROTON MOTIVE FORCE ( / RATIO -ILLER AND 'EN NIS ,ORENCE ET AL IDENTIlED THE THREE HEME PROSTHETIC GROUPS IN THE ENZYME HEME B HEME B AND HEME D 1UINOL OXIDATION IS LIKELY TO BE CARRIED OUT BY HEME B AND OXYGEN BINDING AT HEME D (ATA 4ANAKA ET AL #ARLONE AND ,ASCELLES DEMONSTRATED AN ALTERNATIVE TERMINAL OXIDASE IN # JEJUNI AND ANALYSIS OF THE GENOME SEQUENCE REVEALS THE PRESENCE OF A CYD!" LIKE OPERON ENCOD ING SUBUNITS ) AND )) 3UBUNIT ) K$A IS ENCODED BY CYD! #J AND HAS IDENTITY TO % COLI CYTOCHROME BD OXIDASE SUBUNIT ) 3UBUNIT )) K$A D IS ENCODED BY CYD" #J AND IS SIMILAR TO THE % COLI SUBUNIT )) WITH IDENTITY 0ARKHILL ET AL
-RQDWKDQ'0\HUVDQG'DYLG-.HOO\ 4HE PHYSIOLOGICAL ROLES OF THE TWO OXIDASES IN # JEJUNI ARE UNKNOWN ()ZESPHFOQFSPYJEFBTBO&MFDUSPO"DDFQ UPS (YDROGEN PEROXIDE IS PRODUCED AS A BYPRODUCT IN THE REDUCTION OF MOLECULAR OXYGEN TO WATER )T IS A PARTIALLY REDUCED SPECIES THAT IS TOXIC TO THE CELL (YDROGEN PEROXIDE CAN BE DEGRADED TO (/ AND / BY THE CYTO PLASMIC ENZYME CATALASE BUT IN THE PERIPLASM (/ CAN BE BROKEN DOWN TO WATER ALONE BY A PERIPLASMIC CYTOCHROME C PEROXIDASE WITH THE REQUIREMENT OF REDUCED CYTOCHROME C AS AN ELECTRON DONOR (/ EnCREDD ( A (/ COX #YTOCHROME C PEROXIDASE ##0 HAS BEEN ISOLATED FROM A RANGE OF BACTERIA INCLUDING .ITROSOMONAS EUROPAEA !RCIERO AND (OOPER 2HODOBACTER CAPSULATUS (U ET AL 0ARACOCCUS DENITRIlCANS 'ILMOUR ET AL AND 0SEUDOMONAS AERUGINOSA 2ÚNNBERG ET AL 3TUDIES OF ##0 IN THESE BAC TERIA HAVE REVEALED THAT THE ENZYME HAS A MOLECULAR WEIGHT OF n K$A AND CONTAINS TWO HEMES # ONE HIGH POTENTIAL AND ONE LOW POTENTIAL 4HE HIGH POTENTIAL HEME IS THE SOURCE OF THE SECOND ELECTRON FOR (/ REDUCTION AND THE LOW POTENTIAL HEME ACTS AS A PEROXIDATIC CENTER !NALYSIS OF THE # JEJUNI GE NOME REVEALS TWO SEPARATE GENES ENCODING PUTATIVE CYTOCHROME C PEROXIDASE HOMOLOGUES 0ARKHILL ET AL #JC AND #J ARE K$A AND K$A RESPECTIVELY WITH BOTH PROTEINS CONTAINING TWO CYTOCHROME C HEME BINDING SITE SIGNATURES #JC IS SIMILAR TO THE CYTOCHROME C PEROXIDASE OF .ITRO SOMONAS EUROPAEA AND 0SEUDOMONAS AERUGINOSA WITH AND IDENTITY RESPECTIVELY 0ARKHILL ET AL #J IS SIMILAR TO THE ( PYLORI PUTATIVE CYTOCHROME C PEROXIDASE HOMOLOGUE (0 WITH IDENTITY AND TO THE CYTOCHROME C PEROXIDASE OF 0SEUDOMONAS AERUGINOSA WITH IDENTITY 0ARKHILL ET AL 4HE PHYSIOLOGICAL ROLES AND REGULATION OF EXPRESSION OF THE PEROXIDASES HAVE YET TO BE STUDIED (OWEVER PEROXIDASE HAS BEEN STUDIED IN THE RELATED MICROAEROPHILE #AMPYLOBACTER MUCOSALIS WHERE THE OXIDATION OF FORMATE LEADS TO THE GENERATION OF PERIPLASMIC (/ 4HIS IS REDUCED BY ##0 USING ELECTRONS FROM CYTOCHROME C THAT HAS BEEN REDUCED BY THE BC COMPLEX 4HUS REMOVAL OF (/ FROM THE PERIPLASM LEADS TO NET PROTON EXTRUSION AND ENERGY CONSERVATION 'OODHEW ET AL
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER )3FTQJSBUJPOXJUI"MUFSOBUJWF5FSNJOBM&MFD USPO"DDFQUPSTUP0YZHFO 'VNBSBUFBT&MFDUSPO"DDFQUPS )N THE ABSENCE OF OXYGEN FUMARATE CAN BE USED AS AN ALTERNATIVE TERMINAL ELECTRON ACCEPTOR FOR THE PROTON TRANSLOCATING ELECTRON TRANSPORT CHAIN AND IS IMPORTANT IN !40 GENERATION IN MANY ANAEROBIC BACTERIA ( PYLORI FUMARATE REDUCTASE IS VERY SIMILAR TO THAT OF THE RELATED ANAEROBE 7OLINELLA SUCCINOGENES AND IS ENCODED BY THREE GENES FRD#!" (0 *(0 ENCODING POLYPEPTIDES OF AND K$A RESPECTIVELY 'E AT AL 4OMB ET AL &RD! AND &RD" DISPLAY THE AMINO ACID MOTIFS FOR &!$ AND &E 3 BINDING RESPECTIVELY IN COMMON WITH OTHER FUMARATE REDUCTASES AND ALSO SUCCINATE DEHYDROGENASE ENZYMES 4HE FRD# # GENE ENCODES A HYDROPHOBIC DI HEME CYTOCHROME B WHICH MAY SERVE AS A MEMBRANE ANCHOR FOR &RD! AND " ,IKE THE FUMARATE REDUCTASE OF 7 SUCCINOGENES ACTIVITY WAS LOCALIZED IN THE MEMBRANE FRACTION "IRKHOLZ ET AL 'E ET AL -UTANTS IN FRD! HAVE BEEN GENERATED INDICATING THIS ENZYME IS NOT ESSENTIAL FOR ( PYLORI (OWEVER THE MUTANTS DEMONSTRATED A PRO LONGED LAG PHASE ON STANDARD GROWTH MEDIUM UNDER MICROAEROBIC CONDITIONS 'E ET AL %XPRESSION OF FUMARATE REDUCTASE ACTIVITY APPEARS TO BE CONSTITU TIVE IN ( PYLORI AS UNLIKE % COLI LEVELS OF ACTIVITY DID NOT MARKEDLY CHANGE IN CELLS GROWN UNDER VARYING / CONCENTRATIONS $AVIDSON ET AL !LTHOUGH THE PRESENCE OF FUMARATE REDUCTASE PROVIDES EVIDENCE OF ANAEROBIC TYPE RESPIRATION ( PYLORI HAS NOT BEEN SUCCESSFULLY CULTURED UNDER STRICTLY ANAEROBIC CONDITIONS IN THE PRESENCE OF FU MARATE AS A TERMINAL ELECTRON ACCEPTOR AND 6ERON ET AL REPORTED ANAEROBIC GROWTH WITH FUMARATE TO BE ABSENT IN # JEJUNI 9ET CYTOCHROME B LINKED FUMARATE REDUCTASE ACTIVITY WAS DEMONSTRATED IN THE MEMBRANES OF BOTH # FETUS AND # JEJUNI #ARLONE AND ,ASCELLES (ARVEY AND ,ASCELLES !NALYSIS OF THE # JEJUNI GENOME REVEALS A FRD!"# OPERON ENCODING SUBUNITS SIMILAR TO THE THOROUGHLY INVESTIGATED FUMARATE REDUCTASE OF 7 SUCCINOGENES AND TO ( PYLORI 7HY THEN ARE ( PYLORI AND # JEJUNI APPARENTLY UNABLE TO GROW BY ANAEROBIC RESPIRATION OF FUMARATE 3ELLARS ET AL FOUND THAT ALTHOUGH GROWTH WITH FUMARATE AND OTHER ELECTRON ACCEPTORS SUCH AS NITRATE NITRITE AND 4-!/ WAS INSIGNIlCANT UNDER STRICTLY ANAEROBIC CONDITIONS ELECTRON ACCEP TOR DEPENDENT GROWTH WAS POSSIBLE UNDER SEVERELY
OXYGEN LIMITED CONDITIONS 4HEIR RESULTS INDICATED THAT SOME OXYGEN REQUIRING METABOLIC REACTIONS PREVENTED ANAEROBIC GROWTH !S # JEJUNI ONLY CON TAINS GENES FOR AN OXYGEN REQUIRING CLASS ) TYPE OF RIBONUCLEOTIDE REDUCTASE IT WAS SUGGESTED THAT THE INABILITY TO SYNTHESIZE $.! ANAEROBICALLY IS THE MOST LIKELY EXPLANATION #ONSISTENT WITH THIS CELLS INCUBATED ANAEROBICALLY WITH ELECTRON ACCEPTORS DID NOT DIVIDE PROPERLY BUT FORMED lLAMENTS ANALOGOUS TO THOSE SEEN AFTER TREATMENT OF AEROBIC CELLS WITH THE 2.2 INHIBITOR HYDROXYUREA 3ELLARS ET AL 4HUS # JEJUNI CAN USE ALTERNATIVE ELECTRON ACCEP TORS IN ENERGY CONSERVING REACTIONS BUT ONLY IF SOME OXYGEN IS PRESENT TO SATISFY THE REQUIREMENT FOR DEOXYRIBONUCLEOTIDE PRODUCTION ! SIMILAR EXPLAN ATION FOR ( PYLORI IS LIKELY AS IT TOO ONLY CONTAINS THE CLASS ) 2.2 GENES ! MODEL FOR FUMARATE RESPIRATION COUPLED TO ENERGY CONSERVATION IN # JEJUNI IS SHOWN IN &IG A 4HE ACTIVE SITE OF FUMARATE REDUCTION IS ON THE CYTOPLASMIC SIDE OF THE MEMBRANE BUT ONLY IF QUINOL OXIDATION RELEASES PROTONS INTO THE PERIPLASM WILL FUMARATE REDUCTASE BE AN ELECTROGENIC ENZYME AND CONTRIBUTE TO THE GENERATION OF A PROTON MOTIVE FORCE 4HIS HAS YET TO BE PROVEN (OWEVER THIS IS DElNITELY NOT THE CASE FOR THE OTHER REDUCTASES DIS CUSSED BELOW WHICH ARE ALL PERIPLASMIC IN LOCATION SEE &IG BnD /JUSBUFBT&MFDUSPO"DDFQUPS 4HERE ARE THREE TYPES OF BACTERIAL NITRATE REDUCTASES I SOLUBLE CYTOPLASMIC ASSIMILATORY NITRATE REDUCTASES .!3 II MEMBRANE ASSOCIATED @RESPIRATORY NITRATE REDUCTASES .!2 III SOLUBLE PERIPLASMIC @DISSIMI LATORY NITRATE REDUCTASES .!0 0OTTER ET AL 4HE .!0 CLASS OF NITRATE REDUCTASE IS A TWO SUBUNIT COMPLEX LOCATED IN THE PERIPLASM WHICH IS COUPLED TO QUINOL OXIDATION VIA A MEMBRANE ANCHORED TETRA HEME CYTOCHROME !LTHOUGH ( PYLORI DOES NOT HAVE GENES FOR ANY OF THE KNOWN TYPES OF NITRATE REDUCTASE # JEJUNI IS PREDICTED TO POSSESS A PERIPLASMIC .!0 ENZYME ENCODED BY A NAP!'("$ OPERON 0ARKHILL ET AL +ELLY 3ELLARS ET AL AND NITRATE REDUCTASE ACTIVITY HAS BEEN DEMONSTRATED IN INTACT CELLS BY NITRITE ACCUMULATION ASSAYS 3ELLARS ET AL !S WITH FUMARATE NITRATE DEPENDENT GROWTH IN # JEJUNI CAN ONLY BE DEMONSTRATED UNDER OXYGEN LIMITED NOT STRICTLY ANAEROBIC CONDITIONS 3ELLARS ET AL .AP! IS A ^ K$A CATALYTIC SUBUNIT WHICH BINDS A BIS -'$ CO FACTOR AND A ;&E3= CLUSTER .AP" IS
&IG 0REDICTED TOPOLOGICAL ORGANIZATION AND CONSEQUENCES FOR ENERGY CONSERVATION OF ALTERNATIVE ELECTRON TRANSPORT CHAINS IN # JEJUNI )N EACH CASE FORMATE IS DEPICTED AS THE ELECTRON DONOR TO THE QUINONE POOL THROUGH THE ACTION OF FORMATE DEHYDROGENASE &DH A FUMARATE RESPIRATION MAY RESULT IN THE GENERATION OF A PROTON MOTIVE FORCE BY FUMARATE REDUCTASE &RD IF THE SITES OF QUINOL OXIDATION AND FUMARATE REDUCTION ARE ON OPPOSITE SIDES OF THE MEMBRANE 7ITH 4-!/$-3/ B NITRITE C AND NITRATE D THE TERMINAL REDUCTASES ARE ALL PREDICTED TO BE PERIPLASMIC SO THAT REDUCTION OF THE ELECTRON ACCEPTOR AND QUINOL OXIDATION OCCUR ON THE SAME 0 PHASE SIDE OF THE MEMBRANE )N THESE CASES A PROTON MOTIVE FORCE CAN ONLY BE GENERATED AT THE LEVEL OF THE PRIMARY DEHYDROGENASE
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&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER A ^ K$A ELECTRON TRANSFER SUB UNIT WHICH IN OTHER BACTERIA BINDS TWO C TYPE HEMES 2ICHARDSON ET AL .AP$ K$A IS PROPOSED TO BE INVOLVED IN MATURATION OF .AP! PRIOR TO EXPORT TO THE PERIPLASM "ERKS ET AL 0OTTER AND #OLE 4HE NAP OPERON OF # JEJUNI DOES NOT CONTAIN NAP# ENCODING THE .AP# SUBUNIT .AP# ACTS AS AN ELECTRON DONOR TO THE .AP!" COMPLEX A ROLE WHICH WAS SHOWN TO BE ESSENTIAL IN THE FUNCTION OF NITRATE REDUCTASES IN MANY BACTERIA INCLUDING % COLI AND 0ARACOCCUS PANTOTROPHUS "ERKS ET AL 0OTTER AND #OLE !NALYSIS OF THE # JEJUNI GENOME SHOWS THAT IT DOES ENCODE A .AP# HOMOLOGUE #JC BUT IT IS UPSTREAM OF THE NRF! NITRITE REDUCTASE GENE 4HE PRODUCT OF #JC IS A .AP#.IR4 TYPE CYTO CHROME C HAVING IDENTITY TO THE % COLI + .AP# SUBUNIT 0ARKHILL ET AL $ESPITE THIS THE OPERONAL LOCATION OF #JC IMPLIES THAT IT IS PART OF THE NITRITE REDUCTASE SYSTEM AND THUS MAY NOT BE INVOLVED IN ELECTRON TRANSFER TO THE NITRATE REDUCTASE ! POTENTIAL ROLE OF #JC IN NITRITE REDUCTION IS SUGGESTED BY STUDIES ON THE NRFF OPERON IN 7OLINELLA SUCCINOGENES WHICH ENCODES A .AP# LIKE SUBUNIT .RF( SIMILAR TO THE PRODUCT OF #JC 3IMON ET AL .RF( IS THE MEDIATOR BETWEEN THE QUINONE POOL AND THE CYTOCHROME C NITRITE REDUCTASE .RF! OF 7 SUCCINOGENES 3IMON ET AL 4HIS LEAVES THE QUESTION OF HOW THE .AP!" COMPLEX IS COUPLED TO MENAQUINOL OXIDATION /NE POSSIBILITY IS THAT THE .AP' AND .AP( SUBUNITS COULD FUNCTION IN THIS ROLE 3ELLARS ET AL .AP' K$A IS PREDICTED TO BIND UP TO FOUR &E 3 CLUSTERS .AP( K$A IS PREDICTED TO BE AN INTEGRAL MEMBRANE PROTEIN WITH FOUR TRANSMEMBRANE HELICES WITH BOTH THE . AND # TERMINUS ON THE CYTOPLASMIC SIDE OF THE MEMBRANE ! MODEL FOR NITRATE RESPIRATION IN # JEJUNI IS SHOWN IN &IG D "ECAUSE OF THE PERIPLASMIC LOCATION OF .AP A PROTON MOTIVE FORCE CAN ONLY BE GENERATED AT THE LEVEL OF THE PRIMARY DEHYDROGENASE AND NOT FROM QUINOL OXIDATION 4HERE IS INCREASING EVIDENCE THAT NITRATE RESPIRATION MAY PLAY A SIGNIlCANT ROLE IN THE GROWTH OF HUMAN AND ANIMAL PATHOGENS IN VIVO )N -YCOBACTERIUM BOVIS MEMBRANE BOUND NITRATE REDUCTASE .AR ACTIVITY HAS BEEN SHOWN TO CONTRIBUTE TO VIRULENCE 7EBER ET AL AND NAR MUTANTS WERE UNABLE TO COLONIZE A MOUSE ANIMAL MODEL )N A WIDE RANGE OF 'RAM NEGATIVE BACTERIAL PATHOGENS INCLUDING # JEJUNI PERIPLASMIC NITRATE REDUCTASES ARE MORE COMMONLY PRESENT THAN .AR TYPE ENZYMES 0OTTER ET AL .ITRATE CONCENTRATIONS IN HUMAN BODY mUIDS ARE IN THE
RANGE n - 0OTTER ET AL 3IGNIlCANTLY IT HAS BEEN SHOWN THAT IN % COLI WHICH HAS BOTH .AR AND .AP THE .AP ENZYME HAS A MUCH HIGHER AFlNITY FOR NITRATE COMPARED TO THE MEMBRANE BOUND .AR 0OTTER ET AL MAKING .AP IDEALLY SUITED TO A ROLE IN SCAVENGING THE LOW NITRATE CONCENTRATIONS ENCOUNTERED IN VIVO /JUSJUFBT&MFDUSPO"DDFQUPS 4HREE DISTINCT ENZYMES IN ENTEROBACTERIA CATALYZE THE REDUCTION OF NITRITE TO AMMONIA I THE ANAERO BIC NITRITE REDUCTASE .IR"$ WHICH IS PRESENT IN THE MAJORITY OF ENTEROBACTERIA IS RESPONSIBLE FOR THE REGENERATION OF .!$ AND THE DETOXIlCATION OF NITRITE II ASSIMILATORY NITRITE REDUCTASE .AS" IS FOUND IN ENTEROBACERIA SUCH AS +LEBSIELLA SPP BUT IT IS NOT FOUND IN % COLI + OR 3 TYPHIMURIUM III RESPIRATORY NITRITE REDUCTASE .RF!" FOUND IN % COLI + 'ENNIS AND 3TEWART 4HE NITRITE REDUCING ENZYME OF INTEREST IN # JEJUNI IS CYTOCHROME C NITRITE REDUCTASE .RF! WHICH IS THE TERMINAL ENZYME IN THE SIX ELECTRON DISSIMILATORY REDUCTION OF NITRATE TO AMMONIA ./n ( En A .( (/
4HIS IS A PERIPLASMIC ENZYME WHICH WAS DETECTED IN INTACT CELLS OF # JEJUNI USING METHYL VIOLOGEN AS AN ELECTRON DONOR 3ELLARS ET AL %INSLE ET AL PROPOSED THAT THREE BASIC ELE MENTS ARE REQUIRED TO FORM A FUNCTIONAL PERIPLASMIC NITRITE REDUCTASE COMPLEX I THE .RF! ENZYME II A SYSTEM TO OXIDIZE MEMBRANOUS MENAQUINOL AND TRANSPORT ELECTRONS TO .RF! III A MODIlED HEME LYASE NEEDED FOR THE COVALENT ATTACHMENT OF THE ACTIVE SITE HEME GROUP 4HE CYTOCHROME C NITRITE REDUCTASES .RF! ARE PENTAHEME ENZYMES WITH A MOLECULAR MASS OF n K$A .RF! ACTS AS A HOMODIMER WITH EACH MONOMER PRESUMED TO BE FUNCTIONAL AND TO ACT INDEPENDENTLY 4HE ACTIVE SITE IS LOCATED AT HEME WITH A #A ION IN CLOSE PROXIMITY %INSLE ET AL 4HERE ARE TWO DISTINCT SYSTEMS FOR THE TRANSFER OF ELECTRONS RESULTING FROM MENAQUINOL OXIDATION TO .RF! ENZYME THE .RF( TETRAHEME PROTEIN DESCRIBED BY 3IMON ET AL FOR 7OLINELLA SUCCINOGENES AND THE .RF"#$ SYSTEM OF EG % COLI + IN WHICH THE .RF! IS CONNECTED TO THE QUINOL OXIDASE .RF#$ VIA THE SOLUBLE PENTA HEME CYTOCHROME C .RF" (US SAIN ET AL 4HE PUTATIVE NITRITE REDUCING ENZYME IN # JEJUNI
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#JC AND THE UPSTREAM GENE ENCODING A PUTATIVE .AP#.IR4 TYPE CYTOCHROME C #JC INDICATES SIMILARITY TO THE 7OLINELLA SUCCINOGENES NITRITE REDUC ING SYSTEM IN WHICH .RF! ACCEPTS ELECTRONS FROM A MEMBRANE ANCHORED TETRA HEME CYTOCHROME .RF( 3IMON ET AL .RF( IS A TETRAHEME CYTOCHROME C ACTING AS A QUINOL OXIDASE TO RECEIVE ELECTRONS FROM THE QUINONE POOL AND INTERACTS WITH THE POSITIVELY CHARGED ENVIRONMENT AROUND HEME OF THE .RF! ENZYME %INSLE ET AL .RF! CONTAINS AN UNUSUAL #88#+ MOTIF FOR THE HEME BINDING SITE INSTEAD OF THE #88#( MOTIF FOUND IN MANY HEME CONTAINING ENZYMES 4HIS SUBSTITUTION IN .RF! MAY EXPLAIN THE REQUIREMENT FOR A MODIlED HEME LYASE FOR COVALENT ATTACHMENT OF THE ACTIVE SITE HEME 4HE # JEJUNI .RF! HOMOLOGUE #JC IS SLIGHTLY LARGER AT K$A WHEN COMPARED TO THE 7 SUCCINOGENES .RF! K$A AND CONTAINS A NOVEL LIGATION AT HEME WITH A #88#( MOTIF INSTEAD OF #88#+ 4HIS WOULD EXPLAIN THE ABSENCE IN # JEJUNI OF GENES ENCODING HEME LYASES AND ASSEMBLY PROTEINS THAT ARE FOUND IN OTHER BACTERIA %INSLE ET AL !LTHOUGH THE FUNCTIONAL CONSEQUENCES OF THIS SUBSTITUTION ARE UNKNOWN AT PRESENT IT IS POSSIBLE IT COULD HAVE IM PORTANT IMPLICATIONS FOR THE CATALYTIC ACTIVITY OF THE PROTEIN 4HE POSSIBLE MECHANISM OF NITRITE REDUCTION IN # JEJUNI IS SHOWN IN &IG C 4PS/PYJEFTBT&MFDUSPO"DDFQUPST 4HE MOST COMPREHENSIVE STUDIES OF BACTERIAL $-3/ 4-!/ REDUCTASES ARE THOSE FROM 2HODOBACTER SPP AND %SCHERICHIA COLI 4HE REDUCTION OF 4-!/ AND $-3/ MAY BE CARRIED OUT BY TWO DIFFERENT REDUCTASE ENZYMES AS IN % COLI 'ENNIS AND 3TEWART OR REDUCTION OF BOTH 4-!/ AND $-3/ MAY BE CAR RIED OUT BY A SINGLE REDUCTASE AS IN THE $OR SYSTEM OF 2HODOBACTERR +ELLY ET AL +NABLEIN ET AL )N EITHER CASE THE REDUCTION IS A TWO ELECTRON TRANSFER PROCESS #( 3/ ( En A #( 3 (/
#( ./ ( E A #( . (/
4HE TOR#!$ OPERON IN % COLI ENCODES THE 4-!/ REDUCTASE COMPLEX CONSISTING OF A LARGE PERIPLAS MIC MOLYBDOPROTEIN 4OR! K$A A MEMBRANE ANCHORED PERIPLASMIC CYTOCHROME C 4OR# K$A AND A PRESUMED INTRINSIC MEMBRANE SUBUNIT 4OR$ K$A 'ENNIS AND 3TEWART 4HE $-3/ RE
DUCTASE COMPLEX CONTAINS THREE SUBUNITS CONSISTING OF A LARGE EXTRINSIC MEMBRANE BOUND MOLYBDOPROTEIN FACING THE CYTOPLASM $MS! K$A A SMALLER IRON SULFUR SUBUNIT $MS" K$A AND AN INTRIN SIC MEMBRANE SUBUNIT $MS# K$A "ILOUS AND 7EINER 7EINER ET AL 'ENNIS AND 3TEWART "OTH 4-!/ AND $-3/ REDUCTASE ACTIVITIES CAN BE MEASURED IN INTACT CELLS OF # JEJUNI USING METHYL VIOLOGEN LINKED ASSAYS AND 4-!/ AND $-3/ DEPEN DENT GROWTH UNDER OXYGEN LIMITED BUT NOT ANAEROBIC CONDITIONS HAS BEEN DEMONSTRATED 3ELLARS ET AL !NALYSIS OF THE # JEJUNI GENOME REVEALS A GENE ENCODING A +$A MOLYBDOPROTEIN $OR!4OR! HOMOLOGUE #JC CONTAINING CONSERVED RESIDUES FOR THE BINDING OF A MOLYBDENUM GUANOSINE DINU CLEOSIDE -'$ CO FACTOR AND AN . TERMINAL MOTIF 22+&,+ SIMILAR TO THE @TWIN ARGININE TRANSLOCASE RECOGNITION MOTIF 34228&,+ CHARACTERISTIC OF EXTRACYTOPLASMIC PROTEINS CONTAINING COMPLEX REDOX CO FACTORS "ERKS 4HE #JC GENE WAS MU TATED AND THE MUTANT WAS FOUND TO DElCIENT IN BOTH 4-!/ AND $-3/ REDUCTASE ACTIVITY INDICATING THAT A SINGLE ENZYME IS RESPONSIBLE 3ELLARS ET AL )N ADDITION THERE IS AN UPSTREAM GENE ENCODING A K$A MONO HEME C TYPE CYTOCHROME #JC WITH SIMILARITY TO THE # TERMINUS OF THE MEMBRANE AN CHORED PENTAHEME C TYPE CYTOCHROME 4OR# OF % COLI 'ON ET AL 3ELLARS ET AL 4OR# CONTAINS A # TERMINAL EXTENSION 4OR## WITH AN ADDITIONAL HEME BINDING SITE RESPONSIBLE FOR THE ELECTRON TRANS FER TO 4OR! 4HE TETRAHEME . TERMINUS 4OR#. IS INVOLVED IN THE BINDING OF 4OR# TO 4OR! 'ON ET AL 4HE MONO HEME C TYPE CYTOCHROME #JC OF # JEJUNI MAY BE INVOLVED IN ELECTRON TRANSPORT TO THE $OR!4OR! HOMOLOGUE #JC AS WITH THE # TERMINAL PART OF THE 4OR# SUBUNIT IN % COLI 4HE POS SIBLE TOPOLOGICAL ORGANIZATION OF THE ELECTRON TRANSPORT CHAIN TO 4-!/$-3/ IS SHOWN IN &IG B ALTHOUGH ADDITIONAL REDOX PROTEINS ARE LIKELY TO BE INVOLVED IN ELECTRON TRANSFER FROM QUINOL TO #J ( PYLORI MAY ALSO BE CAPABLE OF 4-!/$-3/ RESPIRATION AS IT CONTAINS A PUTATIVE MOLYBDOPROTEIN HOMOLOGUE OF #J (0 WHICH HAS A TWIN ARGININE MOTIF AT THE . TERMINUS INDICATING A PERIPLASMIC LOCATION SEE &IG A 7$PODMVTJPOT !LTHOUGH CLOSELY RELATED PHYLOGENETICALLY AND SHAR
&KDSWHU 5HVSLUDWLRQLQ (ELICOBACTER DQG#AMPYLOBACTER ING MANY SIMILARITIES # JEJUNI AND ( PYLORI ARE DISTINCT IN SOME MAJOR ASPECTS OF THEIR PHYSIOLOGY ESPECIALLY WITH REGARD TO ELECTRON TRANSPORT # JEJUNI IS EMERGING AS A MORE VERSATILE AND METABOLICALLY ACTIVE PATHOGEN WITH A COMPLETE CITRIC ACID CYCLE AND A COMPLEX AND HIGHLY BRANCHED RESPIRATORY CHAIN ALLOWING THE USE OF A VARIETY OF ALTERNATIVE ELECTRON ACCEPTORS 4HESE PROPERTIES ENABLE IT TO COLONIZE AND SURVIVE IN A NUMBER OF ENVIRONMENTS IN ADDITION TO THE MAMMALIAN OR AVIAN GUT (OWEVER IT SHOULD BE NOTED THAT THERE ARE MANY UN ANNOTATED REDOX PROTEINS IN THE GENOME SEQUENCE WHICH HAVE NOT BEEN REVIEWED HERE THE FUNCTION OF WHICH WILL UNDOUBTEDLY REVEAL MORE ABOUT THE EMERGING METABOLIC VERSATILITY OF # JEJUNI ( PYLORI IS A MORE SPECIALIZED PATHOGEN LARGELY RESTRICTED TO THE HUMAN STOMACH WITH AN INCOMPLETE CITRIC ACID CYCLE A SIMPLE RESPIRATORY CHAIN AND FEW REGULATORY SYSTEMS "OTH BACTERIA SHARE THE PROPERTY OF BEING MICROAEROPHILES BUT THERE ARE DIFFERENCES IN HOW THEY RESPOND TO OXYGEN AND OXIDATIVE STRESS AND WHILE THERE IS LIKELY TO BE NO SINGLE EXPLANATION FOR THEIR OXYGEN SENSITIVITY THE POSSESSION OF SEVERAL OXYGEN SENSITIVE ENZYMES MAY BE ONE IMPORTANT FACTOR (OWEVER THERE ARE ALSO SOME INTERESTING PARALLELS BETWEEN THESE BACTERIA IN TERMS OF ELECTRON TRANSPORT &OR EXAMPLE BOTH HAVE AN AP PARENTLY UNIQUE TYPE OF COMPLEX ) WHICH APPEARS TO USE AN AS YET UNIDENTIlED ELECTRON DONOR 4HIS NOVEL SYSTEM DESERVES FURTHER STUDY "DLOPXMFEHNFOUT 7ORK ON (ELICOBACTERR AND #AMPYLOBACTER IN THE LABORATORY OF $*+ IS SUPPORTED BY THE 5+ "IOTECH NOLOGY AND "IOLOGICAL 3CIENCES 2ESEARCH #OUNCIL INCLUDING A STUDENTSHIP TO *$- 3FGFSFODFT !LDERSON * #LAYTON #, AND +ELLY $* )NVESTIGATIONS INTO THE AEROBIC RESPIRATORY CHAIN OF (ELICOBACTER PYLORI 'UT 3 ! !LM 2! ,ING ,3 -OIR $4 +ING ", "ROWN %$ $OIG 0# 3MITH $2 .OONAN " 'UILD "# DE*ONGE ", #ARMEL ' 4UMMINO 0* #ARUSO ! 5RIA .ICKELSEN - -ILLS $- )VES # 'IBSON 2 -ERBERG $ -ILLS 3$ *IANG 1 4AYLOR $% 6OVIS '& AND 4RUST 4* 'ENOMIC SEQUENCE COMPARISON OF TWO UNRELATED ISOLATES OF THE HUMAN GASTRIC PATHOGEN (ELICOBACTER PYLORI .ATURE n !RCIERO $- AND (OOPER !" ! DI HEME CYTOCHROME C PEROXIDASE FROM .ITROSOMONAS EUROPAEA CATALYTICALLY ACTIVE
IN BOTH THE OXIDIZED AND HALF REDUCED STATES * "IOL #HEM n "AILLON -, VAN 6LIET !( +ETLEY *- #ONSTANINIDOU # AND 0ENN #7 !N IRON REGULATED ALKYL HYDROPEROXIDE REDUCTASE !HP# CONFERS AEROTOLERANCE AND OXIDATIVE STRESS RESISTANCE TO THE MICROAEROPHILIC PATHOGEN #AMPYLOBACTER JEJUNI * "ACTERIOL n "AKER ,-3 2AUDONIKIENE ! (OFFMAN 03 AND 0OOLE ," %SSENTIAL THIOREDOXIN DEPENDENT PEROXIREDOXIN SYSTEM FROM (ELICOBACTER PYLORI 'ENETIC AND KINETIC CHARACTERIZATION * "ACTERIOL n "ERG ", ,I * (EIDER * AND 3TEWART 6 .ITRATE INDUCIBLE FORMATE DEHYDROGENASE IN %SCHERICHIA COLI +n ) .UCLETIDE SEQUENCE OF THE FDN'() OPERON AND EVIDENCE THAT OPAL 5'! ) ENCODES SELENOCYSTEINE * "IOL #HEM n "ERKS "# 2ICHARDSON $* 2EILLY ! 7ILLIS !# AND &ERGUSON 3* 4HE NAP%$!"# # GENE CLUSTER ENCODING THE PERIPLASMIC NITRATE REDUCTASE SYSTEM OF 4HIOSPHAERA PANTOTROPHA "IOCHEM * n "ERKS "# ! COMMON EXPORT PATHWAY FOR PROTEINS BINDING COMPLEX REDOX COFACTORS -OL -ICROBIOL n "ILOUS 04 AND 7EINER *( -OLECULAR CLONING AND EXPRESSION OF THE %SCHERICHIA COLI $-3/ REDUCTASE OPERON * "ACTERIOL n "IRKHOLZ 3 +NIPP 5 ,EMMA % +ROGER ! AND /PFERKUCH 7 &UMARATE REDUCTASE OF (ELICOBACTER PYLORIAN IMMUNOGENIC PROTEIN * -ED -ICROBIOL n "OLTON &* #OATES $ AND (UTCHINSON $. 4HE ABILITY OF #AMPYLOBACTERR MEDIA SUPPLEMENTS TO NEUTRALISE PHOTO CHEMICALLY INDUCED TOXICITY AND HYDROGEN PEROXIDE * !PPL "ACTERIOL n #ARLONE '- AND !NET &!, $ETECTION OF MENAQUINONE AND A NOVEL METHYL SUBSTITUTED MENAQUINONE IN #AMPYLO BACTER JEJUNI AND #AMPYLOBACTER FETUS SUBSP FETUS * 'EN -ICROBIOL n #ARLONE '- AND ,ASCELLES * !EROBIC AND ANAEROBIC RESPI RATORY SYSTEMS IN CAMPYLOBACTER FETUS SUBSP JEJUNI GROWN IN ATMOSPHERES CONTAINING HYDROGEN * "ACTERIOL n #ENSINI 3 ,ANGE # 8IANG :9 #RABTREE *% 'HIRRA 0 "ORODOVSKY - 2APPUOLI 2 AND #OVACCI ! #AG A PATHOGENICITY ISLAND OF (ELICOBACTER PYLORI ENCODES TYPE ) SPECIlC AND DISEASE ASSOCIATED VIRULENCE FACTORS 0ROC .ATL !CAD 3CI 53! n #HANG (4 -ARCELLI 37 $AVISON !! #HALK 0! 0OOLE 2+ AND -ILES 2 * +INETICS OF SUBSTRATE OXIDATION BY WHOLE CELLS AND CELL MEMBRANES OF (ELICOBACTER PYLORI &%-3 -ICROBIOL ,ETT n #HEN - !NDERSEN ,0 :HAI , AND +HARAZMI ! #HARACTER IZATION OF THE RESPIRATORY CHAIN OF (ELICOBACTER PYLORI &%-3 )MMUNOL -ED -ICROBIOL n #OLLINS -$ #OSTAS - AND /WEN *2 )SOPRENOID QUINONE COMPOSITION OF REPRESENTATIVES OF THE GENUS #AMPYLOBACTER !RCH -ICROBIOL n $AVIDSON !! +ELLY $* 7HITE 0* AND #HALK 0! #ITRIC ACID CYCLE ENZYMES AND RESPIRATORY METABOLISM IN ( PYLORI !CTA 'ASTRO %NTEROL "ELG 3 $IXON -& 0ATHOLOGY OF GASTRITIS AND PEPTIC ULCERATION )N -OBLEY (,4 -ENDZ ', AND (AZELL 3, ED (ELICOBACTER PYLORI 0HYSIOLOGY AND 'ENETICS PP n !3- 0RESS 7ASHINGTON $ # $OIG 0 DE *ONG ", !LM 2! "ROWN %$ 5RIA .ICKELSON -
.OONAN " ET AL (ELICOBACTER PYLORI PHYSIOLOGY PREDICTED FROM GENOMIC COMPARISONS OF TWO STRAINS -ICROBIOL -OL "IOL 2EV n %INSLE / 3TACH 0 -ESSERSCHMIDT ! 3IMON * +RÚGER ! (UBER 2 AND +RONECK -( #YTOCHROME C NITRITE REDUCTASE FROM 7OLINELLA SUCCINOGENES * "IOL #HEM n %NOCH (' AND ,ESTER 2, 4HE PURIlCATION AND PROPERTIES OF FORMATE DEHYDROGENASE AND NITRATE REDUCTASE FROM %SCHERICHIA COLI * "IOL #HEM n %VANS $* *R AND %VANS $' )DENTIlCATION OF A FORMATE DEHYDROGENASE ASSOCIATED CYTOCHROME C IN (ELICOBACTER PYLORI 'UT 3 ! &INEL - $OES .!$( PLAY A CENTRAL ROLE IN ENERGY ME TABOLISM IN (ELICOBACTER PYLORI 4RENDS "IOCHEM 3CI n &RIEDMAN #2 .EIMAN * 7EGENER (# AND 4AUXE 26 %PIDEMIOLOGY OF #AMPYLOBACTER JEJUNI IN THE 5NITED 3TATES AND OTHER INDUSTRIALIZED NATIONS )N .ACHAMKIN ) AND "LASER -* ED #AMPYLOBACTER ND %D PP n !3- 0RESS 7ASHINGTON $# &RIEDRICH 4 4HE .!$(UBIQUINONE OXIDOREDUCTASE COMPLEX ) FROM %SCHERICHIA COLI "IOCHIM "IOPHYS !CTA n &U (! )UCHI 3 AND ,IN %## 4HE REQUIREMENT OF !RC! AND &NR FOR PEAK EXPRESSION OF THE CYDD OPERON IN %SCHERICHIA COLI UNDER MICROAEROBIC CONDITIONS -OL 'EN 'ENET n 'E : *IANG 1 +ALISIAK -3 AND 4AYLOR $% #LONING AND FUNCTIONAL CHARACTERIZATION OF (ELICOBACTER PYLORI FUMARATE REDUCTASE OPERON COMPRISING THREE STRUCTURAL GENES CODING FOR SUBUNITS # ! AND " 'ENE n 'ENNIS 2" AND 3TEWART 6 2ESPIRATION )N .EIDHARDT &# ED %SCHERICHIA COLI AND 3ALMONELLA TYPHIMURIUM ND %D 6OL PP n !3- 0RESS 7ASHINGTON $# 'ILBERT *6 2AMAKRISHNA * 3UNDERMAN *R &7 7RIGHT ! AND 0LAUT !' 0ROTEIN (PN #LONING AND CHARACTERISATION OF A HISTIDINE RICH METAL BINDING POLYPEPTIDE IN (ELICOBACTER PYLORI ANDD (ELICOBACTER MUSTELAE )NFECT )MMUN n 'ILMOUR 2 'OODHEW #& 0ETTIGREW '7 0RAZERES 3 -OURA ) AND -OURA ** 3PECTROSCOPIC CHARACTERIZATION OF CYTO CHROME C PEROXIDASE FROM 0ARACOCCUS DENITRIlCANS "IOCHEM * n 'ON 3 'UIDICI /RTICONI -4 -EJEAN 6 AND )OBBI .IVOL # %LECTRON TRANSFER AND BINDING OF THE C TYPE CYTOCHROME 4OR# TO THE TRIMETHYLAMINE . OXIDE REDUCTASE IN %SCHERICHIA COLI * "IOL #HEM n 'OODHEW #& %L+URDI !" AND 0ETTIGREW '7 4HE MICRO AEROPHILIC RESPIRATION OF #AMPYLOBACTER MUCOSALIS "IOCHIM "IOPHYS !CTA n 'REEN '. &ANG ( ,IN 2 * .EWTON ' -ATHER - 'EORGIOU #$ AND 'ENNIS 2" 4HE NUCLEOTIDE SEQUENCE OF THE CYD D LOCUS ENCODING THE TWO SUBUNITS OF THE CYTOCHROME D TERMINAL OXIDASE COMPLEX OF %SCHERICHIA COLI * "IOL #HEM n (ARVEY 0 AND ,EACH 3 !NALYSIS OF COCCAL CELL FORMATION BY #AMPYLOBACTER JEJUNI USING CONTINUOUS CULTURE TECHNIQUES AND THE IMPORTANCE OF OXIDATIVE STRESS * !PPL -ICROBIOL n (ARVEY 3 AND ,ASCELLES * 2ESPIRATION SYSTEMS AND CYTO CHROMES IN #AMPYLOBACTER FETUS SUBSP INTESTINALIS * "ACTERIOL n
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4VNNBSZ !CETIC ACID BACTERIA ARE OBLIGATE AEROBES AND WELL KNOWN TO HAVE A STRONG ABILITY TO OXIDIZE ETHANOL SUGARS OR SUGAR ALCOHOLS TO PRODUCE THE CORRESPONDING SUGAR ACIDS 4HESE OXIDATION REACTIONS OF SUGARS AND ALCOHOLS IN ACETIC ACID BACTERIA ARE UNIQUELY CARRIED OUT BY PRIMARY DEHYDROGENASES LOCATED IN THE PERIPLASMIC SIDE OF THE CYTOPLASMIC MEMBRANE AND LINKED TO THE TERMINAL UBIQUINOL OXIDASES VIA UBIQUINONE IN THE RESPIRATORY CHAIN 4HE TERMINAL UBIQUINOL OXIDASES WORKING IN THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA COULD BE CLASSIlED INTO FOUR DIFFERENT TYPES CYTOCHROME O CYTOCHROME A CYTOCHROME D AND #. RESISTANT BYPASS OXIDASE WHICH ARE NOT UNIQUE BUT FOUND IN OTHER BACTERIAL SPECIES WHEREAS THE PRIMARY DEHYDROGENASES WORKING IN THE PERI PLASMIC SUGAR AND ALCOHOL RESPIRATIONS INCLUDE MANY UNIQUE QUINOPROTEINS AND QUINOPROTEIN CYTOCHROME C COMPLEXES AND mAVOPROTEIN CYTOCHROME C COMPLEXES 3UCH PERIPLASMIC SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA ARE INVOLVED IN THE ACCUMULATION OF THE OXIDIZED PRODUCTS OF SUGARS OR ALCOHOLS BUT NOT IN THE ASSIMILATION OF THESE SUBSTRATES AT LEAST IN THEIR EARLY GROWTH PHASE 4HESE SUGAR AND ALCOHOL RESPIRATIONS SEEM
!UTHOR FOR CORRESPONDENCE EMAIL KAZUNOBU AGRYAMAGUCHI UACJP $AVIDE :ANNONI ED 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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NOT TO GENERATE SO MUCH ENERGY BUT INSTEAD TO WORK FOR RAPID OXIDATION TO PRODUCE A LARGE AMOUNT OF OXIDIZED PRODUCTS WHICH ARE USUALLY VERY HARD TO BE UTILIZED BY OTHER ORGANISMS OR CONFER VERY HARMFUL CONDITIONS FOR OTHER ORGANISMS TO KEEP ALIVE 4HUS THE SPECIlC RESPIRATIONS OF ACETIC ACID BACTERIA SEEM TO HAVE EVOLVED TO THEIR OWN LIVING STRATEGY FOR COMPETING WITH OTHER MICROORGANISMS **OUSPEVDUJPO !CETIC ACID BACTERIA ARE OBLIGATE AEROBES AND ARE WELL KNOWN TO HAVE A STRONG ABILITY TO OXIDIZE ETHANOL SUGARS AND ALCOHOLS TO PRODUCE THE CORRESPONDING SUGAR ACIDS 3UCH OXIDATION REACTIONS ARE TRADITIONALLY CALLED @OXIDATIVE FERMENTATION SINCE THEY INVOLVE INCOMPLETE OXIDATION OF THESE COMPOUNDS "ACTERIA CAPABLE OF EFFECTING SUCH OXIDATIVE FERMENTATION ARE CALLED @OXIDATIVE BACTERIA OF WHICH THE MOST PROMINENT ONES ARE ACETIC ACID BACTERIA 4HESE OXIDA TIVE BACTERIA DO NOT OXIDIZE SUCH SUGARS OR ALCOHOLS COMPLETELY TO CARBON DIOXIDE AT LEAST IN THEIR EARLY CULTURE PHASE AND THUS ACCUMULATE THE CORRESPONDING INCOMPLETE OXIDATION PRODUCTS IN LARGE AMOUNTS IN THE GROWTH MEDIUM 4HESE OXIDATION REACTIONS OF SUGARS AND ALCOHOLS BY ACETIC ACID BACTERIA ARE UNIQUELY CARRIED OUT BY MEMBRANE BOUND DEHYDROGENASES LINKED TO THE RESPIRATORY CHAIN LOCATED IN THE PERIPLASMIC SIDE OF THE CYTOPLASMIC MEMBRANE OF THE ORGANISMS 3UCH UNIQUE PERIPLASMIC RESPIRATIONS ARE INVOLVED IN ONLY THE PARTIAL OXIDATION REACTIONS OF THESE SUGARS AND ALCOHOLS BUT NOT IN THE COMPLETE OXIDATION AND THE ASSIMILATION OF THE SUBSTRATES 3INCE THE ASSIMILATION REACTION CAN BE CARRIED OUT BY CYTOPLASMIC ENZYMES LINKED TO THE .!$( RESPIRATION THE REACTION PROD UCTS MUST BE TRANSPORTED INTO THE CYTOPLASM FROM THE PERIPLASMIC REACTION SITE 4HUS THE PERIPLASMIC RESPIRATION OF ACETIC ACID BACTERIA SEEMS REMINISCENT OF IRON OR HYDROGEN RESPIRATION IN CHEMOLITHOTROPHS AND THUS HAS A UNIQUE POSITION IN HETEROTROPHIC ENERGY METABOLISM )N ADDITION SINCE THESE PERIPLASMIC RESPIRATION SYSTEMS ARE INVOLVED IN THE ACCUMULATION OF A LARGE AMOUNT OF OXIDATION PRODUCTS SUCH AS ACETIC ACID OR , SORBOSE IN THE CULTURE MEDIA ACETIC ACID BACTERIA !BBREVIATIONS +'$( n KETOGLUCONATE DEHYDROGENASE !$(n ALCOHOL DEHYDROGENASE !,$( n ALDEHYDE DEHYDROGE NASE &$( n FRUCTOSE DEHYDROGENASE '!$( n GLUCONATE DE HYDROGENASE '$( n GLUCOSE DEHYDROGENASE ',$( n GLYCEROL DEHYDROGENASE 011 n PYRROLOQUINOLINE QUINONE 3$( n SORBOSE DEHYDROGENASE 3,$( n SORBITOL DEHYDROGENASE 3.$( n SORBO SONE DEHYDROGENASE 51 n UBIQUINONE 51N n UBIQUINONE N EG UBIQUINONE
ARE ALSO IMPORTANT FOR FERMENTATION INDUSTRIES TO PRODUCE USEFUL BIOMATERIALS !CETIC ACID BACTERIA HAVE BEEN SHOWN TO BELONG TO _ 0ROTEOBACTERIA AND THE CLOSEST RELATED SPECIES ARE 2HODOPHILA GLOBIFORMIS AND !CIDIPHILIUM SPECIES IN THE BACTERIAL PHYLOGENETIC TREE 3IEVERS ET AL !CETIC ACID BACTERIA ARE CLASSIlED INTO TWO GENERA 'LUCONOBACTERR AND !CETOBACTER OF THE &AMILY !CETOBACTERACEAE 4HE CLASSIlCATION HAS ALSO BEEN CONlRMED BY PHYLOGENETIC ANALYSIS IN WHICH ALL 'LU CONOBACTER SPECIES FORM A CLOSELY RELATED CLUSTER AND ARE WELL SEPARATED FROM !CETOBACTERR SPECIES WHEREAS !CETOBACTER SPECIES ARE RELATIVELY DIVERSE AND ARE lRST R SEPARATED INTO TWO GROUPS THE lRST GROUP INCLUDING !CETOBACTER ACETI AND !CETOBACTER PASTEURIANUS AND THE OTHER THE REMAINING !CETOBACTERR SPECIES 4HE REMAINING SPECIES SEEM TO BE FURTHER SUBDIVIDED INTO TWO GROUPS THE SECOND GROUP INCLUDING !CETOBACTER LIQUEFACIENS AND !CETOBACTER DIAZOTROPHICUS AND THE THIRD GROUP INCLUDING !CETOBACTER EUROPAEUS AND !CETOBACTER XYLINUM 2ECENTLY THE LATTER TWO GROUPS WERE PROPOSED TO BE INCLUDED IN THE GENUS 'LUCO NOACETOBACTERR 9AMADA ET AL !MONG THESE !CETOBACTERR ANDOR 'LUCONOACETOBACTER SPECIES !CETOBACTER METHANOLICUS HAS A SOMEWHAT DIFFERENT LOCATION IN BETWEEN THE lRST GROUP AND THE SECOND OR THIRD GROUPS &IG (OWEVER IN THE PRESENT CHAPTER ALL @!CETOBACTER SPECIES ARE OPERATIONALLY DElNED AS THOSE INCLUDED IN THE GENUS !CETOBACTER 'LUCONOACETOBACTER AND R !CIDOMONAS 3UCH A PHYLOGENETIC RELATIONSHIP AMONG ACETIC ACID BACTERIA SEEMS TO BE SOMEWHAT RELATED TO THE SUGAR AND ALCOHOL RESPIRATIONS 'LUCONOBACTERR SPECIES EX HIBIT HIGHLY ACTIVE OXIDATION REACTIONS ON SUGARS OR SUGAR ALCOHOLS SUCH AS $ GLUCOSE $ GLUCONIC ACID $ SORBITOL GLYCEROL AND SO ON IN ADDITION TO ETHANOL )N CONTRAST !CETOBACTER SPECIES HAVE A HIGHLY ACTIVE R ETHANOL OXIDIZING ACTIVITY BUT FEWER OTHER SUGAR OR SUGAR ALCOHOL OXIDIZING ACTIVITIES &URTHERMORE !CETOBACTERR SPECIES UNLIKE 'LUCONOBACTERR SPECIES ARE ABLE TO OXIDIZE ACETATE THE OXIDATION PRODUCT OF ETHANOL COMPLETELY TO #/ AND WATER UNDER SOME SPECIlC GROWTH CONDITIONS 4HUS ALTHOUGH THE TWO GENERA EXHIBIT A CRITICAL DIFFERENCE IN THEIR OXIDIZ ING ABILITY THESE SUGAR AND ALCOHOL RESPIRATIONS
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IN BOTH GENERA SEEM TO FUNCTION BY LINKING TIGHTLY TO THE AEROBIC RESPIRATORY CHAINS OF THE ORGANISMS -ATSUSHITA ET AL !LTHOUGH A CONSIDERABLE AMOUNT OF EARLIER WORK HAD BEEN DONE ON THE SUGAR METABOLISM OF ACETIC ACID BACTERIA !SAI LITTLE IS KNOWN ABOUT THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA AND OF THE RELATION WITH THE SUGAR OR ALCOHOL OXIDATION REACTIONS 4HE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA NOT ONLY HAS MANY COMMON PRINCIPLES BUT ALSO SOME DIVERSITY )N THIS CHAPTER THE GENERAL ASPECTS IN THE RESPIRATORY CHAINS AND ALSO THE CHARACTERISTIC PERI PLASMIC SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA ARE DESCRIBED **3FTQJSBUPSZ$IBJOTPG"DFUJD"DJE #BDUFSJB "(FOFSBM"TQFDUTJOUIF3FTQJSBUPSZ$IBJO PG"DFUJD"DJE#BDUFSJB "ACTERIAL RESPIRATORY CHAINS HAVE BEEN CLASSIlED INTO TWO CATEGORIES BASED ON THE TERMINAL OXIDASE ONE IS A CYTOCHROME C OXIDASE AND THE OTHER A UBIQUINOL OXIDASE )N THIS RESPECT THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA HAS BEEN SHOWN TO BE CLASSIlED INTO ONE HAVING UBIQUINOL OXIDASE AT THE TERMINAL END OF
THE RESPIRATORY CHAIN 4HUS THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA IS RATHER SIMPLE WITH RESPECT TO THEIR ARRANGEMENT OF THE RESPIRATORY COMPONENTS LIKE %SCHERICHIA COLI !NRAKU !S SHOWN IN &IG THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA SIMPLY CONSISTS OF PRIMARY DEHYDRO GENASES AND TERMINAL UBIQUINOL OXIDASES BOTH OF WHICH ARE CONNECTED BY UBIQUINONE 51 4HE 51 PRESENT IN THE RESPIRATORY CHAIN OF ACETIC ACID BAC TERIA IS EITHER UBIQUINONE 51 OR UBIQUINONE 51 WHICH VARIES DEPENDING ON THE BACTERIAL R SPECIES SPECIES AND IS MAINLY 51 IN !CETOBACTER STRICTLY DElNED AS !CETOBACTER SPECIES IN THIS CASE R OR 51 IN 'LUCONOBACTER SPECIES AND ALSO IN THE RE R
CENTLY DElNED 'LUCONOACETOBACTER AND R !CIDOMONAS SPECIES !LTHOUGH BOTH THE PRIMARY DEHYDROGENASE AND THE TERMINAL OXIDASE PARTS ARE VERY DIVERGENT IN THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA THE DIVERSITY OF THE TERMINAL OXIDASE IS NOT UNUSUAL WHILE THE PRIMARY DEHYDROGENASE IS ESPECIALLY DIVERGENT IN THE RESPIRATORY CHAIN )N ACETIC ACID BACTERIA MANY PECULIAR PERIPLASMIC DEHYDROGENASES ARE WORK ING FOR THE SPECIlC SUGAR ALCOHOL OR SUGAR ALCOHOL OXIDATION SYSTEMS 4HE DIVERSITY AND PECULIARITY OF THE PERIPLASMIC OXIDATION SYSTEMS IN THE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA WILL BE DESCRIBED IN THE NEXT SECTION
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#5FSNJOBMPYJEBTFTJOUIF3FTQJSBUPSZ $IBJOTPG"DFUJD"DJE#BDUFSJB 3EVERAL DIFFERENT SPECIES OF UBIQUINOL OXIDASE ARE KNOWN IN THE RESPIRATORY CHAIN OF ACETIC ACID BACTE RIA WITH RESPECT TO THE CYTOCHROME SPECIES AS SUM MARIZED IN 4ABLE /RIGINALLY "ÊCHI AND %TTLINGER HAVE DIVIDED ACETIC ACID BACTERIA INTO THREE GROUPS BASED ON THE _ ABSORPTION PEAK IN THE LONGER WAVELENGTH REGION OVER NM OF CYTOCHROMES !CETOBACTERR STRAINS 0EROXYDANS GROUP EXHIBITING BOTH PEAKS AT ^ NM AND ^ NM ANOTHER !CE TOBACTERR SPECIES /XYDANS AND -ESOXYDANS GROUPS HAVING ONLY THE PEAK AT ^ NM AND 'LUCONO BACTER STRAINS WHICH DO NOT SHOW ANY CORRESPONDING R PEAKS 4HE ORIGINAL CLASSIlCATION CAN NOW BE REALIZED AS THE PRESENCE OF THE DIFFERENT SPECIES OF THE TERMINAL OXIDASES IN DIFFERENT CLASSES OF ACETIC ACID BACTERIA !CETOBACTER SPECIES EXHIBITING BOTH _ PEAKS AT D BD OXIDASE D AND NM SEEM TO HAVE CYTOCHROME D WHICH IS SHOWN TO EXHIBIT BOTH B TYPE NM AND D D TYPE NM ABSORPTION PEAKS DUE TO THE PRES
ENCE OF HEME B AND HEME D RESPECTIVELY WHEREAS OTHER !CETOBACTERR SPECIES HAVING ONLY THE NM PEAK IN THE REGION ARE SHOWN TO HAVE CYTOCHROME A BA WHICH EXHIBITS AN _ ABSORPTION PEAK AROUND NM DUE TO THE PRESENCE OF HEME A IN ADDITION TO A CYTOCHROME B COMPONENT 'LUCONOBACTERR SPECIES ARE SHOWN TO HAVE ONLY CYTOCHROME O AS A TYPICAL TERMINAL OXIDASE WHICH DOES NOT HAVE ANY TYPICAL _ PEAKS IN SUCH A LONG WAVELENGTH REGION 4HUS BY ADDING A #. RESISTANT BYPASS OXIDASE THE COMPONENT OF WHICH HAS NOT BEEN IDENTIlED THE TERMINAL OXIDASES OF ACETIC ACID BACTERIA SEEM TO BE DIVIDED INTO FOUR DIFFERENT TYPES 4ABLE 4HESE TERMINAL OXIDASES ARE
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AND CONTAINS HEME B HEME O AND COPPER ATOM AS THE PROSTHETIC GROUP ,ATER CYTOCHROME O WAS FOUND IN ONE SPECIES OF !CETOBACTER ACETI 2 STRAIN WHICH CAN GROW ON STATIC CULTURE BY mOATING ON THE MEDIUM SURFACE BY PRODUCING PELLICLE POLYSACCHARIDE -AT SUSHITA ET AL B 4HE CYTOCHROME O OF ! ACETI HAS ALSO BEEN SHOWN TO CONSIST OF ONE HEME B AND ONE HEME O AND COPPER ATOM AND TO BE THE SAME PROTEIN ENTITY AS CYTOCHROME A PURIlED FROM ! ACETI 3 STRAIN AS DESCRIBED BELOW #YTOCHROME O AND ALSO CYTOCHROME A HAS BEEN SHOWN TO PRODUCE A PROTON MOTIVE FORCE BY RECONSTITUTING INTO PROTEOLIPOSOMES -ATSUSHITA ET AL D #YTOCHROME O MIGHT BE PRESENT AND FUNCTION AS AT LEAST ONE OF THE TERMINAL OXIDASES IN ALMOST ALL ACETIC ACID BACTERIA SINCE CYTOCHROME O HAS ALSO BEEN DETECTED IN OXYGEN SUFlCIENT ! PASTEURIANUS HAV ING CYTOCHROME DD AS THE TERMINAL OXIDASE 7ILLIAMS AND 0OOLE AND ALSO IN ! METHANOLICUS UNDER NORMAL GROWTH CONDITIONS HAVING AN ETHANOL OXIDASE RESPIRATORY CHAIN -ATSUSHITA ET AL C IN ADDI TION TO ! ACETI AND 'LUCONOBACTER SPECIES %$ZUPDISPNFB BT"OPUIFS.BKPS5FSNJOBM 0YJEBTFJO"DFUPCBDUFS4QFDJFT #YTOCHROME A WAS ORIGINALLY FOUND IN !CETOBACTER SPECIES BY 7ARBURG AS A CYTOCHROME EXHIBITING A WEAK ABSORPTION PEAK AT NM WHICH IS INTENSIlED IN THE PRESENCE OF #. AND LATER IDENTIlED AS A TERMINAL OXIDASE BY #HANCE SEE -ATSUSHITA ET AL !L THOUGH THE PRESENCE OF SUCH A CYTOCHROME OXIDASE WAS DOUBTED LATER A CYTOCHROME A LIKE TERMINAL OXIDASE WAS FOUND IN THE MEMBRANES OF ! ACETI GROWN ON SHAKING CULTURES -ATSUSHITA ET AL ! ACETI PRODUCES EITHER ONE OF THE TWO DIFFERENT TERMINAL OXIDASES DEPENDING ON THE CULTURE CONDITIONS OR THE CELL TYPE &IG ! #ELLS 3 TYPE STRAIN PRODUCING CYTOCHROME A PREDOMINATE ON THE SHAKING CULTURE WHILE CYTOCHROME O IS PREDOMINANTLY PRESENT IN CELLS 2 TYPE STRAIN IN STATIC CULTURE 4HE CYTOCHROME A WAS CHARACTERIZED AS CYTOCHROME BA TYPE UBIQUINOL OXIDASE CONSISTING OF FOUR SUBUNITS AND CONTAINING MOL EACH OF HEME A HEME B AND COPPER ION -ATSU SHITA ET AL !LTHOUGH NO STRUCTURAL DIFFERENCE COULD BE SEEN BETWEEN CYTOCHROME A AND CYTOCHROME O OF ! ACETI BOTH ENZYMES HAVE A DIFFERENCE IN THEIR OXYGEN AFlNITY TIMES HIGHER IN CYTOCHROME A AND #. SENSITIVITY TIMES HIGHER IN CYTOCHROME O -ATSUSHITA ET AL A 4HE GENES CYA OPERON ENCODING SUBUNITS OF CYTOCHROME A WERE CLONED FROM
! ACETI CHROMOSOMES AND THE DEDUCED AMINO ACID SEQUENCES OF THESE SUBUNITS ESPECIALLY SUBUNIT ) SHOWED A GREAT SIMILARITY TO THE CYTOCHROME O OF % COLI &UKAYA ET AL &URTHERMORE 3OUTHERN HYBRIDIZATION ANALYSIS SHOWED THAT ONLY ONE SET OF THE GENE EXISTS FOR CYTOCHROME A AND CYTOCHROME O IN ! ACETI AND THUS THE TWO OXIDASES SHARE AN IDENTICAL PROTEIN MOIETY + -ATSUSHITA ET AL UNPUBLISHED 4HUS THE DIFFERENCES BETWEEN TWO UBIQUINOL OXIDASES ARE SOLELY DUE TO A PERIPHERAL GROUP OF THE HEME AT THE BINUCLEAR CENTER SITE SUBSTITUTION OF THE METHYL GROUP AT POSITION OF HEME O WITH A FORMYL GROUP #OMPARISON OF THE BINUCLEAR SITE STRUCTURE OF BOTH ENZYMES USING RESONANCE 2AMAN &4 )2 AND %02 SPECTROSCOPIES SHOWED THAT THE OVERALL ARCHITECTURE AND THE ELECTRONIC CONlGURATIONS OF THE BINUCLEAR CENTER SEEM TO BE WELL CONSERVED IN THEIR OXIDIZED STATE BUT THAT THE BINUCLEAR CENTER IN CYTOCHROME A HAS CONFORMATIONAL mEXIBILITY IN THE REDUCED STATE WHICH MAY INDUCE A HYDROGEN BOND NETWORK FORMA TION AMONG THE FORMYL GROUP OF HEME A WATER AND THE SURROUNDING AMINO ACID RESIDUES 4SUBAKI ET AL 4HIS NETWORK IS LIKELY THE ORIGIN OF THE HIGHER AFlNITY FOR OXYGEN AND THE LOWER SENSITIVITY TO #. OF CYTOCHROME A #YTOCHROME A HAS BEEN FOUND IN ANOTHER !CETO BACTER SPECIES BUT NOT IN R 'LUCONOBACTERR SPECIES )N ADDITION TO ! ACETI 2 STRAIN !CETOBACTER DIAZOTROPHI CUS HAS RECENTLY BEEN SHOWN TO PRODUCE CYTOCHROME A WHEN THE CELLS ARE GROWN UNDER HIGHLY AERATED AND DIAZOTROPHIC GROWTH CONDITIONS &IG " WHERE A HIGH ENERGY STATE IS REQUIRED FOR THE . lXATION &LORES %NCARNACØN ET AL &$ZUPDISPNFEBTUIF"EEJUJPOBM5FSNJOBM 0YJEBTFJO"DFUPCBDUFS4QFDJFT #YTOCHROME D IS ONE OF THE UBIQUINOL OXIDASES AND CONTAINS TWO HEMES B AND ONE HEME D AS THE PROSTHETIC D GROUPS AND THE BINUCLEAR CENTER FOR OXYGEN REACTION IS CONSTITUTED OF ONE OF THE HEMES B AND THE HEME D (ILL ET AL #YTOCHROME D HAS BEEN SHOWN TO D BE PRODUCED UNDER LIMITED OXYGEN CONDITIONS AND TO HAVE LOW PROTON MOTIVE FORCE GENERATING ABILITY DIFFERENT FROM CYTOCHROME A OR O #YTOCHROME D WAS FOUND IN !CETOBACTER PER R OXYDANS BY "ÊCHI AND %TTLINGER AND LATER IN ! PASTEURIANUS SEEMINGLY THE SAME STRAIN AS ! PEROXYDANS BY 7ILLIAMS AND 0OOLE ! PASTEURIANUS PRODUCES CYTOCHROME O IN AIR SUFlCIENT GROWTH CONDITIONS WHILE CYTOCHROME D IS PRODUCED D
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PROTEOLIPOSOMES WITH !$( 51 AND CYTOCHROME O SOME OTHER COMPONENTS MUST BE REQUIRED TO PRO DUCE #. RESISTANT BYPASS OXIDASE IN ADDITION TO THE CYTOCHROME SUBUNIT OF !$( &IG $ 7HEN THE MEMBRANES OF ' SUBOXYDANS ARE TREATED WITH A RELATIVELY LOW CONCENTRATION OF DETERGENT !$( CAN BE SOLUBILIZED WHILE GLUCOSE DEHYDROGENASE '$( IS RETAINED IN THE MEMBRANE RESIDUES !L THOUGH BOTH '$( AND !$( CAN REDUCE FERRICYANIDE IN THE NATIVE MEMBRANES '$( LOSES ITS ABILITY TO REACT WITH THE ARTIlCIAL DYE AFTER THE SOLUBILIZATION BUT !$( DOES NOT 4HE FERRICYANIDE REDUCTASE ACTIV ITY OF '$( CAN BE REPRODUCED IN PROTEOLIPOSOMES RECONSTITUTED FROM '$( !$( AND FURTHER 51 ALL THREE COMPONENTS OF WHICH ARE INDISPENSABLE 3HINAGAWA ET AL 4HUS THE ELECTRON TRANSFER FROM '$( TO FERRICYANIDE SEEMS TO BE MEDIATED BY 51 AND !$( IN THE MEMBRANES OF ' SUBOXYDANS 3INCE '$( AND !$( ARE ABLE TO REACT DIRECTLY WITH 51 IN THE PHOSPHOLIPID BILAYER IT IS SUGGESTED THAT '$( DONATES ELECTRONS TO 51 lRST AND THEN THE RESULTANT UBIQUINOL REACTS DIRECTLY WITH !$( WHICH SUBSEQUENTLY REDUCES FERRICYANIDE !CTUALLY !$( HAS BEEN SHOWN TO HAVE AN ADDITIONAL FUNCTION TO ACCEPT ELECTRONS FROM UBIQUINOL -ATSUSHITA ET AL SUGGESTING THAT !$( IS ABLE TO MEDIATE ELECTRON TRANS FER FROM UBIQUINOL TO FERRICYANIDE IN VITRO AND ALSO TO THE BYPASS OXIDASE SYSTEM IN VIVO &IG $ 4HE SAME TYPE OF #. RESISTANT BYPASS OXIDASE INVOLVING SOME CYTOCHROME C COMPONENTS HAS BEEN SHOWN IN THE RESPIRATORY CHAIN OF ! DIAZOTROPHICUS WHERE MOST OF THE CYTOCHROME C WAS SOLUBILIZED WITH 4RITON 8 AND THE RESIDUAL MEMBRANE RETAINED A FULLY ACTIVE GLUCOSE OXIDASE ACTIVITY WHICH IS SIGNIlCANTLY MORE SENSITIVE TO #. &LORES %NCAR NACIØN ET AL 4HUS THE #. RESISTANT BYPASS OXIDASE MAY WORK NOT ONLY IN 'LUCONOBACTER SPECIES BUT ALSO IN SOME !CETOBACTER SPECIES R ***1FSJQMBTNJD4VHBSBOE"MDPIPM3FTQJSB UJPOT "1FSJQMBTNJD0YJEBTF4ZTUFNT 4HE PERIPLASM AND THE PERIPLASMIC SURFACE OF THE CYTOPLASMIC MEMBRANE HAVE BEEN RECOGNIZED AS THE IMPORTANT LOCATION FOR METABOLISM ESPECIALLY FOR ELECTRON TRANSPORT OF 'RAM NEGATIVE BACTERIA AND MANY ELECTRON TRANSPORT PROTEINS HAVE BEEN ASSIGNED TO WORK IN THIS REGION 3UCH PERIPLASMIC PRIMARY
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IN THE METHANOL OR METHYLAMINE OXIDIZING SYSTEM OF METHYLOTROPHS OR IN THE ALCOHOL OR AMINE OXIDIZING SYSTEM OF SOME OXIDATIVE BACTERIA SUCH AS 0SEUDO MONAS PUTIDA OR 0ARACOCCUS DENITRIlCANS SEE # !NTHONY #HAPTER 6OL WHEREAS THE OTHERS HAVE MEMBRANE BOUND DEHYDROGENASES LINKED TO THE RESPI RATORY CHAIN VIA 51 -ANY MEMBRANE BOUND PRIMARY DEHYDROGENASES ARE FOUND IN THE PERIPLASMIC OXIDASE SYSTEMS OF ACETIC ACID BACTERIA AND CAN BE DIVIDED INTO QUINOPROTEINS AND mAVOPROTEINS WHICH HAVE PYR ROLOQUINOLINE QUINONE 011 AND COVALENTLY BOUND mAVIN ADENINE DINUCLEOTIDE &!$ AS THE PROSTHETIC GROUPS RESPECTIVELY 4ABLE 4HE mAVOPROTEINS ARE UNIQUELY FOUND IN THE SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA EXCEPT FOR THE GLUCONATE OXIDASE SYSTEM ALSO FOUND IN PSEUDOMONADS AND SOME ENTERIC BACTERIA WHEREAS IN ADDITION TO THE SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA 011 QUINOPRO TEINS ARE ALSO FOUND IN THE METHANOL OXIDASE SYSTEM OF METHYLOTROPHS IN THE ALCOHOL OXIDASE SYSTEM OF SOME OXIDATIVE BACTERIA AND ALSO IN THE GLUCOSE
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LIPIDS !$( CONSISTS OF THREE SUBUNITS AND SUBUNIT ) CONTAINS 011 AND HEME C MOIETY AND SUBUNIT )) HAS HEME C MOIETIES 4ABLE -ATSUSHITA ET AL 4HE PERIPLASMIC LOCATION OF ALL THE SUBUNITS ADH!" AND ADH3 THE PRESENCE OF 011 AND HEME 3 C IN SUBUNIT ) ADH! AND HEMES C IN SUBUNIT )) ADH" WERE CONlRMED BY GENETIC DATA SEE +ONDO AND (ORI NOUCHI !,$( CATALYZING THE NEXT STEP FROM ALDEHYDE TO ACETIC ACID IS ALSO A THREE SUBUNIT COMPLEX 4ABLE WHICH IS ALSO SUPPORTED BY GENETIC DATA 4HURNER ET AL !LL THREE SUBUNITS ALD&'( ( SEEM TO HAVE A SIGNAL SEQUENCE WHICH CORRESPONDS TO THE PRESENCE OF !,$( IN THE PERIPLASM !LTHOUGH 011 WAS PROPOSED AS A COFACTOR FOR THIS ENZYME THE SEQUENCE DATA OF THE LARGEST SUBUNIT ALD( SUG (
GEST THAT THE SUBUNIT HAS NO SEQUENCE HOMOLOGY TO QUINOPROTEIN BUT INSTEAD SHOWS HIGH HOMOLOGY TO MOLYBDOPTERIN ENZYME WHICH IS CONSISTENT WITH THE lNDINGS THAT A 011 DElCIENT !CETOBACTER STRAIN IS R STILL ABLE TO PRODUCE ACTIVE !,$( 4AKEMURA ET AL 4HE SECOND SUBUNIT ALD& SIMILAR TO SUBUNIT & )) OF !$( IS ALSO SUGGESTED TO HAVE THREE HEME C BINDING MOTIFS !$( HAS BEEN SHOWN TO DONATE ELECTRONS TO 51 EMBEDDED IN THE MEMBRANE PHOSPHOLIPIDS AND THEN TO THE TERMINAL OXIDASE -ATSUSHITA AND !DACHI !LTHOUGH NO !$( SUBUNITS HAVE A TRANSMEM BRANOUS DOMAIN SUBUNIT )) CYTOCHROME SUBUNIT HAS BEEN SHOWN TO HAVE A 51 REACTING SITE -ATSUSHITA ET AL AND TO HAVE TWO AMPHIPHILIC _ HELICES AS THE POSSIBLE MEMBRANE ANCHOR + -ATSUSHITA ET AL UNPUBLISHED 3INCE !$( HAS lVE PROSTHETIC GROUPS ONE 011 AND FOUR HEME C MOIETIES INSIDE THE THREE SUBUNIT COMPLEX AN INTRAMOLECULAR ELECTRON TRANSFER REACTION SHOULD OCCUR THROUGH THESE MOLECULES TO REDUCE 51 &ROM SEVERAL PIECES OF CIRCUMSTANTIAL EVI DENCE IT HAS BEEN SUGGESTED THAT ELECTRONS EXTRACTED FROM ETHANOL AT THE 011 SITE COULD BE TRANSFERRED VIA THE HEME C SITE IN SUBUNIT ) TO EITHER OF THREE HEME C SITES IN THE CYTOCHROME SUBUNIT AND THE ELECTRONS ARE PASSED TO 51 THROUGH TWO OF THE HEMES C -AT SUSHITA ET AL &RÏBORTOVÈ ET AL 4HUS 51 REDUCTION IN !$( MAY OCCUR VIA ELECTRON TRANSFER FROM 011 THROUGH THREE OF THE FOUR HEME C MOIETIES PRESENT !LTHOUGH NO DATA HAVE BEEN PRESENTED IN THE CASE OF !,$( THE ENZYME ALSO SEEMS TO BIND TO THE MEMBRANE AND THUS TO TRANSFER THE REDUCING EQUIVALENT TO 51 VIA THE CYTOCHROME SUBUNIT !N ACTIVE ETHANOL OXIDASE RESPIRATORY CHAIN COULD BE REPRODUCED IN AN ARTIlCIAL PROTEOLIPOSOME WHEN BOTH !$( AND UBIQUINOL OXIDASE EITHER CYTOCHROME
A OR CYTOCHROME O WERE RECONSTITUTED INTO LIPOSOMES CONTAINING 51 OR 51 -ATSUSHITA ET AL D 4HE RECONSTITUTED RESPIRATORY CHAIN HAS A REASONABLE ELECTRON TRANSFER TURNOVER FROM ETHANOL TO OXYGEN ^ OF THE NATIVE RESPIRATORY CHAIN AND ALSO A MEMBRANE POTENTIAL GENERATION ABILITY DURING THE ELECTRON TRANSFER ^ M6 INSIDE NEGATIVE 3INCE !,$( HAS 51 REDUCTASE ACTIVITY SIMILAR TO !$( ACETALDEHYDE MAY ALSO BE OXIDIZED BY TERMINAL UBI QUINOL OXIDASE VIA 51 IN THE ACETALDEHYDE RESPIRATORY CHAIN 4HUS @ETHANOL RESPIRATION CAN BE EXPECTED TO OCCUR IN THE RESPIRATORY CHAINS CONSISTING OF TWO PRI MARY DEHYDROGENASES !$( AND !,$( AND TERMINAL UBIQUINOL OXIDASE CYTOCHROME A OR CYTOCHROME O BOTH OF WHICH ARE CONNECTED VIA 51 TO 51 AS SHOWN IN &IG $(MVDPTF3FTQJSBUJPO &IGURE SHOWS THE POSSIBLE DEHYDROGENASES TAKING PART IN GLUCOSE METABOLISM IN ACETIC ACID BACTERIA !LTHOUGH THERE ARE .!$0 DEPENDENT ENZYMES IN THE CYTOPLASM SUCH AS $ GLUCOSE DEHYDROGENASE AND KETO $ GLUCONATE REDUCTASE MEMBRANE BOUND .!$0 INDEPENDENT ENZYMES SUCH AS '$( GLUCONATE DEHYDROGENASE '!$( AND KETO $ GLUCONATE DEHYDROGENASE +'$( ARE EXTENSIVELY INVOLVED IN THE DIRECT OXIDATION OF $ GLUCOSE TO $ GLUCONATE $ GLUCONATE TO KETO $ GLUCONATE AND FURTHER TO DIKETO $ GLUCONATE AT THE CELL SURFACE OF 'LUCONOBACTER SPECIES -ATSUSHITA ET AL 4HUS .!$0 INDEPENDENT ENZYMES '$( '!$( AND +'$( WORK AS THE PRIMARY DEHYDROGENASES OF EACH GLUCOSE GLUCONATE AND KETO $ GLUCONATE RESPIRATORY CHAIN RESPECTIVELY CONSTITUTING @GLUCOSE RESPIRATION OF ACETIC ACID BACTERIA /F THESE PRIMARY DEHYDROGENASES '$( AND '!$( ARE NOT UNIQUE IN ACETIC ACID BACTERIA BUT FOUND IN A WIDE VARIETY OF 0ROTEOBACTERIA INCLUDING FACULTATIVE ANAEROBES SUCH AS ENTERIC BACTERIA AND STRICTLY AEROBIC BACTERIA SUCH AS PSEUDOMONADS WHEREAS +'$( IS UNIQUELY FOUND ONLY IN ACETIC ACID BACTERIA ESPECIALLY 'LUCONOBACTER SPECIES '$( IS A WELL KNOWN QUINOPROTEIN HAVING 011 AS THE PROSTHETIC GROUP SEE #HAPTER IN 6OL UME WHILE '!$( AND +'$( HAVE A COVALENTLY BOUND mAVIN WHICH IS &!$ BOUND VIA THE METHYL GROUP AT THE # SITE WITH THE . SITE OF A HISTIDINE RESIDUE -C)NTIRE ET AL '$( WAS PURIlED CLONED AND THEN CHARACTER IZED FROM THE MEMBRANES OF ' SUBOXYDANS AND ' OXYDANS 4HE STRUCTURAL AND FUNCTIONAL PROPERTIES
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&IG 'LUCOSE RESPIRATION OF 'LUCONOBACTER SPECIES CONSISTING OF GLUCOSE GLUCONATE AND KETOGLUCONATE RESPIRATORY CHAINS 1UINO R
PROTEIN $ GLUCOSE DEHYDROGENASE '$( AND mAVOPROTEINS $ GLUCONATE DEHYDROGENASE '!$( KETO $ GLUCONATE DEHYDROGENASE +'$( ARE LOCATED ON THE OUTER SURFACE OF THE CYTOPLASMIC MEMBRANE AND WORK AS THE PRIMARY DEHYDROGENASES OF EACH RESPIRATORY CHAINS .!$ DEPENDENT GLUCOSE DEHYDROGENASE .!$ '$( AND .!$0 DEPENDENT KETOGLUCONATE REDUCTASE +'2 ARE WORKING IN THE CYTOPLASM FOR PARTIAL ASSIMILATION OF GLUCOSE GLUCONATE AND KETOGLUCONATE
ARE SHOWN TO BE ALMOST THE SAME AS '$( FROM OTHER BACTERIAL STRAINS SUCH AS 0SEUDOMONAS SP % COLI !CINETOBACTER CALCOACETICUS -ATSUSHITA AND !DACHI '$( IS A SINGLE PEPTIDE QUINOPROTEIN 4ABLE OF WHICH THE . TERMINAL REGION HAS A HYDROPHOBIC lVE MEMBRANE SPANNING _ HELIX AND THE RESIDUAL # TERMINAL PART IS A 011 CONTAINING CATALYTIC DOMAIN LOCATED AT THE PERIPLASMIC SIDE 9AMADA ET AL !LTHOUGH THE ENZYME IS ABLE TO DONATE ELECTRONS TO LONG CHAIN 51 51 OR 51 AS WELL AS SHORT CHAIN HOMOLOGUES 51 OR 51 -ATSUSHITA ET AL B ONLY THE # TERMINAL REGION HAS RECENTLY BEEN SHOWN TO BE SUFlCIENT TO CARRY OUT THE ELECTRON TRANSFER TO 51 %LIAS ET AL 5NLIKE '$( BOTH '!$( AND +'$( ARE THREE SUBUNIT COMPLEXES INCLUDING mAVOPROTEIN AND CYTOCHROME C 4ABLE !LTHOUGH SOME 'LUCONOBACTER STRAINS HAVE BEEN REPORTED TO R PRODUCE KETO $ GLUCONATE AS WELL AS KETO $ GLUCONATE 3HINAGAWA ET AL '!$( IS ONLY RESPONSIBLE FOR KETO $ GLUCONATE PRODUCTION AS DESCRIBED BELOW 4HE '!$( GENE HAS NOT BEEN CLONED FROM ACETIC ACID BACTERIA BUT FROM %RWINIA CYPRIPEDII 9UM ET AL 4HE DATA HAVE SHOWN THAT ALL THE THREE SUBUNITS HAVE A TYPICAL SIGNAL SEQUENCE AND THAT THE LARGEST SUBUNIT HAS A &!$ BINDING MOTIF AND THE SECOND SUBUNIT HAS THREE HEME C BINDING MOTIFS AND RELATIVELY HIGH IDENTITY ^ TO SUBUNIT )) OF !$( OF ACETIC ACID BACTERIA
!S IN THE CASE OF !$( THE GLUCOSE OXIDASE RESPIRA TORY CHAIN COULD BE RECONSTITUTED IN A PROTEOLIPOSOME HAVING '$( 51 AND CYTOCHROME O -ATSUSHITA ET AL B IN WHICH THE ELECTRON TRANSFER RATE FROM GLUCOSE TO OXYGEN IS REASONABLY HIGH ^ OF THE NATIVE SYSTEM AND ABLE TO GENERATE A REASONABLE ELECTROCHEMICAL PROTON GRADIENT n M6 P( UNITS !LTHOUGH THE 51 REDUCTASE ACTIVITY WAS NOT EXAMINED IN '!$( AND +'$( OF 'LUCONOBACTER SPECIES '!$( OF 0SEUDOMONAS SPECIES HAS ALSO BEEN SHOWN TO DONATE ELECTRONS TO 51 AND ALSO TO REPRODUCE GLUCONATE OXIDASE ACTIVITY ONLY AFTER ADDING 51 OR 51 WHEN THE PURIlED ENZYME WAS ADDED TO THE '!$( DEPLETED MEMBRANE -ATSUSHITA ET AL 4HUS '!$( AND +'$( ARE ALSO EXPECTED TO LINK TO THE REST OF THE RESPIRATORY CHAIN VIA 51 AS IN THE CASE OF '$( !S SHOWN IN &IG THUS @GLU COSE RESPIRATION OF ACETIC ACID BACTERIA IS EFFECTED BY THREE DIFFERENT PERIPHERAL PRIMARY DEHYDROGENASES '$( '!$( AND +'$( LOCATED ON THE PERIPLASMIC SIDE OF THE MEMBRANE AND THE TERMINAL UBIQUINOL OXIDASES PRESENT INSIDE THE MEMBRANE TOGETHER WITH MEMBRANOUS 51 %4VHBS"MDPIPM3FTQJSBUJPOBOE0UIFS 4VHBS3FTQJSBUJPOT !LMOST ALL 'LUCONOBACTER SPECIES ARE KNOWN TO HAVE R
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A HIGH @OXIDATIVE FERMENTATION ABILITY AGAINST SEVERAL DIFFERENT SUGAR ALCOHOLS ESPECIALLY GLYCEROL BESIDES ALCOHOL AND SUGAR 4HE MOST TYPICAL OXIDATION IS DIHYDROXYACETONE PRODUCTION FROM GLYCEROL )N ADDI TION 'LUCONOBACTERR SPECIES ARE ABLE TO OXIDIZE SUGAR ALCOHOLS SUCH AS $ SORBITOL $ MANNITOL $ ARABITOL $ RIBITOL OR MESO ERYTHRITOL TO THE CORRESPONDING SUGARS , SORBOSE $ FRUCTOSE $ XYLULOSE $ RIBU LOSE OR , ERYTHRULOSE RESPECTIVELY &IG 2ECENTLY SOME OF THESE SUGAR ALCOHOL DEHYDROGENASES HAVE BEEN PURIlED FROM THE MEMBRANES OF SEVERAL DIFFER ENT 'LUCONOBACTERR SPECIES ARABITOL DEHYDROGENASE AND ALSO SORBITOL DEHYDROGENASE HAVE BEEN PURIlED FROM ' SUBOXYDANS )&/ AND ' SUBOXYDANS )&/ RESPECTIVELY !DACHI ET AL 3UGISAWA AND (OSHINO "OTH ENZYMES EXHIBITED SIMILAR MOLECULAR PROPERTIES AND SIMILAR BROAD SUBSTRATE SPECIlCITY WHICH WAS ALSO FOUND TO BE VERY SIMILAR TO THOSE OF GLYCEROL DEHYDROGENASE PREVIOUSLY PURIlED PARTIALLY FROM 'LUCONOBACTER INDUSTRIUS !MEYAMA ET AL 4HUS THE SIMILARITY OF THESE SUGAR ALCOHOL DEHYDROGENASES WERE CONlRMED BY A GENE DISRUPTION BASED ON THE ISOLATED GENE SLD!" OF SORBITOL DEHY DROGENASE -IYAZAKI ET AL WHICH SHOWED THAT ALL THESE SUGAR ALCOHOL DEHYDROGENASES ARE THE SAME ENZYME BASED ON THE SAME GENETIC ORIGIN 3HINJOH ET AL )N ADDITION ANOTHER OXIDATION REACTION OF $ GLUCONATE TO KETO $ GLUCONATE WAS ALSO SHOWN TO BE CARRIED OUT BY THE SAME ENZYME -ATSUSHITA ET AL 4HUS AS SHOWN IN &IG MANY DIFFERENT OXIDATION REACTIONS HAVE BEEN SHOWN TO BE DONE BY GLYCEROL DEHYDROGENASE ',$( OF WHICH THE NAME IS SELECTED BECAUSE GLYCEROL IS THE LEAST REACTIVE MOL ECULE 4HIS ENZYME HAS BEEN SHOWN BIOCHEMICALLY TO BE A QUINOPROTEIN IN BOTH GLYCEROL AND ARABITOL DEHY DROGENASES WHICH IS ALSO SUPPORTED BY THE lNDING THAT THE QUINOPROTEINnTYPE STRUCTURE IS WELL CONSERVED IN 3LD! 4HE ENZYME HAS BEEN SHOWN TO HAVE 51 REDUCTASE ACTIVITY IN ARABITOL DEHYDROGENASE AND TO HAVE A HYDROPHOBIC MEMBRANE SPANNING SUBUNIT 3LD" SIMILAR TO THE . TERMINAL MEMBRANE SPANNING DOMAIN OF '$( SUGGESTING THAT ',$( WORKS AS THE PRIMARY DEHYDROGENASE FOR SUGAR ALCOHOL RESPIRATION TOGETHER WITH 51 AND THE TERMINAL UBIQUINOL OXIDASE AS IN THE CASE OF '$( /THER SUGAR OR SUGAR ALCOHOL OXIDIZING SYSTEMS ARE ALSO SHOWN IN &IG ONE OF WHICH IS A DIFFERENT KIND OF ALCOHOL OXIDIZING SYSTEM TO OXIDIZE , SORBOSE DEHYDROGENASE 3$( AND , SORBOSONE DEHYDRO GENASE 3.$( TO PRODUCE KETO , GULONATE THE PRECURSOR FOR 6ITAMIN # 4HESE ALCOHOL AND ALDEHYDE
DEHYDROGENASES ARE DIFFERENT FROM !$( AND !,$( OF ACETIC ACID BACTERIA AND HAVE BEEN SHOWN TO BE A MEMBRANE BOUND SINGLE PEPTIDE mAVOPROTEIN 3UGI SAWA ET AL 3AITO ET AL AND A MEMBRANE BOUND ENZYME HAVING . TERMINAL HYDROPHOBIC STRETCH BUT AN UNKNOWN TYPE COFACTOR 3HINJOH ET AL RESPECTIVELY )N &IG ANOTHER SORBITOL DEHYDROGENASE 3,$( DIFFERENT FROM ',$( IS SHOWN WHICH IS A mAVOPROTEIN CYTOCHROME C COMPLEX 4ABLE 4HE 3,$( HAS RECENTLY BEEN SHOWN TO PRODUCE $ FRUC TOSE UNLIKE ',$( PRODUCING , SORBOSE / !DACHI ET AL UNPUBLISHED 4HE SAME TYPE OF mAVOPROTEIN CYTOCHROME C COMPLEX $ FRUCTOSE DEHYDROGENASE &$( HAVING A THREE SUBUNIT COMPLEX IS PRESENT IN SOME 'LUCONOBACTER R SPECIES WHERE $ FRUCTOSE IS OXIDIZED FURTHER TO KETO $ FRUCTOSE &IG 4HESE SUGAR OR SUGAR ALCOHOL DEHYDROGENASES ARE ALL MEMBRANE BOUND AND SEEM TO BE PRESENT ON THE PERIPLASMIC SIDE OF THE CYTOPLASMIC MEMBRANE !LSO ALTHOUGH THERE IS NO DIRECT EVIDENCE THESE OXIDORE DUCTASES WORK AS PRIMARY DEHYDROGENASES BY LINKING TO THE TERMINAL UBIQUINOL OXIDASE VIA 51 AS IN THE CASE OF OTHER RESPIRATIONS *71IZTJPMPHJDBM'VODUJPOBOE.FBOJOHT PG4VHBSBOE"MDPIPM3FTQJSBUJPOTPG"DF UJD"DJE#BDUFSJB "&OFSHFUJD"TQFDUPG$/SFTJTUBOU#Z Z QBTT0YJEBTFJO(MVDPOPCBDUFS3FTQJSBUPSZ $IBJOT !S DESCRIBED ABOVE SINCE THE RESPIRATORY CHAIN OF 'LUCONOBACTER SPECIES BRANCHES WITH A #. SENSITIVE CYTOCHROME O AND A #. RESISTANT BYPASS OXIDASE THE #. RESISTANT BYPASS OXIDASE MAY HAVE SOME PHYSIOLOGICAL MEANINGS ESPECIALLY FROM THE ENERGETIC POINT OF VIEW !S SHOWN IN &IG THE ( / RATIO IN THE RESTING CELLS OF ' SUBOXYDANS HAS BEEN SHOWN TO VARY FROM TO DEPENDING ON THE RESPIRATORY SUBSTRATES AND ON THE EXTRACELLULAR P( OF THE GROWTH MEDIUM -ATSUSHITA ET AL A #. SENSITIVE CYTOCHROME O OF ' SUBOXYDANS HAS BEEN SHOWN TO GENERATE AN ELECTROCHEMICAL PROTON GRADIENT BY THE OXIDATION OF UBIQUINOL -ATSUSHITA ET AL )N GENERAL CYTOCHROME O CATALYZES A SCALAR PROTON RELEASE FROM UBIQUINOL OUTSIDE THE MEMBRANE AND A CONCOMITANT PROTON UPTAKE INSIDE THE MEMBRANE TO REDUCE OXYGEN / TO WATER (/ WHERE THE SCALAR REACTION PRODUCES A ( / RATIO OF -ATSUSHITA ET
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&IG /XIDATION PATHWAYS OF SEVERAL SUGAR ALCOHOLS SUGARS AND SUGAR ACIDS IN 'LUCONOBACTER SPECIES 1UINOPROTEIN GLYCEROL DEHYDRO R
GENASE ',$( IS ABLE TO OXIDIZE $ GLUCONATE $ SORBITOL $ MANNITOL $ ARABITOL GLYCEROL TO KETOGLUCONATE , SORBOSE $ FRUCTOSE $ XYLULOSE AND DIHYDROXYACETONE RESPECTIVELY $ 'LUCONATE IS ALSO OXIDIZED TO KETOGLUCONATE THEN TO DIKETOGLUCONATE BY A mAVOPROTEIN GLUCONATE DEHYDROGENASE '!$( AND KETOGLUCONATE DEHYDROGENASE +'$( RESPECTIVELY , 3ORBOSE IS OXIDIZED TO , SORBOSONE THEN TO KETOGULONATE BY A mAVOPROTEIN SORBOSE DEHYDROGENASE 3$( AND AN UNIDENTIlED ENZYME SORBOSONE DEHYDROGENASE 3.$( RESPECTIVELY $ &RUCTOSE IS OXIDIZED TO KETOFRUCTOSE BY A mAVOPROTEIN FRUCTOSE DEHYDROGENASE &$( WHEREAS $ 3ORBITOL IS OXIDIZED TO $ FRUCTOSE BY A mAVOPROTEIN SORBITOL DEHYDROGENASE 3,$(
AL AND FURTHER IT HAS AN ADDITIONAL ABILITY TO PUMP PROTONS WITH A ( E RATIO OF 0UUSTINEN ET AL 4HUS ALTHOUGH CYTOCHROME O OF ' SUB OXYDANS IS EXPECTED TO EXHIBIT A ( / RATIO OF A ( E RATIO OF THE OBSERVED ( / RATIO IS LOWER THAN THE VALUE EXPECTED WITH CYTOCHROME O ONLY WORKING AS THE TERMINAL OXIDASE &URTHERMORE A MEMBRANE POTENTIAL GENERATED DURING THE OXIDATION OF GLUCOSE OR ETHANOL HAS BEEN SHOWN TO BE DISSIPATED COMPLETELY
WITH M- +#. ABLE TO BLOCK CYTOCHROME O COM PLETELY IN THE MEMBRANE VESICLES OF ' SUBOXYDANS -ATSUSHITA ET AL A 4HUS IT IS SUGGESTED THAT THE #. RESISTANT BYPASS OXIDASE HAS NO ABILITY TO PRODUCE AN ELECTROCHEMICAL PROTON GRADIENT 4HIS NOTION IS HIGHLY CONSISTENT WITH THE lNDINGS THAT THE ( / RATIO IN CELLS GROWN AT LOW EXTRACELLULAR P( WHERE #. RESISTANT BYPASS OXIDASE IS INCREASED IS LOWER THAN THAT IN THE CELLS GROWN AT HIGHER P(
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&IG %FFECT OF GROWTH P( ON CYANIDE SENSITIVITY AND ( / RATIO OF ETHANOL AND GLUCOSE RESPIRATIONS OF 'LUCONOBACTER SUBOXYDANS 2IGHT PANEL SHOWS THE CYANIDE SENSITIVITY ON ETHANOL GLUCOSE AND .!$( OXIDASE ACTIVITIES OF THE MEMBRANES PREPARED FROM THE CELLS GROWN ON P( S AND P( D ,EFT PANEL SHOWS THE MEASUREMENT OF ENDOGENOUS RESPIRATION DEPENDENT ( / RATIO IN THE RESTING CELLS OBTAINED FROM P( AND P( CULTURES AND ALSO THE ( / VALUES OBTAINED FROM ENDOGENOUS ETHANOL AND GLUCOSE RESPIRATIONS
&IG BUT THE MEMBRANE POTENTIAL GENERATION IN THE MEMBRANE VESICLES OF ' SUBOXYDANS IS SENSITIVE TO #. REGARDLESS OF THE EXTERNAL P( DURING GROWTH OF THE CELLS 4HUS NON ENERGY GENERATING #. RESISTANT BYPASS OXIDASE MAY CONTRIBUTE OR MORE TO THE RESPIRATORY ACTIVITY OF ' SUBOXYDANS UNDER THE LOWER EXTRACELLULAR P( P( 5NDER THE SAME ACIDIC GROWTH CONDITIONS WHERE #. RESISTANT BYPASS OXIDASE IS INCREASED IT WAS ALSO FOUND THAT THE!$( CONTENT AND THUS THE CYTOCHROME C CONTENT IN THE MEMBRANES OF ' SUBOXYDANS WAS LARGELY INCREASED BUT THE ACTIVITY DID NOT CHANGE MUCH SUGGESTING THAT SUCH A CONDITION PRODUCES AN INACTIVE FORM OF !$( INACTIVE !$( -ATSUSHITA ET AL !LTHOUGH THE INACTIVE !$( COULD NOT BE DISTINGUISHED FROM THE ACTIVE !$( WITH RESPECT TO STRUCTURE IT EXHIBITED TEN TIMES LOWER 51 REDUCTASE ACTIVITY THAN THE ACTIVE !$( !S DESCRIBED ABOVE !$( IS ABLE TO ACCEPT ELECTRONS FROM UBIQUINOL AND THUS SEEMS TO MEDIATE ELECTRON TRANSFER FROM UBIQUINOL TO THE BYPASS OXIDASE SYSTEM IN VIVO 3INCE INACTIVE !$( WAS SHOWN TO EXHIBIT HIGHER UBIQUINOL OXIDASE ACTIVITY THAN THE ACTIVE ENZYME -ATSUSHITA ET AL INACTIVE !$( MIGHT HAVE A HIGHER ABILITY TO DONATE ELECTRONS TO THE BYPASS OXIDASE 4HUS INACTIVE !$( IS PRODUCED UNDER CONDITIONS OF LOW P( AND HIGH AERATION WHERE THE BYPASS OXIDASE ACTIVITY IS HIGHLY
ELEVATED 4HEREFORE SUCH AN ALTERNATIVE ELECTRON mOW IN THE RESPIRATORY CHAIN MIGHT BE GENERATED BY SWITCHING !$( FROM ACTIVE TO INACTIVE 3INCE OTHER MEMBRANE BOUND DEHYDROGENASES SUCH AS '!$( OR 3,$( ALSO HAVE A TRIHEME CYTOCHROME C SUBUNIT HOMOLOGOUS TO SUBUNIT )) OF !$( AS DESCRIBED PRE VIOUSLY SUCH A SPECIlC ELECTRON TRANSFER MAY ALSO BE CARRIED OUT BY THE CYTOCHROME SUBUNIT OF THESE DEHYDROGENASES 'ENERATION OF INACTIVE !$( AND ALSO THE INCREASE OF THE BYPASS OXIDASE ACTIVITY OCCUR UNDER ACIDIC P( GROWTH CONDITIONS WHICH CAN BE GENERATED AS A RESULT OF A LARGE ACCUMULATION OF OXIDATION PRODUCTS 4HUS IT IS REASONABLE TO SPECULATE THAT INACTIVE !$( CONFERS A HIGHER NON ENERGY GENERATING BYPASS OXIDASE ACTIVITY ON THE RESPIRATORY CHAIN WHICH KEEPS THE OXIDATION REACTIONS AT A HIGH LEVEL IN ANY GROWTH CONDITIONS #"DFUJD"DJE3FTJTUBODFBOE&UIBOPM3FT QJSBUJPOTPG"DFUPCBDUFS4QFDJFT !CETIC ACID RESISTANCE IS A CRUCIAL FACTOR TO STABLY PRODUCE LARGE AMOUNTS OF ACETIC ACID BY !CETOBACTER SPECIES WHICH ARE IMPORTANT STRAINS FOR VINEGAR FERMENTATION )N THESE !CETOBACTER SPECIES SPONTA R
NEOUS MUTATION IS OBSERVED AT HIGH FREQUENCIES AND SOMETIMES LEADS TO SIMULTANEOUS DEFECTS IN BOTH
&KDSWHU 5HVSLUDWRU\&KDLQRI$FHWLF$FLG%DFWHULD ACETATE PRODUCING ETHANOL OXIDIZING AND ACETIC ACID RESISTANCE ABILITIES /HMORI ET AL 4AKEMURA ET AL 3INCE ALL THESE MUTANTS ALSO SHOW A COMPLETE LOSS OF !$( ACTIVITY !$( SEEMS TO BE INVOLVED IN ACETIC ACID RESISTANCE 7HEN ACETIC ACID SENSITIVE MUTANTS WERE ISOLATED BY .4' MUTAGENESIS HOWEVER ONLY THE GENES AAR!"# OPERON RELATED TO # THE ENZYMES FOR ACETATE UTILIZATION INCLUDING CITRATE SYNTHASE WERE OBTAINED AS THE TARGET GENE WHICH COULD NOT COMPLEMENT THE DElCIENCY OF ACETATE RESISTANCE IN THE SPONTANEOUS MUTANTS DESCRIBED ABOVE SEE "EPPU )N ETHANOL CULTURE OF ! ACETI THERE ARE THREE GROWTH PHASES &IG ! ACETI lRST GROWS BY OXIDIZ ING ETHANOL COMPLETELY TO ACETIC ACID ETHANOL OXIDA TION PHASE THEN STOPS THE GROWTH AND REMAINS FOR A LONG TIME WITH THE VIABLE CELL NUMBER BEING DECREASED lRST STATIONARY PHASE AND lNALLY STARTS TO GROW AGAIN BY UTILIZING THE ACCUMULATED ACETIC ACID THE PHASE OF WHICH IS CALLED AS @OVEROXIDATION OF ACETATE )T IS THUS CONCEIVABLE THAT !CETOBACTERR SPECIES HAVE TWO DIFFERENT PHASES RELATED TO ACETIC ACID RESISTANCE THE ETHANOL OXIDATION AND THE lRST STATIONARY PHASES WHERE THE STRAINS RESIST AGAINST ACETIC ACID ACCUMULATED IN THE CULTURE MEDIUM WITHOUT UTILIZING THE ACETATE AND THE OVEROXIDATION PHASE WHERE THE STRAINS JUST UTILIZE
THE ACETATE FOR CELL GROWTH 3INCE IT HAS BEEN SHOWN IN THE OVEROXIDATION PHASE THAT ACETIC ACID UTILIZATION ENZYMES SUCH AS 4#! CYCLE ENZYMES ACETYL #O! SYNTHASE INVOLVED IN THE SUPPLY OF ACETYL #O! AND PHOSPHOENOLPYRUVATE CARBOXYLASE PROVIDING OXALO ACETATE ARE INCREASED &IG 3AEKI ET AL THE AAR GENES ISOLATED AS @ACETATE RESISTANCE GENES ARE R CONSIDERED AS ONE OF THE ACETATE UTILIZATION SYSTEMS IN THE OVEROXIDATION PHASE WHEREAS THESE ACETATE UTILIZING ENZYMES SEEM TO BE SUPPRESSED DURING THE ETHANOL OXIDATION AND THE lRST STATIONARY PHASES IN WHICH ANOTHER MECHANISM MUST BE WORKING TO RESIST A HIGH CONCENTRATION OF ACETIC ACID PRODUCED BY THEMSELVES 3UCH A RESISTANT MECHANISM MAY BE AN ENERGY REQUIRING PROCESS OF WHICH THE ENERGY MAY BE PRODUCED BY ETHANOL RESPIRATION 4HUS SINCE !$( DElCIENCY CAUSES THE DEFECT IN ETHANOL RESPIRATION CELLS HAVING A MUTATION IN !$( SEEM NOT TO RESIST ACETIC ACID 4HUS THE ETHANOL RESPIRATION IS ALWAYS EXPOSED TO @UNCOUPLING WHICH MAY BE INDUCED BY ACETIC ACID ITSELF OR BY THE MECHANISM OF ACETATE RESISTANCE AND THUS THE ETHANOL OXIDATION REACTION BY THE RESPIRATORY CHAIN SEEMS TO BE VERY RAPID BUT NOT TO BE DISTURBED BY THE ELECTROCHEMICAL PROTON GRADIENT GENERATED BY THE RESPIRATION
&IG 'ROWTH PATTERNS OF !CETOBACTER SPECIES IN ETHANOL CULTURE R !CETOBACTER STRAIN EXHIBITS A BIPHASIC GROWTH CURVE IN ETHANOL CULTURE R WHERE THE lRST PHASE HAS AN ETHANOL OXIDATION TO PRODUCE ACETIC ACID AND THE SECOND PHASE AN OVEROXIDATION OF ACETIC ACID ASSIMILA TION
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$&DPMPHJDBM"TQFDUTPG4VHBSBOE"MDPIPM 3FTQJSBUPSZ1SPDFTTFTJO"DFUJD"DJE#BDUF SJB$PNQFUJUJPO.FDIBOJTN !CETIC ACID BACTERIA ARE ABLE TO ACCUMULATE LARGE AMOUNTS OF OXIDATION PRODUCTS OUTSIDE THE CELLS 4HE OXIDATION REACTIONS ARE PERFORMED BY SUGAR AND ALCOHOL RESPIRATIONS OF WHICH THE ELECTRON TRANSFER GENERATES AN ELECTROCHEMICAL PROTON GRADIENT ACROSS THE MEMBRANES THAT IS USEFUL FOR !40 GENERATION OR BIOENERGETIC EVENTS FOR CELL GROWTH !S A GENERAL PRINCIPLE HOWEVER THE PROTON GRADIENT THUS GENER ATED MAY SUPPRESS THE ELECTRON TRANSFER BY A FEEDBACK CONTROL WHICH MAY TURN OUT TO DISTURB THESE OXIDATION REACTIONS 4HUS A RAPID OXIDATION BY SUCH A RESPIRATORY CHAIN DISTURBS THE ELECTRON TRANSFER BY ITSELF WHICH MAY BE UNFAVORABLE FOR ORGANISMS THAT ARE REQUIRED TO PRODUCE LARGE AMOUNTS OF OXIDATION PRODUCTS BY THE RESPIRATORY CHAIN 4HERE SEEMS TO BE TWO WAYS TO OVERCOME SUCH A SELF CONTRADICTION ONE IS THAT AT LEAST A PART OF THE RESPIRATORY CHAIN HAS A ROUTE WITH NO ENERGY GENERATION AND THE OTHER IS THAT AN ELECTRON TRANSFER AND AN ENERGY GENERATION IN THE RESPIRATORY CHAIN ARE UNCOUPLED IN SOME WAY 4HE FORMER INSTANCE CAN BE SEEN IN 'LUCONOBACTER SPECIES WHERE THERE IS A NON ENERGY GENERATING #. INSENSITIVE BYPASS SYSTEM IN THE RESPIRATORY CHAIN AND THUS A RAPID ALCOHOL OR SUGAR OXIDATION CAN BE CARRIED OUT WITH THE GENERATION OF A LITTLE ENERGY WHICH MAY NOT INTERFERE WITH THE ELECTRON TRANSFER SO MUCH 4HE LATTER INSTANCE UNCOUPLING OF ELECTRON TRANSFER AND ENERGY GENERATION COULD BE EXPECTED IN !CETOBACTER SPECIES WHICH DO NOT HAVE A NON ENERGY GENERATING
BYPASS OXIDASE UNLIKE 'LUCONOBACTER 4HE ORGANISMS GROW IN AN ENVIRONMENT OF A HIGH CONCENTRATION OF ACETIC ACID WHICH IS KNOWN IN GENERAL TO BE A POWERFUL UNCOUPLER AND THUS A WELL KNOWN BACTERICIDAL AGENT AND ALSO RESIST ACETIC ACID BY USING A HIGH LEVEL OF ENERGY 4HEREFORE THE ETHANOL RESPIRATION AND ALSO OTHER RESPIRATIONS OF !CETOBACTER SPECIES MAY BE AFFECTED BY ACETIC ACID WHICH MAY UNCOUPLE ENERGY GENERATION AND ELECTRON TRANSFER OF THE RESPIRATORY CHAIN AND THUS INCREASE THE RATE OF ALCOHOL AND SUGAR OXIDATION !CETIC ACID BACTERIA INHABIT THE SURFACE OF mOWERS PISTIL FRUITS AND THEIR FERMENTED PRODUCTS SUCH AS VINEGAR SAKE WINE OR BEER !SAI AND THUS SEEM TO BE EVOLVED IN ADAPTING TO SUCH A SPECIlC ENVIRONMENT WHERE HIGH CONCENTRATIONS OF SUGARS ALCOHOLS OR SUGAR ALCOHOLS EXIST IN HIGHLY AEROBIC CONDITIONS )N SUCH A SPECIlC ENVIRONMENT ACETIC ACID BACTERIA SEEM TO ARRANGE A SPECIlC RESPIRATORY CHAIN TO PERFORM A RAPID OXIDATION OF HIGH CONCEN TRATIONS OF SUGARS ALCOHOLS OR SUGAR ALCOHOLS 4HUS 'LUCONOBACTER R SPECIES ARE ABLE TO RAPIDLY OXIDIZE SUGAR ALCOHOLS TO SUGARS THEN SUGARS TO SUGAR ACIDS AND THUS TO lNALLY ACCUMULATE SUGAR ACIDS AT A HIGH CONCENTRATION IN THEIR CULTURE MEDIA WHILE !CETO BACTER R SPECIES MAINLY OXIDIZE ETHANOL TO PRODUCE A HIGH CONCENTRATION OF ACETIC ACID 4HESE REACTION PRODUCTS SUGAR ACIDS INCLUDING ACETIC ACID CAUSE DECREASED P( AND AN EVEN MORE BACTERICIDAL EFFECT AND ALSO MAY BE HARD TO UTILIZE BY OTHER ORGANISMS SO THAT THESE SUGAR ACIDS MAY DISTURB THE GROWTH OF OTHER BACTERIA AND YEASTS LIVING IN THE SAME HABITAT &IG !S SHOWN IN THE PHENOMENON OF @ACETIC ACID
&IG #OMPETITION MECHANISM OF ACETIC ACID BACTERIA !CETIC ACID BACTERIA ARE ABLE TO OXIDIZE SEVERAL SUGARS AND SUGAR ALCOHOLS RAPIDLY AND THEN TO ACCUMULATE A LARGE AMOUNT OF THE CORRESPONDING SUGAR ACIDS OUTSIDE THE CELLS 4HESE OXIDATION REACTIONS CREATE ACIDIC ENVI RONMENT WHICH IS HARMFUL FOR OTHER COMPETING BACTERIA OR YEASTS !T THE SAME TIME SUCH A RAPID OXIDATION OF SUGARS OR SUGAR ALCOHOLS TO THE SUGAR ACIDS ALSO DISTURB THE GROWTH OF OTHER COMPETITIVE MICROORGANISMS BECAUSE OF THE DEPLETION OF AVAILABLE SUGARS OR SUGAR ALCOHOLS FROM THEIR ENVIRONMENT
&KDSWHU 5HVSLUDWRU\&KDLQRI$FHWLF$FLG%DFWHULD RESISTANCE &IG ! ACETI STRAINS DO NOT UTILIZE ANY ACETIC ACID ACCUMULATED IN THE CULTURE MEDIUM DESPITE THE CELLS DYING IN THE lRST STATIONARY PHASE 4HEREAFTER WHEN THE CELL NUMBER IS DECREASED TO SOME THRESHOLD LEVEL TO BE ALIVE THE CELLS START TO UTILIZE THE ACETIC ACID TO MAINTAIN THEIR POPULATION 4HUS IT IS CONCEIVABLE THAT !CETOBACTER SPECIES ARE PATIENT FOR A LONG TIME IN A HIGH CONCENTRATION OF ACETIC ACID WHICH IS A MORE SEVERE CONDITION FOR OTHER BACTERIA AND MICROORGANISMS !LTHOUGH THE lRST STATIONARY PHASE IS NOT AS LONG AS IN THE CASE OF ! ACETI IN ETHANOL CUL R TURE 'LUCONOBACTER SPECIES ALSO HAVE THE SAME TYPE OF BIPHASIC GROWTH WHERE THEY CAN lRST ACCUMULATE THE SUGAR ACIDS AT A HIGH CONCENTRATION AND UTILIZE SUCH SUGAR ACIDS LATER 4HUS THESE SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA ABLE TO CARRY OUT RAPID OXIDATION OF SUGAR AND ALCOHOL SEEM TO HAVE EVOLVED AS A COMPETITION MECHANISM AGAINST OTHER MICROORGANISMS TOGETHER WITH THE DELAYED UTILIZATION STRATEGY OF SUCH REACTION PRODUCTS !CETIC ACID BACTERIA EFFECT THESE SPECIlC SUGAR AND ALCOHOL RESPIRATIONS MAINLY FOR THE OXIDATION OF EXTRACELLULAR SUBSTRATES TO ACCUMULATE THE REACTION PRODUCTS OUTSIDE OF THE CELLS AND PARTLY FOR THE ENERGY GENERATION !ND AT LEAST IN THE lRST STATIONARY PHASE THESE OXIDATION REACTIONS SEEM NOT TO BE UTILIZED FOR THE ASSIMILATION OF THE SUBSTRATES 4HUS SUCH SUGAR AND ALCOHOL RESPIRATIONS OF ACETIC ACID BACTERIA SEEM TO BE RELATED PARTLY TO THE PERIPLASMIC OXIDATIONS OF CHEMOLITHOTROPHS SUCH AS HYDROGEN OR IRON OXIDATION SEE #HAPTERS AND IN THIS VOLUME 3FGFSFODFT !DACHI / &UJII 9 'HALY -& 4OYAMA ( 3HINAGAWA % AND -AT SUSHITA + -EMBRANE BOUND QUINOPROTEIN $ ARABITOL DEHYDROGENASE OF 'LUCONOBACTER SUBOXYDANS )&/ ! VERSATILE ENZYME FOR THE OXIDATIVE FERMENTATION OF VARIOUS KETOSES "IOSCI "IOTECHNOL "IOCHEM n !MEYAMA - 3HINAGAWA % -ATSUSHITA + AND !DACHI / 3OLUBILIZATION PURIlCATION AND PROPERTIES OF MEMBRANE BOUND GLYCEROL DEHYDROGENASE FROM 'LUCONOBACTER INDUSTRIUS !GRIC "IOL #HEM n !MEYAMA - -ATSUSHITA + 3HINAGAWA % AND !DACHI / 3UGAR OXIDIZING RESPIRATORY CHAIN OF 'LUCONOBACTER SUBOXY DANS %VIDENCE FOR A BRANCHED RESPIRATORY CHAIN AND CHARAC TERIZATION OF RESPIRATORY CHAIN LINKED CYTOCHROMES !GRIC "IOL #HEM n !NRAKU 9 "ACTERIAL ELECTRON TRANSPORT CHAINS !NNU 2EV "IOCHEM n !SAI 4 !CETIC !CID "ACTERIA #LASSIlCATION AND "IOCHEMI CAL !CTIVITIES 4OKYO 5NIVERSITY 0RESS 4OKYO "ÊCHI " AND %TTLINGER , #YTOCHROME DIFFERENCE SPECTRA OF
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-ATSUSHITA + 3HINAGAWA % !DACHI / AND !MEYAMA - B 2EACTIVITY WITH UBIQUINONE OF QUINOPROTEIN $ GLUCOSE DEHY DROGENASE FROM 'LUCONOBACTER SUBOXYDANS * "IOCHEM n -ATSUSHITA + 3HINAGAWA % !DACHI / AND !MEYAMA - #YTOCHROME A OF !CETOBACTER ACETI IS A CYTOCHROME BA FUNCTIONING AS UBIQUINOL OXIDASE 0ROC .ATL !CAD 3CI 53! n -ATSUSHITA + .AGATANI 9 3HINAGAWA % !DACHI / AND !MEYAMA - 2ECONSTITUTION OF THE ETHANOL OXIDASE RESPIRATORY CHAIN IN MEMBRANES OF QUINOPROTEIN ALCOHOL DEHYDROGENASE DElCIENT 'LUCONOBACTER SUBOXYDANS SUBSP _ STRAINS * "ACTERIOL n -ATSUSHITA + %BISUYA ( AND !DACHI / A (OMOLOGY IN THE STRUCTURE AND THE PROSTHETIC GROUPS BETWEEN TWO DIFFERENT TERMINAL UBIQUINOL OXIDASES CYTOCHROME A AND CYTOCHROME O OF !CETOBACTER ACETI * "IOL #HEM n -ATSUSHITA + %BISUYA ( !MEYAMA - AND !DACHI / B #HANGE OF THE TERMINAL OXIDASE FROM CYTOCHROME A IN SHAKING F CULTURES TO CYTOCHROME O IN STATIC CULTURES OF !CETOBACTER ACETI * "ACTERIOL n -ATSUSHITA + 4AKAHASHI + 4AKAHASHI - !MEYAMA - AND !DACHI / C -ETHANOL AND ETHANOL OXIDASE RESPIRATORY CHAINS OF THE METHYLOTROPHIC ACETIC ACID BACTERIUM !CETOBACTER METHANOLICUS * "IOCHEM n -ATSUSHITA + 4AKAKI 9 3HINAGAWA % !MEYAMA - AND !DACHI / D %THANOL OXIDASE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA 2EACTIVITY WITH UBIQUINONE OF PYRROLOQUINOLINE QUINONE DEPENDENT ALCOHOL DEHYDROGENASES PURIlED FROM !CETOBACTER ACETI AND 'LUCONOBACTER SUBOXYDANS "IOSCI "IOTECH "IOCHEM n -ATSUSHITA + 4OYAMA ( AND !DACHI / 2ESPIRATORY CHAIN AND BIOENERGETICS OF ACETIC ACID BACTERIA )N 2OSE !( AND 4EMPEST $7 ED !DVANCES IN -ICROBIAL 0HYSIOLOGY 6OL PP n !CADEMIC 0RESS ,ONDON -ATSUSHITA + 9AKUSHI 4 4AKAKI 9 4OYAMA ( AND !DACHI / 'ENERATION MECHANISM AND PURIlCATION OF AN INACTIVE FORM CONVERTIBLE IN VIVO TO THE ACTIVE FORM OF QUINOPROTEIN ALCOHOL DEHYDROGENASE IN 'LUCONOBACTER SUBOXYDANS * "AC TERIOL n -ATSUSHITA + 9AKUSHI 4 4OYAMA ( 3HINAGAWA % AND !DACHI / &UNCTION OF MULTIPLE HEME C MOIETIES IN INTRAMOLECULAR ELECTRON TRANSPORT AND UBIQUINONE REDUCTION IN THE QUINOHE MOPROTEIN ALCOHOL DEHYDROGENASE CYTOCHROME C COMPLEX OF 'LUCONOBACTER SUBOXYDANS * "IOL #HEM n -ATSUSHITA + 9AKUSHI 4 4OYAMA ( !DACHI / -IYOSHI ( 4A GAMI % AND 3AKAMOTO + 4HE QUINOHEMOPROTEIN ALCOHOL DEHYDROGENASE OF 'LUCONOBACTER SUBOXYDANS HAS UBIQUINOL OXIDATION ACTIVITY AT A SITE DIFFERENT FROM THE UBIQUINONE REDUC TION SITE "IOCHIM "IOPHYS !CTA n -ATSUSHITA + &UJII 9 !NO 9 4OYAMA ( 3HINJOH - 4OMIYAMA . -IYAZAKI 4 3UGISAWA 4 (OSHSINO 4 AND !DACHI / +ETO $ GLUCONATE PRODUCTION IS CATALYZED BY A QUINOPROTEIN GLYCEROL DEHYDROGENASE MAJOR POLYOL DEHYDROGENASE IN 'LU CONOBACTER SP !PPL %NVIRONM -ICROBIOL n -C)NTIRE 7 3INGER 40 !MEYAMA - !DACHI / -ATSUSHITA + AND 3HINAGAWA % )DENTIlCATION OF THE COVALENTLY BOUND mAVINS OF $ GLUCONATE DEHYDROGENASE FROM 0SEUDOMONAS AERUGINOSA AND 0SEUDOMONAS mUORESCENS AND OF KETO $ GLUCONATE DEHYDROGENASE FROM 'LUCONOBACTER MELANOGENUS "IOCHEM * n
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7ILLIAMS ($ AND 0OOLE 2+ 4HE CYTOCHROMES OF !CETO BACTER PASTEURIANUS .#)" %VIDENCE OF A ROLE FOR A CYTO CHROME A LIKE HAEMOPROTEIN IN ELECTRON TRANSFER TO CYTOCHROME OXIDASE D * 'EN -ICROBIOL n 7ILLIAMS ($ AND 0OOLE 2+ #YTOCHROMES OF !CETOBACTER PASTEURIANUS .#)" 2EACTION WITH OXYGEN OF CYTOCHROME O IN CELLS MEMBRANES AND NONSEDIMENTABLE SUBCELLULAR FRAC TIONS #URR -ICROBIOL n 9AMADA - 3UMI + -ATSUSHITA + AND !DACHI / 4OPO LOGICAL ANALYSIS OF QUINOPROTEIN GLUCOSE DEHYDROGENASE IN %SCHERICHIA COLI AND ITS UBIQUINONE BINDING SITE * "IOL #HEM n 9AMADA 9 (OSHINO + AND )SHIKAWA 4 4HE PHYLOGENY OF ACETIC ACID BACTERIA BASED ON THE PARTIAL SEQUENCES OF 3 RIBO SOMAL 2.! 4HE ELEVATION OF SUBGENUS 'LUCONOACETOBACTERR TO THE GENERIC LEVEL "IOSCI "IOTECH "IOCHEM n 9UM $9 ,EE 90 AND 0AN *' #LONING AND EXPRESSION OF A GENE CLUSTER ENCODING THREE SUBUNITS OF MEMBRANE BOUND GLUCONATE DEHYDROGENASE FROM %RWINIA CYPRIPEDII !4## IN %SCHERICHIA COLI * "ACTERIOL n
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4VNNBSZ .ITROGEN lXATION ALLOWS A DIVERSE ARRAY OF BACTERIA EITHER FREE LIVING IN THE ENVIRONMENT OR IN SYMBIOSIS WITH PLANTS TO GROW IN AREAS WHERE lXED . IS DElCIENT 4HIS CONFERS THEM A LARGE ADVANTAGE OVER THEIR NON . lXING COMPETITORS .EVERTHELESS . lXATION IS AN ENERGY DEMANDING PROCESS SO THAT ENERGY GENERATING RESPIRATION IS OFTENTIMES CLOSELY ASSOCIATED WITH EFlCIENT . lXING SYSTEMS 4HE ROOT NODULE BACTERIA HAVE MANY / BINDING TERMINAL OXIDASES THAT DIFFER IN EXPRESSION DEPENDING ON WHETHER THE BACTERIUM EXISTS IN FREE LIVING OR IN THE SYMBIOTIC STATE -ANY OF THE AEROBIC . lXERS CONTAIN A VERY HIGH / AFlNITY TERMINAL OXIDASE LIKE ONE THAT CAN FUNCTION AT FREE / LEVELS AS LOW AS N- WHEN THE BACTERIA ARE SURROUNDED IN LEGHEMOGLOBIN WITHIN LEGUME ROOT NODULES 4HE PREVIOUSLY DESCRIBED @UNCOUPLED NATURE OF SOME OXIDASES OF . lXING BACTERIA MAY NOT BE THE CASE FROM MORE RECENT STUDIES 4HE . lXING ENZYME NITROGENASE IS LABILE TO / EXPOSURE THIS CREATES A PHYSIOLOGICAL PROBLEM FOR AEROBIC DIAZOTROPHS 4HE MANY STRATEGIES TO OVERCOME THIS PROBLEM MUST ENSURE A STEADY SUPPLY OF !40 DURING NITROGENASE FUNCTION YET PERMIT THE ENZYME TO BE PROTECTED FROM / INACTIVA TION 3TRATEGIES INCLUDE LIVING IN AGGREGATES OR WITHIN VISCOUS SLIMY SHEATHS OR IN / RESTRICTED ROOT NODULE BARRIERS OR FORMING SPECIALIZED CELLS LACKING OXYGENIC / EVOLVING PHOTOSYNTHETIC MACHINERY 4HE TEMPORAL SEPARATION OF . lXATION FROM / EVOLVING PHOTOSYNTHESIS IS A MECHANISM USED BY SOME MARINE lLAMENTOUS
%MAIL RMAIER ARCHESUGAEDU $AVIDE :ANNONI ED 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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CYANOBACTERIA %MPLOYING VIGOROUS RESPIRATORY ACTIVITY AT THE MEMBRANE SO THAT THE INSIDE OF THE CELL IS NEARLY ANAEROBIC OR THE USE OF A SMALL REDOX ACTIVE IRON SULFUR PROTEIN THAT BINDS TO AND PROTECTS NITROGENASE FROM / INACTIVATION ARE WELL STUDIED MECHANISMS -OST OF THE PROTECTION MECHANISMS EVEN THE EXPRESSION OF TERMINAL OXIDASE ACTIVITIES ARE GENERALLY CO REGULATED WITH . lXATION GENES AND SOME OF THESE PROTECTION SYSTEMS MAY CONCOMITANTLY COMBAT OXIDATIVE STRESS 4HIS ADDITIONAL STRESS IS RELATED TO REACTIVE OXYGEN SPECIES PRODUCED FROM . lXATION RELATED PROTEINS
**OUSPEVDUJPOBOE1FSTQFDUJWF /XYGEN DEPENDENT RESPIRATION IS A KEY PROCESS FOR THE AEROBIC FUNCTIONING OF MANY BACTERIA AS THE EN ERGETIC ADVANTAGES OF RESPIRATORY METABOLISM MAKE IT BENElCIAL FOR THE SURVIVAL OF ORGANISMS HAVING ENERGETICALLY DEMANDING FEATURES /NE ENERGY DE MANDING BACTERIAL PROCESS IS NITROGEN lXATION A KEY PROCESS FOR MAINTAINING A BALANCED BIOGEOCHEMICAL NITROGEN CYCLE ON EARTH .ITROGEN lXATION IS CARRIED OUT BY AN !40 UTILIZING . REDUCING ENZYME NITRO GENASE %NERGY GENERATING RESPIRATORY METABOLISM IS THEREFORE A REQUIREMENT TO FUEL NITROGENASE IN MANY . lXERS )NDEED THE BULK OF . lXING BACTERIA IN NATURE ARE BELIEVED TO BE / RESPIRING ONES -AN CINELLI 4HE ENERGY INPUT INTO . lXATION VIA RESPIRATORY ELECTRON TRANSPORT IS WELL WORTH THE COST AS THE . lXING PROCESS ALLOWS THE ORGANISM TO GROW IN ENVIRONMENTS WHERE lXED NITROGEN SOURCES ARE LACKING OR DElCIENT .ITROGEN IS FREQUENTLY A LIMITING NUTRIENT FOR GROWTH OF ORGANISMS AND ONLY A LIMITED NUMBER OF BACTERIA HAVE THE ABILITY TO INCORPORATE OR @lX ATMOSPHERIC . .EVERTHELESS THEY ARE A HIGHLY DIVERSE GROUP OF BACTERIA WITH . lXING MEMBERS OCCUPYING ALL OF THE MAJOR TAXONOMIC GROUPS OF PROKARYOTES &UCHS &URTHERMORE MANY OF THEM HAVE EVOLVED COMPLEX INTERPLAY SYSTEMS WITH HIGHER ORGANISMS IN THE FORM OF SYMBIOSES IN ORDER TO INHABIT A SANCTUARY FROM THE MORE COMPETITIVE ENVIRONMENT OF FREE LIVING LIFE 4HE NEED FOR NITROGEN FOR ALL CELLS AS A COMPONENT OF VITAL MACROMOLECULES MEANS THESE BACTERIA CAN THEN GROW WHERE THEIR NON . lXING COMPETITORS CANNOT .OT ONLY ARE THE ENERGETIC DEMANDS OF THE . REDUCING PROCESS GREAT BUT THE / LABILITY OF THE . lXING ENZYMES CREATES A NEED FOR A NEAR ANAEROBIC ENVIRONMENT WITHIN THE CELLS OF DIAZOTROPHIC AEROBES 4HEREFORE THE REQUIREMENT FOR / REMOVAL TO PERMIT !BBREVIATIONS !V) n COMPONENT ) OF NITROGENASE #YC- n CYTO CHROME C- .!# n NITROGEN ASSIMILATION CONTRO 0(" n POLY ` HYDROXY BUTYRATE 3/$ n SUPEROXIDE DISMUTASE &E3)) n IRON SULFUR )) PROTEIN
. lXATION ADDS ANOTHER VALUABLE ROLE TO THE OVERALL IMPORTANCE OF / CONSUMING RESPIRATION )NDEED IN SOME CASES THE BASIS FOR FORMING A SYMBIOTIC REFUGE IS UNDOUBTEDLY RELATED TO THE NEED FOR A LOW LEVEL OF OXYGEN SUPPLY TO OPTIMALLY lX . 4HERE IS NO DISAGREEMENT THAT BIOLOGICAL NITROGEN lXATION THE REDUCTION OF . TO FORM AMMONIUM BY ORGANISMS IS AN ENERGY INTENSIVE PROCESS 4HE AMOUNT OF !40 USED PER . lXED CAN BE AS HIGH AS ACCORDING TO SOME GROWTH YIELD MEASUREMENTS 3TAM ET AL AND UNDER PHYSIOLOGICAL CONDITIONS IT IS COMMONLY REPORTED AS n !40 PER DINITROGEN MOLECULE REDUCED "URRIS 3IMILARLY . lXING BACTERIA GROW MORE SLOWLY WHEN lXING . THAN WHEN PROVIDED WITH A lXED NITROGEN SOURCE /NE REASON FOR THE CONSIDERABLE EXTRA COST TO THE CELL IS DUE TO THE INEFlCIENT ALLOCATION OF ELECTRONS TO SUBSTRATES BY NITROGENASE &IRST OF ALL NITROGENASE ALLOCATES VALU ABLE REDUCTANT FOR THE PURPOSE OF PROTON REDUCTION IN ADDITION TO PASSING ELECTRONS TO CARRY OUT . REDUC TION &URTHER NITROGENASE DOES NOT EFlCIENTLY ALLOCATE ELECTRONS TO PROTONS VERSUS DINITROGEN &OR EXAMPLE IN SOME CONDITIONS THE NITROGENASE REACTION IN . lXING ROOT NODULES CAN GREATLY FAVOR THE PRODUCTION OF AN UNDESIRABLE PRODUCT NAMELY ( -AIER AND 4RIPLETT 5NDER PHYSIOLOGICAL CONDITIONS n OF THE ENERGY INPUT INTO . lXATION IS LOST AS ( EVOLUTION FROM THE ROOT NODULES 3CHUBERT AND %VANS (YDROGEN PRODUCTION DEPLETES THE !40 AND REDUCING EQUIVALENTS NORMALLY USED FOR THE PRODUCTION OF THE DESIRED END PRODUCT .( )N THIS WAY AMMONIUM FORMATION FROM . CAN COST CONSIDERABLY MORE !40 THAN THE IDEAL . REDUCTION REACTION SHOWN BELOW %VANS ET AL -AIER AND 4RIPLETT 5NDER IDEAL CONDITIONS . lXATION VIA NITROGENASE PROCEEDS ACCORDING TO THE FOLLOWING REACTION . ( E< !40 A .( ( !$0
0I .OTE THAT EVEN UNDER IDEAL CONDITIONS THE ENERGETIC COSTS IN TERMS OF !40 IS HIGH 7HEN ONE CONSIDERS THAT
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO THE LOW POTENTIAL REDUCTANT USED THE EIGHT ELECTRONS IN THE REACTION ABOVE IS USUALLY POISED AT THE FERREDOXIN OR EQUIVALENT REDOX LEVEL THE OVERALL ENERGETIC COSTS IS VERY DEMANDING TO THE CELL #ONSEQUENTLY MICRO ORGANISMS THAT lX . OFTENTIMES EXHIBIT HIGH CARBON USAGE OR HIGH PHOTOSYNTHETIC ACTIVITIES CONCOMITANTLY WITH HIGH RESPIRATORY RATES /THER COSTS TO THE CELL THAT CANNOT BE READILY MEASURED WOULD INCLUDE THE MAINTENANCE AND SYNTHESIS OF THE NITROGENASE STRUC TURAL MATURATION AND THE REGULATION RELATED PROTEINS 4HESE PROCESSES CAN BE HIGHLY COMPLEX AND MULTI COMPONENT EVEN FOR A SINGLE PROCESS WITHIN THE OVERALL MECHANISM OF MATURATION OR REGULATION 9UVANIYAMA ET AL -ASEPOHL ET AL .EVERTHELESS . lXATION BY NITROGENASE ACCOUNTS FOR MILLIONS OF TONS OF . lXED ON EARTH ANNUALLY AND IT IS A VITALLY IMPORTANT PROCESS FOR BALANCED NITROGEN TRANSFORMATIONS ON THE PLANET 4HIS CHAPTER WILL REVIEW THE PHYSIOLOGICALLY LINKED PROCESSES OF / CONSUMING RESPIRATION AND . lXATION )T IS APPROPRI ATE THAT THE TWO PROCESSES BE CONSIDERED AS A SINGLE SUBJECT TOGETHER AS THE BENElT IN TERMS OF ENERGY PRODUCTION OF ONE IS TIGHTLY ASSOCIATED PHYSIOLOGICALLY WITH THE ENERGY DEMANDS OF THE OTHER 4HIS CHAPTER WILL FOCUS PRIMARILY ON THE DIAZOTROPHIC AEROBES ESPECIALLY 2HIZOBIUM AND !ZOTOBACTER AS THESE ARE AEROBIC . lXERS FOR WHICH WE HAVE A CONSIDERABLE AMOUNT OF RESPIRATORY METABOLISM INFORMATION 4HEIR RELATIONSHIP WITH / EXTENDS FAR BEYOND JUST THE USE OF / AS A TERMINAL ELECTRON ACCEPTOR FOR ENERGY GENERATION IT INCLUDES EVOLVED RESPONSES TO OXYGEN AS BOTH A HINDRANCE TO AND A REQUIREMENT FOR EFlCIENT . lXATION **/JUSPHFO'JYBUJPOBOE0YZHFO5PMFSBODF .ITROGENASE IS WELL DOCUMENTED TO BE AN / LABILE ENZYME AND . lXING BACTERIA DISPLAY A WIDE VARIA TION IN THEIR TOLERANCE TO / (ILL 'ALLON 4HIS IS NOT SURPRISING AS THEY OCCUPY A WIDE RANGE OF ENVIRONMENTS AND THEY EMPLOY DIFFERENT MECHANISMS TO ENSURE THAT NITROGENASE REMAINS STABLE )N SPITE OF DOCUMENTED / PROTECTION MECHANISMS MOST . lXERS HAVE OPTIMUM . lXATION RATES IN MICROAEROBIC RATHER THAN IN ATMOSPHERIC / LEVELS (ILL THUS THE PROTECTION MECHANISMS MAY NOT BE TOTALLY ADEQUATE IN MANY ENVIRONMENTS COMMONLY ENCOUN TERED !VOIDANCE OF / OR lXING OF . ONLY UNDER FERMENTATIVE CONDITIONS 'ALLON IS SOMETIMES CONSIDERED TO BE STRATEGIES TO PROTECT NITROGENASE
4HE / LABILITY OF THE PURIlED NITROGENASE PROTEINS DOES NOT VARY MUCH BETWEEN ORGANISMS AND THIS IS PROBABLY DUE TO THE HIGHLY CONSERVED NATURE AND STRUCTURE OF THE NITROGENASE COMPONENTS 4HEREFORE THE OBSERVED DIFFERENCES IN / TOLERANCE AMONG DIAZOTROPHS MUST BE EXPLAINED BY PHYSIOLOGICAL MECHANISM DIFFERENCES ! CONVENIENT EXAMPLE TO DEMONSTRATE THIS PHYSIOLOGICAL VARIABILITY IS THE BACTEROID RESPONSE TO / "ACTEROIDS THE TERM FOR THE MATURE . lXING BACTERIA IN ROOT NODULES FROM THE NODULES OF DIFFERENT LEGUMES SHOW A CONSIDER ABLY WIDE RANGE OF SENSITIVITIES TO / &OR EXAMPLE " JAPONICUM BACTEROIDS HAVE NO NITROGENASE ACTIV ITY AT - / BUT !ZORHIZOBIUM CAULINODANS HAS HIGH ACTIVITY AT THIS / TENSION /"RIAN AND -AIER !LSO LUPIN BACTEROIDS HAVE NITROGENASE ACTIV ITY WHEN ISOLATED FROM NODULES AEROBICALLY WHEREAS NITROGENASE IN SOYBEAN NODULE BACTEROIDS IS ABOLISHED WHEN THE BACTEROIDS ARE EXPOSED TO AIR " JAPONICUM BACTEROIDS HAVE AN / OPTIMUM CLOSE TO N- FOR NITROGENASE ACTIVITY "ERGERSEN AND 4URNER WHILE 2 LEGUMINOSARUM BACTEROIDS HAVE AN / OPTI MUM OF N- WITH REGARD TO THE GREATEST MEASURED NITROGENASE ACTIVITIES !GAIN THE EXPLANATION FOR THIS CANNOT LIE IN NITROGENASE PER SE "ASED ON THE OBSER VATIONS IT SEEMS THAT AEROBIC . lXERS MUST BALANCE THEIR RELATIONSHIP WITH / TO INCLUDE AEROBIC LIFE AS BOTH HINDRANCE TO AND A REQUIREMENT FOR EFlCIENT . lXATION 4HIS BALANCE MUST BE PRECISELY MAINTAINED FOR THE ORGANISM TO COMPETE IN ENVIRONMENTS LACKING IN lXED NITROGEN )N ADDITION TO PREVENTING / ACCESS TO NITROGENASE DIAZOTROPHS NEED TO KEEP METABOLICALLY ACTIVE IN TERMS OF REDUCTANT AND ENERGY GENERATION #LEAR EXAMPLES OF LOSS OF NITROGENASE PEPTIDES BY CARBON STARVATION OF CELLS ARE EVIDENT -OSHIRI ET AL /ELZE AND THIS COULD BE EXPLAINED BY A LACK OF ELECTRON mUX TO THE NITROGENASE PEPTIDES )T APPEARS THAT THE NITROGENASE COMPLEX IS MORE SENSITIVE TO OXYGEN INACTIVATION UPON CARBON STARVATION OF CELLS DUE TO THE LOWERED mUX OF ELECTRONS TO THE COMPLEX +UHLA AND /ELZE )T IS WELL KNOWN THAT THE ELECTRONIC NATURE OF THE METALLOCENTER CLUSTERS OF NITROGENASE ARE ALTERED BY OXIDATION REDUCTION AND THESE METAL CENTERS AT LEAST FOR THE -O&E PROTEIN OF NITROGENASE ARE THE MOST / LABILE AREAS TOO !LSO IF !40 LEVELS FOR NITROGENASE ARE DEPLETED THE ENZYMES REDUCTION SHOULD BE AFFECTED AS THE TRANSFER OF ELECTRONS FROM THE &E PROTEIN TO THE -O&E PROTEIN REQUIRES !40 "URRIS
***5IF/àYJOH3IJ[PCJVNMFHVNF4ZN CJPTJT 4HE . lXING ROOT NODULE OF LEGUMINOUS PLANTS IS FORMED FROM THE SYMBIOSIS OF VARIOUS 2HIZOBIUM BACTERIA WITH A PARTICULAR PLANT HOST 4HESE INCLUDE "RADYRHIZOBIUM -ESORHIZOBIUM 2HIZOBIUM AND 3INORHIZOBIUM ALL MEMBERS WITHIN THE FAMILY 2HIZOBIACEAE 4HE NITROGENASE ENZYME THAT CONSUMES !40 AND THE ASSOCIATED REDUCTANT PROTEINS ARE LOCATED IN THE DIFFERENTIATED BACTEROIDS WITHIN ROOT NODULES 4HESE SPECIALIZED BACTERIAL CELLS ARE ALSO THE SOURCE OF THE RESPIRATORY MACHINERY AND !40 SYNTHESIS THE LATTER MOLECULE DRIVES THE . REDUCING REACTION 4HE ENERGY FOR NITROGEN lXATION IS ULTIMATELY DERIVED FROM PLANT HOST PHOTOSYNTHATE AND HIGH CONCENTRATIONS OF CARBOHYDRATE BOTH AS STORED AND IMMEDIATE CARBON SOURCES ARE FOUND IN THE NODULES 0OOLE AND !LLA WAY 4HE CARBON SOURCES DRIVING SYMBIOTIC NITROGEN lXATION AND TRANSPORTED INTO THE BACTEROIDS ARE PRIMARILY # DICARBOXYLIC ACIDS 0OOLE AND !LLAWAY 2ESULTS FROM BACTEROID RESPIRATION EXPERIMENTS PHYSIOLOGICAL STUDIES ON MUTANTS IN DICARBOXYLIC TRANSPORT SYSTEMS AND LABELING STUDIES HAVE SHOWN THE IMPORTANCE OF THE # DICARBOXYLIC ACIDS IN INTERMEDIARY METABOLISM FOR . lXATION EN ERGY INPUT )N ADDITION TO PROVIDING CARBON SKELETONS FOR SYNTHESIS OF MACROMOLECULES THE CATABOLISM OF THESE CARBON SOURCES IS LINKED TO ENERGY GENERATION VIA RESPIRATION 4HE BACTEROID IS IN ESSENCE CARRYING OUT RIGOROUS RESPIRATORY METABOLISM TO DRIVE . lXATION )TS HABITAT THE ROOT NODULE PROVIDES PHOTOSYNTHATE CARBON AND A FREE / LEVEL OF LESS THAT N- "/PEVMFT -FHIFNPHMPCJO !LTHOUGH THEY FUNCTION AS AEROBES BACTEROID ME TABOLISM WITHIN ROOT NODULES DEPENDS BOTH ON TISSUE STRUCTURES AND MACROMOLECULES SURROUNDING THE BAC TEROIDS THAT ARE DESIGNED TO MINIMIZE THE BACTEROIDS EXPOSURE TO / 4HE IMPORTANT STEPS OF BACTEROID DIFFERENTIATION AND SYNTHESIS OF NITROGENASE AND OTHER ENZYMES ALL REQUIRE A MICROAEROBIC ENVIRON MENT "ATUT AND "OISTARD &ISHER 4HIS MICROAEROBIC ENVIRONMENT AT THE BACTEROID SURFACE IS PRIMARILY ACHIEVED BY AN INTRIGUING PROTEIN CALLED LEGHEMOGLOBIN A HEME PROTEIN RESPONSIBLE FOR SERV ING AS AN / BUFFER FOUND IN THE ROOT NODULES 3IMILAR HEMOGLOBINS OF PLANT ORIGIN HAVE ALSO BEEN IDENTIlED
3PCFSU+.BJFS IN THE NON LEGUME SYMBIOSES OF "RADYRHIZOBIUM SP AND IN THE ACTINORHIZAL SYMBIOSES OF &RANKIA WITH VARIOUS PLANT HOSTS 4HE LEGHEMOGLOBIN APOPROTEIN IS A PLANT PRODUCT AND HEME SYNTHESIS FOR THE SYM BIOSIS IS IN PART CONTRIBUTED BY THE BACTERIAL SYMBIONT /"RIAN ET AL A #HAUHAN AND /"RIAN 4HE AMINO ACID SEQUENCE OF LEGHEMOGLOBIN SHOWS IT IS HOMOLOGOUS TO ANIMAL GLOBINS IN CRITICAL REGIONS 4HIS PROMPTED !PPLEBY TO SPECULATE THAT THE ORIGIN OF LEGHEMOGLOBIN ALONG WITH ANIMAL HEMOGLOBIN ARE DESCENDED FROM A COMMON ANCESTOR /"RIAN AND -AIER )NTERESTINGLY THE ENGINEERED ALTERA TION OF " JAPONICUM !,! DEHYDRATASE FROM A -G DEPENDENT ENZYME TO A :N DEPENDENT ONE DOES NOT ALTER THE ENZYME ABILITY TO SUPPLY HEME IN SYMBIOSIS #HAUHAN AND /"RIAN ,EGHEMOGLOBIN FOUND IN ROOT NODULES FACILITATES THE DIFFUSION OF / TO THE BACTEROIDS AND BUFFERS THE FREE / CONCENTRATION IN THE NODULE 4HE DISSOCIATION BINDING CONSTANT OF SOYBEAN LEGHEMOGLOBIN IS N- SO FREE / LEVELS IN SOYBEAN NODULES IS N- WHEN THE ROOT NODULE IS AT PARTIAL OXYGENATION !PPLEBY 4HIS VALUE IS IN THE RANGE OF THE APPARENT DISSOCIATION BINDING CONSTANT ESTIMATED FOR THE EFlCIENT BACTEROID OXIDASE "ERGERSEN AND 4URNER +EEFE AND -AIER 0REISIG ET AL THUS EFlCIENT RESPIRATION CAN OCCUR AT THE VERY LOW / TENSION DEEP WITHIN THE NODULE )N OTHER WORDS THE / CAN BE POISED AT A LOW BUT READILY AVAILABLE CONCENTRATION FOR RESPIRATION IN NODULES BECAUSE LEGHEMOGLOBIN HAS A VERY HIGH AFlNITY FOR / &URTHERMORE AS THE LEGHEMOGLOBIN CONCENTRATION IN SOYBEAN NODULE CY TOSOL IS M- /"RIAN AND -AIER THE FREE / MUST BE LARGELY UNAVAILABLE EXCEPT TO THE MOST HIGH / AFlNITY OXIDASE !PPLEBY AT LEAST ONE SUCH OXIDASE IS PRESENT IN BACTEROIDS SEE BELOW 4HE HIGH AFlNITY OF LEGHEMOGLOBIN FOR / IS DUE TO A VERY FAST ASSOCIATION RATE FOR / /"RIAN AND -AIER AND A VERY SLOW / DISSOCIATION RATE 4HE / DISSOCIATION RATE CONSTANT IS NEVERTHELESS FAST ENOUGH FOR SOYBEAN LEGHEMOGLOBIN TO BE KINETICALLY COMPETENT TO CARRY / TO THE BACTEROIDS 4HIS SHOULD BE SO EVEN DURING VIGOROUS RESPIRATORY METABOLISM WHICH IS THE CASE DURING HIGH PHOTOSYNTHATE SUPPLY &ACILITATED / DIFFUSION TO PERMIT BACTEROID RES PIRATION AND !40 SYNTHESIS WAS CORRELATED TO A FUNCTION OF LEGHEMOGLOBIN WHEN 7ITTENBERG ET AL FOUND THAT THE ADDITION OF LEGHEMOGLOBIN TO ISOLATED BACTEROIDS RESULTED IN A TEN FOLD INCREASE IN NITROGENASE ACTIVITY WITH ONLY A SMALL INCREASE IN / UPTAKE ACTIVITY 4HE PROPERTIES OF THE ROOT NODULE WITH
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO ASSOCIATED / BINDING PROTEINS RESULTS IN A REMARKABLE / BUFFERING SYSTEM THE LOW FREE / CONCENTRATION CAN BE MAINTAINED DURING BOTH RAPID OR SLUGGISH BAC TEROID TERMINAL OXIDASE ACTIVITY 4HE INCREASED LOCAL FREE / GRADIENT AT THE BACTEROID SURFACE PROVIDED BY LEGHEMOGLOBIN OR OTHER / BINDING PROTEINS WAS POSTULATED TO STIMULATE / UPTAKE OF THE @HIGH AFlNITY OXIDASE /"RIAN AND -AIER 7E NOW KNOW THIS OXIDASE IS PRIMARILY A CBB TYPE SYMBIOTIC SPECIlC OXIDASE 0REISIG ET AL SEE ALSO #HAPTERS AND 6OL AND USE OF THIS OXIDASE PRESUMABLY STIMULATES ENERGY CONSERVING ELECTRON mOW /PEVMF3FTQJSBUJPOBOE0 1FSNFBCJMJUZ 0RESUMABLY TO PROTECT NITROGENASE FROM / DAMAGE LEGUME NODULES ALSO HAVE MECHANISMS TO REGULATE THEIR PERMEABILITY TO / 7ITTY ET AL (UNT AND ,AYZELL 4HE INNER CORTEX OF THE ROOT NODULE IS AN INITIAL BARRIER TO GAS DIFFUSION 4HEN IN THE RHIZO BIUM INFECTED PLANT CELLS THE / TENSION IS MAINTAINED AT n N- COMPARED WITH CELLS IN EQUILIBRIUM WITH AIR AT - +ING AND ,AYZELL $ENISON ET AL 4HIS IS NOT SOLELY ATTRIBUTED TO LEGHEMOGLO BIN BUT TO INTERCONNECTED GAS lLLED SPACES BETWEEN THE CELLS ! PHYSIOLOGICAL REGULATION OF THE VOLUME OF THESE INTERCELLULAR SPACES MAY HELP CONTROL GAS PERMEABILITY ,AYZELL ET AL &OR INTACT NODULES NITROGENASE ACTIVITY AND NODULE RESPIRATION CAN BE STIMULATED BY GRADUALLY INCREASING THE EXTERNAL PARTIAL PRESSURE (UNT ET AL OF / SO THE CAPABILITY FOR INCREASED RESPIRATION AND !40 SYNTHESIS IS EVIDENT 3PECTROSCOPIC STUDIES ON SOYBEAN NODULES INDICATES THAT THE OXYGEN BARRIER CAN LIMIT THE NITROGENASE ACTIVITY ,AYZELL ET AL 7HEN PHOTOSYNTHATE SUPPLY IS REDUCED EXPERIMENTALLY $ENISON ET AL DE ,IMA ET AL NODULE METABOLISM CAN BE MARKEDLY INCREASED BY RAISING THE EXTERNAL PARTIAL PRESSURE OF / )T IS CONCLUDED +UZMA ET AL THAT NODULE METABOLISM IS CONTINUALLY LIMITED BY / SUPPLY AND THAT NODULES CONTAIN BARRIER MECHANISMS TO DECREASE THE / SUPPLY TO INFECTED CELLS /XYGEN CONSUMPTION CAN PRESUMABLY CONTINUE EVEN WHEN PHOTOSYNTHATE IS DEPLETED DUE TO LARGE CARBON STORAGE RESERVOIRS IN BACTEROIDS $UE TO LIMITATIONS ON THE / AVAILABILITY TO BACTE ROIDS A PRESUMABLY NODULE REGULATED PROCESS ,AYZELL ET AL AEROBIC RESPIRATION IS LIMITED SO THAT !40 SUPPLY TO NITROGENASE IS IN TURN LIMITED 7ER NER +UZMA ET AL $UE TO THE LOW +OF " JAPONICUM BACTEROIDS FOR / THE HIGH AFlNITY
BACTEROID RESPIRATION WOULD LIMIT THE / AVAILABLE FOR RESPIRATION BY MITOCHONDRIA OF THE NODULE SO THAT !40 LIMITATIONS WOULD BE @SEEN IN THE PLANT CELLS BEFORE THE BACTERIAL ONES 4HE +- OF THE TERMINAL OXIDASES OF NODULE MITOCHONDRIA OF n N- 2AWSTHORNE AND ,A2UE -ILLAR ET AL IS CERTAINLY GREATER THAN FOR THE BACTERIAL TERMINAL OXIDASES 4HE ACTUAL SITE OF / LIMITATION IN NODULES IS THEREFORE OF INTEREST "Y MEASURING THE ADENYLATE POOLS AS A MEASURE OF THE HYPOXIC METABOLIC STATE OF ROOT NODULES +UZMA ET AL CONCLUDED THAT THE BACTEROIDS ARE THE SITE OF / LIMITATION FOR NODULE RESPIRATORY METABOLISM !LTHOUGH RESEARCHERS HAD PREDICTED THAT BACTEROIDS WERE THE SITE OF THE / LIMITATION OF NODULE METABO LISM $ILWORTH 7ERNER THE LATEST STUDY +UZMA ET AL PROVIDES EVIDENCE THAT THIS IS SO WITHIN INTACT NODULES 4HE MECHANISMS BY WHICH NITROGENASE ACTIVITY IS LIMITED BY / MAY BE DUE TO A LIMITATION CAUSED BY THE !40 NEEDS OF THE NITROGENASE REACTION OR DUE TO AN INHIBITION OF NITROGENASE BY ACCUMULATION OF !$0 +UZMA ET AL #&MFDUSPO5SBOTQPSUJO3IJ[PCJB $VMUVSFE$FMMT !LTHOUGH SOME RHIZOBIA ARE CAPABLE OF NITRATE RESPIRATION THE MAJORITY OF RESPIRATION BY RHIZOBIA IS THOUGHT TO BE / DEPENDENT FOR BOTH FREE LIVING AND NODULE ASSOCIATED CELLS 4HE TERMINAL OXIDASES USED BY RHIZOBIA AND OTHER AEROBES MAY DIFFER IN THE REDUCTANTS USED / AFlNITIES TYPES OF HEME AND METAL REQUIREMENTS BUT THEY ARE NEVERTHELESS RELATED MEMBERS OF A HEME COPPER SUPERFAMILY OF OXIDASES 'ARCIA (ORSMAN ET AL VAN DER /OST ET AL 4HE CHARACTERIZED TERMINAL OXIDASES OF 2HI ZOBIUM ARE WITHIN THIS FAMILY (OWEVER THE RELATIVELY LARGE NUMBER OF THEM IN " JAPONICUM IN PARTICULAR IS UNUSUAL )NITIAL SPECTRAL AND INHIBITOR STUDIES ON MEMBRANES ISOLATED FROM FREE LIVING AND BACTEROID FORMS OF " JAPONICUM IMPLICATED THE EXISTENCE OF A NUMBER OF TERMINAL OXIDASES /"RIAN AND -AIER 4HE SPECTRAL STUDIES WERE RIGHTLY INTERPRETED CAUTIOUSLY AS ARTIFACTS OF #/ BINDING COMPONENTS FOR EXAMPLE CAN COMPLICATE CLEAR INTERPRETATIONS !PPLEBY A B 7ILLIAMS ET AL 4HE TERMINAL OXIDASES CLEARLY IDENTIlED IN " JAPONICUM INCLUDE AN AA TYPE CYTOCHROME C OXIDASE A HEME B CONTAINING UBIQUINOL OXIDASE AN ALTERNATIVE HEME COPPER AEROBICALLY FUNCTIONING CYTOCHROME C OXIDASE AND A HIGH / AFlNITY CBB TYPE SYMBIOSIS SPECIlC
FUNCTIONING OXIDASE %VIDENCE FOR OTHER POSSIBLE OXIDASES THAT MAY NOT EVEN CONTAIN CYTOCHROME HAS BEEN PRESENTED /"RIAN AND -AIER &RUSTACI ET AL %LECTRON TRANSPORT IN "RADYRHIZOBIUM HAS BEEN STUDIED MOST INTENSIVELY IN " JAPONICUM AND TO A LESSER EXTENT IN "RADYRHIZOBIUM SP ,UPINUS AND IN 2 LEGUMINOSARUM $ELGADO ET AL AND 2 ETLI "ARQUERA ET AL A B 4HE COMPLEMENT OF CYTOCHROMES EXPRESSED IS SIGNIlCANTLY DIFFERENT IN BACTEROIDS COMPARED WITH CELLS GROWN IN CULTURE " JAPONICUM CULTURED CELLS CONTAIN B AND C TYPE CYTOCHROMES AND AS TERMINAL OXIDASES CYTOCHROMES AA AND O "ASED ON INHIBITOR AS WELL AS PHYSIOLOGICAL STUDIES OF MUTANTS IT IS LIKELY THAT OTHER OXIDASES PERHAPS THE ONES LACKING HEME PEPTIDES ARE PROBABLY EXPRESSED IN CULTURED CELLS !PPLEBY &RUSTACI ET AL #YTOCHROMES O AND AA FORM COMPLEXES WITH CARBON MONOXIDE IN A #/ / ATMOSPHERE VV BUT RESPIRATION IS NOT COMPLETELY INHIBITED UNDER THESE CONDITIONS !LSO IT MUST BE KEPT IN MIND THAT HEME O WAS NOT DETECTED IN " JAPONICUM MEMBRANES SO THE SPECTRAL CHARACTERISTICS ASSIGNED TO CYTOCHROME O MAY ACTUALLY BE DUE TO A BB TYPE TERMINAL OXIDASE 3URPIN ET AL 4HE AA TYPE TERMINAL OXIDASE PATHWAY EXISTS IN AEROBICALLY GROWN CELLS OF MANY RHIZOBIA )T IS SIMILAR TO THAT OF MITOCHONDRIA AND MANY AEROBIC BACTERIA 4HE PATHWAY IS DEHYDROGENASE A 1 A &E3 PROTEINBC COMPLEX A #YT AA A / ! " JAPONICUM MUTANT THAT LACKED #YT C AND #YT AA HAD RESPIRATORY PROPERTIES THAT WERE LIKE A MUTANT STRAIN LACKING ONLY THE #YT AA COMPONENT /"RIAN ET AL THIS IS SUGGESTIVE THAT CYTOCHROMES C AND AA PARTICIPATED IN THE SAME BRANCH OF A SINGLE TERMINAL OXIDASE PATHWAY /THER STUDIES ON OXIDASE DElCIENT MUTANTS ALSO INDICATED THAT #YT AA AND A MEMBRANE BOUND #YT C SHARED A TERMINAL RESPIRA TORY BRANCH TO / "OTT ET AL 3UBSEQUENTLY A MEMBRANE BOUND C TYPE CYTOCHROME OF K$A CALLED #YC- WAS POSTULATED TO BE THE INTERMEDI ARY HEME ELECTRON TRANSFER COMPONENT BETWEEN THE BC COMPLEX AND THE #YT AA FOR BOTH " JAPONICUM AND 2 LEGUMINOSARUM BIOVARR VICIAE $ELGADO ET AL "OTT ET AL 4HE PRESENCE OF #YC- WOULD HELP EXPLAIN WHY THE ISOLATION OF " JAPONICUM CYTOCHROME MUTANTS BASED ON SCREENING FOR A RESPI RATORY DElCIENCY ALWAYS YIELDED LESIONS IN BOTH THE CYTOCHROME C AND AA PART OF THE ELECTRON TRANSPORT SYSTEM /"RIAN ET AL 4HE ELECTRON TRANSPORT SYSTEM OF FREE LIVING 2 TRIFOLII IS SIMILAR TO THAT OF " JAPONICUM WITH THE BRANCH POINT AT CYTOCHROME
3PCFSU+.BJFS B AND WITH AA AND C TYPE CYTOCHROMES FORMING A BRANCH SEPARATE FROM THAT OF CYTOCHROME O $E(OL LANDER AND 3TOUTHAMER ,IKE " JAPONICUM 2 LEGUMINOSARUM BIOVAR VICIAE CONTAINS #YC- AND MUTATIONS IN THAT GENE ALSO CAUSED A LACK OF DETECTABLE #YT AA 7U ET AL 4HE #YC- OF 2 LEGUMINOSARUM 7U ET AL IS NOT NECESSARY FOR A FUNCTIONING SYMBIOSIS AND A FUNCTION FOR #YC- AS A MEMBRANE ANCHOR FOR ANOTHER HEME COMPLEX IS INDICATED !LSO 2 LEGUMI NOSARUM CAN APPARENTLY COMPENSATE FOR LOSS OF THE #YT C AND AA PATHWAY BY INCREASING THE LEVEL OF AN ALTERNATIVE OXIDASE CYTOCHROME D 7U ET AL #YTOCHROMES B C AA AND O HAVE BEEN OBSERVED IN MANY AEROBICALLY GROWN 2HIZOBIUM INCLUDING 2HIZOBIUM TRIFOLII $E(OLLANDER AND 3TOUTHAMER AND 2 LEGUMINOSARUM $ELGADO ET AL "OTH MICROORGANISMS AND 2 ETLI AS WELL "ARQUERA ET AL A EXPRESS THE UNUSUAL TERMINAL OXIDASE CYTOCHROME DD WHEN / LIMITED INTERESTINGLY " JAPONICUM DID NOT EXPRESS A CYTOCHROME D SIGNAL UNDER THE SAME CONDITIONS $ELGADO ET AL -ICROAEROBIC INCUBATION CONDITIONS CAN MIMIC THE ROOT NODULE ENVIRONMENT AND PERHAPS THE LACK OF #YT D IN " JAPONICUM IS RELATED TO THE OBSERVED DIFFER ENCES IN OPTIMAL / LEVELS FOR " JAPONICUM VERSUS 2 LEGUMINOSARUM BACTEROIDS 2HIZOBIUM TROPICI MUTANTS WITH ELEVATED TERMINAL OXIDASE ACTIVITY -ARROQUI ET AL HAD INCREASED LEVELS OF #YC- AS WELL AS OF C TYPE CYTOCHROMES AND #YT AA 4HE STRAINS HAD SUPERIOR SYMBIOTIC PERFOR MANCES ON BEAN PLANTS SO THIS TERMINAL OXIDASE PATH WAY CAN BE IMPORTANT TO THE SYMBIOSIS 4HE ENHANCED RESPIRATION AND SYMBIOTIC PERFORMANCE WERE DUE TO A MUTATION IN GLG! ENCODING A GLYCOGEN SYNTHETASE 4HESE RESULTS UNDERSCORE THE IMPORTANCE TO THE CELLS OF REGULATING CYTOCHROME OXIDASE PATHWAYS IN RE SPONSE TO CARBOHYDRATE METABOLISM OR AVAILABILITY 3UCH REGULATION IS LIKELY OCCURRING CONTINUALLY IN THE BACTEROID AS PHOTOSYNTHATE SUPPLY OR STORED CARBON RESERVES mUCTUATE IN LEVEL /THER CYTOCHROME OXIDASE MUTANTS OF 2HIZOBIUM HAVE BEEN REPORTED TO HAVE INCREASED SYMBIOTIC PERFORMANCE AS WELL 3OBERON ET AL 9URGEL ET AL 4HE ADAPTIVE RESPONSES NEEDED FOR AN ORGANISM TO ADJUST TO AN OXYGEN TENSION CHANGE FROM - AIR TO N- THE LEGHEMOGLOBIN BUFFERED CONCENTRATION AT THE BACTEROID SURFACE MUST BE NUMEROUS .OT ONLY MUST THE NEW HIGHER AFlNITY OXIDASES BE SYNTHESIZED BUT ALSO IT WOULD BE OF BENElT TO THE ORGANISM TO DOWN REGULATE THOSE OXIDASES THAT FUNCTION AT -
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO / LEVELS #ONSTITUTIVE SYNTHESIS OF THE LATTER WOULD DEPLETE VALUABLE METALS AND AMINO ACIDS NEEDED FOR OTHER SYNTHESIS /XYGEN DEPENDENT REGULATION OF TERMINAL OXIDASE SPECIlCALLY #YT AA EXPRESSION WAS DEMONSTRATED FOR " JAPONICUM THE AMOUNT OF COX! TRANSCRIPTION BASED ON PROBING 2.! SAMPLES WITH COX! BY CELLS GROWN IN PARTIAL PRESSURE / WAS FOLD LESS THAN IN FULLY AEROBIC AIR GROWN CELLS 3OME COX! MESSAGE WAS DETECTED IN BACTEROIDS TOO BUT IT WAS ONLY ABOUT OF THE LEVEL OBSERVED FOR EVEN THE LOW / ADAPTED FREE LIVING CULTURES 'ABEL AND -AIER 0ERHAPS THIS RESIDUAL MESSAGE IS DUE TO THE YET UNDIFFERENTIATED CELLS IN THE DEVELOPING ROOT NODULE STILL EXPRESSING SOME COX! MESSAGE )N CONTRAST TO " JAPONICUM THE SAME AUTHORS OBSERVED NO / DEPENDENT REGULATION OF COX! MESSAGE OR #YT AA ACTIVITY OF 2 TROPICI AND BEAN NODULE BACTEROIDS FROM 2 TROPICI CONTAINED OF THE COX! MESSAGE AS FULLY AEROBIC GROWN CELLS 'ABEL ET AL )T IS LIKELY THAT THE 2 TROPICI BEAN NODULE BACTEROIDS USE #YT AA FOR RESPIRATION AS THE #YT AA LEVELS DE TERMINED EITHER BY DIFFERENCE SPECTRA OR BY ENZYME ACTIVITY AS WELL AS THE LEVEL OF THE TRANSCRIPT WERE ALL HIGHLY SIGNIlCANT IN AMOUNT )N THIS REGARD IT IS INTERESTING TO NOTE THAT THE LEGHEMOGLOBIN BUFFERING CAPACITY OF BEAN NODULES IS LESS THAN FOR SOYBEAN SO THAT THE FREE / LEVEL WILL BE HIGHER AT THE BACTEROID SURFACE OF BEAN NODULES &OR FREE LIVING " JAPONICUM COX! MESSAGE WAS NOT INmUENCED BY COPPER ADDITION TO THE MEDIUM EVEN WHEN COMPARED TO MEDIUM TREATED TO REMOVE COPPER 'ABEL ET AL 4HIS WAS OBSERVED FOR BOTH " JAPONCIUM AND 2 TROPICI BUT A POST TRANSLATIONAL AFFECT OF COPPER ADDITION FOR BOTH OF THE FREE LIVING BACTERIA WAS OBSERVED " JAPONICUM LACKED SPECTRALLY DETECT ABLE #YT AA IN MEDIUM NEARLY DEVOID OF COPPER LESS THAN G PER L OF COPPER AND 2 TROPICI RETAINED ABOUT OF THE NORMAL COPPER SUPPLEMENTED LEVEL CYTOCHROME AA DETECTED BY DIFFERENCE SPECTRA OF MEMBRANES 7HETHER THIS DIFFERENCE BETWEEN THE TWO BACTERIA IS DUE TO A SPECIlC AFFECT ON PROVISION OF COPPER TO THE TERMINAL OXIDASE OR TO DIFFERENCES IN THE EFlCIENCY OF COPPER SEQUESTERINGTRANSPORT AT THE CELL WALL OR MEMBRANE IS NOT KNOWN 4HE SYMBIOTIC DEVELOPMENT ALSO RESULTS IN AN INCREASED OVERALL DEMAND FOR CYTOCHROME SYNTHESIS BY " JAPONICUM !CCORDINGLY / LIMITATION HAS BEEN SHOWN TO UP REGULATE THE HEME " GENE FOR !,! DEHYDRATASE IN " JAPONICUM #HAUHAN AND /"RIAN 3UCH REGULATION IS CONNECTED TO GLOBAL REGULATORS THAT CONTROL MANY OTHER GENES RELATED
TO BACTERIAL DIFFERENTIATION AND A FUNCTIONAL SYMBIOSIS 4HIS RESULT ILLUSTRATES THE KEY ROLE PLAYED BY HEME SYNTHESIS IN THE OVERALL FUNCTION OF THE VIGOROUSLY RESPIRING BACTEROID 3YMBIOTIC EXPRESSION OF CYTOCHROME AA IN SOME EFFECTIVE " JAPONICUM STRAINS WAS REPORTED AND PERHAPS THE TERMINAL OXIDASE IS REGULATED DIFFER ENTLY IN THE DIFFERENT RHIZOBIAL STRAINS -ARROQUI ET AL (IRSCH ET AL /XYGEN MAY NOT BE THE SOLE REGULATOR OF THE EXPRESSION OF THIS OXIDASE AS THE DIFFERENT STRAINS MUST PRESUMABLY EXIST AT THE SAME LEGHEMOGLOBIN BUFFERED / TENSION AS THE STRAINS WHICH DO NOT EXPRESS CYTOCHROME AA SYM BIOTICALLY &UNCTIONAL CYTOCHROMES AA HAS NOT BEEN DEMONSTRATED IN SOYBEAN NODULES AND A CYTOCHROME AA DElCIENT MUTANT AS WELL AS A COX-./0 0 THE ALTERNATIVE HEME COPPER #YT C OXIDASE DElCIENT MUTANT OF " JAPONICUM STRAIN STILL PRODUCE EFFECTIVE NODULES !LSO MUTATIONS IN CYC- THE PROPOSED ELEC TRON DONOR TO #YT AA WERE UNAFFECTED IN SYMBIOSIS "OTT ET AL 4HEREFORE IT CAN BE ASSUMED THAT OTHER OXIDASES MUST FUNCTION IN THE VARIOUS STAGES OF THE SYMBIOSIS SEE 3ECTION )))" !N INTEGRATED PICTURE OF " JAPONICUM SYMBIOTIC RESPIRATION MUST ACCOUNT FOR THE ROLES OF THE MULTIPLE TERMINAL OXIDASE COMPLEXES 4HUS FAR WE CAN ASSIGN ONE " JAPONICUM OXIDASE SOLELY TO A ROLE IN THE SYMBIOSIS AND TWO OXIDASES SPECIlCALLY TO THE FREE LIVING CONDITION 4HE OXIDASES IDENTIlED THUS FAR AS GENE CLUSTERS ARE lX ./10 0REISIG ET AL 0 COX-./0 0 "OTT ET AL COX"! "OTT ET AL : 3URPIN ET AL 'ABEL AND -AIER AND COX789: 4HE ROLES OF THESE BASED ON ANALYSIS OF MUTANTS ARE GIVEN IN THE TABLE COX"! ENCODES TWO SUBUNITS OF THE CYTOCHROME AA OXIDASE COMPLEX AND IS EXPRESSED ONLY UNDER CONDITIONS OF HIGH AERATION 'ABEL AND -AIER 'ABEL ET AL 4HE OXIDASES THAT HAVE BEEN SHOWN TO PLAY A ROLE IN FREE LIVING BUT LOW / DEPENDENT GROWTH LESS THAN - / WITH ( AS SUBSTRATE ARE THE PRODUCTS OF GENES COX-./0 0 AND COX789: : 3URPIN AND -AIER 4HESE OXIDASE GENES ENCODE COMPLEXES WITH SIMILARITIES TO #UU! CONTAINING CYTOCHROME C OXIDASES "OTT ET AL AND B TYPE UBIQUINOL OXIDASES 3URPIN ET AL RESPECTIVELY "ASED ON THE PRE DICTED PROPERTIES OF #OX789: AS WELL AS THE LACK OF DETECTABLE HEME O IN " JAPONICUM MEMBRANES IT WAS CONCLUDED THAT #OX789: IS PROBABLY A BB TYPE UBIQUINOL OXIDASE &ROM CYANIDE INHIBITION TITRATION EXPERIMENTS 3URPIN AND -AIER IT WAS CON CLUDED THAT THE OXIDASE IS EXPRESSED MICROAEROBICALLY
4HE ROLES PLAYED BY #OX789: AND #OX-./0 IN MICROAEROBIC FREE LIVING GROWTH WITH ( AS THE ELEC TRON DONOR WAS ESTABLISHED BY STUDYING MUTANTS IN THE TWO OXIDASES ! MUTANT IN EACH OF THE OXIDASES WAS STILL ABLE TO GROW VIA MICROAEROBIC RESPIRATION BUT A DOUBLE MUTANT WAS DElCIENT IN THIS GROWTH MODE AND IN (/ RESPIRATION !PPARENTLY EACH OXIDASE COULD SUBSTITUTE FOR THE OTHER 4HE PUTATIVE " JAPONICUM UBIQUINOL OXIDASE #OX789: WAS STILL FOUND TO BE IMPORTANT IN SYMBIOSIS SYMBIOTIC NITROGEN lXATION ACTIVITIES OF THE COX8 MUTANT WERE TO LESS THAN FOR THE WILD TYPE 3URPIN AND -AIER 4HE VIABLE NUMBER OF BACTERIA THAT COULD BE RE CULTURED FROM CRUSHED ROOT NODULES WAS LESS FOR THE COX8 MUTANT THAN FOR THE WILD TYPE SO A ROLE FOR THIS OXIDASE IN SURVIVAL OF THE BACTEROIDS IS IMPLICATED )T IS POSSIBLE THAT THIS OXIDASE IS EXPRESSED DURING EARLY STAGES OF THE SYMBIOSIS WHEN / CONCENTRATIONS AROUND THE DEVELOPING BACTERIAL CELLS ARE HIGH RELATIVE TO LEGHE MOGLOBIN BATHED CELLS )NTERESTINGLY FREE LIVING 2 ETLI EXPRESSES A mAVOPROTEIN OXIDASE "ARQUERA ET AL B )T IS NOT EXPRESSED IN BACTEROIDS AND HAS A LOW / AFlNITY 4ABLE SHOWS THE TERMINAL OXIDASES PRESENT IN SOME OF THE 2HIZOBIA 4HEIR KNOWN ROLES ALONG WITH THEIR BRANCH POINT IN THE TERMINAL OXIDASE SPECIlC PATHWAY IS NOT NOTED AS WELL #BDUFSPJET 4HE ROOT NODULE BACTERIA "RADYRHIZOBIUM -ESORHI
ZOBIUM 2HIZOBIUM AND 3INORHIZOBIUM WITHIN THE FAMILY 2HIZOBIACEAE HAVE THE UNIQUE ABILITY TO FORM A NITROGEN lXING SYMBIOSIS ON LEGUME ROOTS $UE TO THE SYNTHESIS OF BOTH BACTERIAL AND PLANT OXYGEN BINDING PROTEINS WITHIN THE NODULE A HIGHLY UNIQUE AND VARY ING OXYGEN ENVIRONMENT MUST BE CONSIDERED WHEN ADDRESSING RHIZOBIAL RESPIRATORY METABOLISM )NDEED THE TERMINAL OXIDASES WITH VARYING / AFlNITIES OF THE BACTERIAL PARTNER ARE LIKELY TO BE EXPRESSED AT DIF F FERENT TIMES DURING THE PROGRESSION OF THE SYMBIOSIS 4HE HIGH AFlNITY OXIDASE OF THE CBB TYPE WITH A +- FOR / OF ABOUT N- APPARENTLY ACCOUNTS FOR A LARGE PORTION OF RESPIRATORY ACTIVITY OF BACTEROIDS THAT FUNCTION IN VERY LOW FREE / LEVELS )N !ZOTOBACTERR SPECIES A SMALL REDOX ACTIVE &E3 PROTEIN PROTECTS NITROGENASE FROM / DAMAGE IN CONDITIONS WHEN THE / TENSION BECOMES ELEVATED DUE TO INSUFlCIENT RESPIRATION CAUSED BY INSUFlCIENT OXIDIZABLE CARBON SOURCE AVAILABILITY )NTERESTINGLY BRIEF TREATMENT OF INTACT SOYBEAN ROOTS WITH ATM /
3PCFSU+.BJFS DIMINISHES ACTIVITY OF NITROGENASE BUT THIS ACTIVITY IS PARTIALLY RESTORED UPON EXPOSURE TO AIR 0ATTERSON ET AL &ROM THESE AND OTHER STUDIES !PPLEBY THE POSSIBILITY THAT AN / PROTECTION MECHANISM INVOLVING ONE OR MORE PROTEINS BINDING TO NITROGENASE HAS BEEN CONSIDERED FOR THE ROOT NODULE SYMBIOSES (OWEVER USING THE CLONEDD ! VINELANDII PROTECTIVE PROTEIN AS A PROBE EVEN USING LOW STRINGENCY HY BRIDIZATION CONDITIONS -OSHIRI ET AL FOUND NO EVIDENCE FOR THE EXISTENCE OF THE SAME PROTEC TIVE TYPE &E3)) PROTEIN IN " JAPONICUM "RADYRHIZOBIUM JAPONICUM BACTEROIDS EXPRESSED CARBON MONOXIDE REACTIVE CYTOCHROMES C C 0 AND 0 THEY ARE POSSIBLY PUTATIVE OXIDASES !PPLEBY A /"RIAN AND -AIER $ELGADO ET AL (OWEVER GENETIC AND BIOCHEMI CAL STUDIES SHOWED THAT MOST OF THESE COMPONENTS ARE NOT TOO IMPORTANT TO THE SYMBIOSIS AS MUTATIONS IN THESE GENES HAVE NO EFFECT OF NITROGEN lXATION 3IMILARLY A SOLUBLE CYTOCHROME C A NON #/ RE ACTIVE CYTOCHROME WAS SHOWN NOT TO BE IMPORTANT FOR THE SYMBIOSIS BUT FOR ./ DEPENDENT RESPIRATION 4ULLY ET AL "OTT ET AL $ELGADO ET AL "RADYRHIZOBIUM SP ,UPINUS BACTEROIDS ALSO T EXPRESS CYTOCHROME 0 AS DO TO A LESSER EXTENT 2 LEGUMINOSARUM STRAIN 02% AND CULTURED CELLS OF "RADYRHIZOBIUM SP 3TRAIN ( INDUCED FOR NITROGE NASE ACTIVITY !PPLEBY 4HIS HEME CONTAINING OXIDASE COULD BE A TERMINAL OXIDASE BUT MORE LIKELY FUNCTION AS AN OXIDASE FOR OTHER METABOLIC PURPOSES -ORE RECENT BIOCHEMICAL AND GENETIC EVIDENCE FOR SOME OF THE VARIABLE / AFlNITY OXIDASES INCLUD ING A VERY HIGH / AFlNITY ONE THAT WAS POSTULATED TO EXIST MANY YEARS AGO HAS BEEN PRESENTED 4HIS IS THE OXIDASE COMPLEX ENCODED BY THE GENE CLUSTER lX ./10 0REISIG ET AL SEE BELOW 0 4HE IDENTIlCATION OF THE GENE CLUSTERS FOR TERMINAL OXIDASES AND THE GENERATION OF THE CORRESPONDING MUTANTS DO NOT MEAN WE ARE lNISHED IDENTIFYING THE SPECIlC COMPONENTS OF THE COMPLEX TERMINAL RESPI RATORY SYSTEM "ERGERSEN AND 4URNER ORIGINALLY IDENTIlED FOUR / AFlNITY STATES OF " JAPONICUM BACTEROID RESPIRATION WHICH PRESUMABLY CORRESPOND TO FOUR OXIDASES OR TO FOUR / BINDING SITES WITH DIFFERENT AFlNITIES FOR / 4HE HIGHEST / AFlNITY STATE SHOWED AN APPARENT +S VALUE OF N"ERGERSEN AND 4URNER THIS FREE / IS NEAR THAT ESTIMATED FOR THE / CONCENTRATION WITHIN A SOYBEAN NODULE !PPLEBY 4HE FACT THAT THE BACTEROID &IX./10 OXIDASE HAS A +- OF N- BOLSTERS THE ORIGINAL OBSERVATIONS OF "ERGERSEN AND 4URNER 4HERE
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#YT BO 1 5NKNOWN 4HE INDIVIDUAL #YT BDD AND #YT CBB OF !ZORHIZOBIUM CAULINODANS KNOCK OUT MUTANTS WERE BOTH REDUCED IN SYMBIOTIC . lXATION ACTIVITIES +AMINISKI ET AL "RADYRHIZOBIUM JAPONICUM KNOCK OUT MUTANTS IN COX789: AND IN : CBB lX./1/ WERE REDUCED IN SYMBIOTIC . lXATION BY ABOUT AND RESPEC TIVELY 4HE @BRANCH POINT REPRESENTS THE ELECTRON CARRIER PRIOR TO THE INDICATED TERMINAL OXIDASE THAT INITIATES AN ELECTRON mOW PATHWAY UNIQUE TO THAT OXIDASE !BBREVIATIONS BV BIOVAR 1 UBIQUINONE &E3 IRON SULFUR PROTEIN &OR A DISCUSSION ON THE FEATURES AND ROLES SEE TEXT e
FORE OTHER / AFlNITY STATES MAY ALSO BE RELEVANT TO AND CORRELATED TO IDENTIlCATION OF SPECIlC OXIDASES " JAPONICUM BACTEROID MEMBRANES WERE SHOWN TO CONTAIN THREE CYANIDE BINDING SITES /"RIAN AND -AIER SUPPORTING THE #/ BINDING DATA AND THE RESPIRATION KINETICS EXPERIMENTS FOR MULTIPLE OXI DASES .!$( DEPENDENT / UPTAKE BY " JAPONICUM THAT HAD BEEN INCUBATED MICROAEROBICALLY MAXIMUM PARTIAL PRESSURE / AND CORRESPONDING TO LESS THAN - DISSOLVED / HAD THREE #.n INHIBITION PHASES 3URPIN AND -AIER /NE OF THESE WAS ATTRIBUTED TO #YT AA AND ANOTHER INHIBITION AREA WAS COMMON TO BOTH #OX-./0 AND #OX789: 3TILL AT LEAST ONE OTHER #.n INHIBITED BUT UNIDENTIlED / UPTAKE ACTIVITY REMAINED 3URPIN AND -AIER 3TUDIES ON RESPIRATION OF WHOLE CELLS OF THE " JA PONICUM #OX MUTANTS MICROAEROBICALLY INCUBATED INDICATE THAT AT LEAST ONE MORE OXIDASE NEEDS TO BE
IDENTIlED 4HE DOUBLE MUTANT COX789:COX-./0 WAS CAPABLE OF SIGNIlCANT RESPIRATION WHEN TESTED IN - / LEVELS WHERE #YT AA WOULD BE EXPECTED TO BE NON FUNCTIONAL 4HE REMAINING OXIDASE ACTIVITY COULD BE SYMBIOSIS RELATED AS THE CELLS WERE INCU BATED INITIALLY IN VERY LOW / CONDITIONS LESS THAN - / AND WITH ( AS THE REDUCTANT FOR DAYS 3URPIN AND -AIER %VEN ANAEROBICALLY GROWN " JAPONICUM EXHIBITED TWO DISTINCT BINDING PHASES FOR / AND ONLY ONE WAS ATTRIBUTED TO THE CBB TYPE &IX./10 OXIDASE 0REISIG ET AL B'JY/021 "RADYRHIZOBIUM JAPONICUM GENES RELATED TO A lX. REGION LINKED TO NITROGENASE GENES OF 2 MELILOTI WERE CHARACTERIZED "ATUT ET AL 0REISIG ET AL 4HAT THIS ENCODED AN OXIDASE DESIGNED FOR
SYMBIOTIC RESPIRATION WAS INDICATED BY THE FOLLOWING " JAPONICUM MUTANTS WITH INSERTIONS IN lX./10 WERE . lXATION NEGATIVE AND THE OPERON WAS INDUCED IN FREE LIVING CULTURE UNDER MICROAEROBIC CONDITIONS OR ANAEROBICALLY WITH ./n AS TERMINAL ACCEPTOR 4WO C TYPE CYTOCHROME COMPONENTS OF THE COMPLEX ARE PRESUMABLY &IX/ AND &IX0 &IX1 IS LIKELY A HY DROPHOBIC MEMBRANE ANCHOR AND &IX. IS A SUBUNIT ) TYPE HEME COPPER OXIDASE 4HE OXIDASE FALLS INTO A SUBFAMILY OF THE HEME COPPER OXIDASE SUPERFAMILY VAN DER /OST ET AL 'ARCIA (ORSTMAN ET AL )T IS PRESENT IN ALL RHIZOBIA THUS FAR EXAMINED $ELGADO ET AL 4HE CBB COMPLEX ENCODED BY A lX./10 0 OPERON IS WELL SUITED TO FUNCTION MICROAEROBICALLY IN THE ROOT NODULE AND IT ACCEPTS ELECTRONS FROM &E3BC :UFFEREY ET AL .OT ALL OF THE #YT C OXIDASE ACTIVITY OF MEMBRANES FROM ANAEROBICALLY GROWN CELLS CAN BE ATTRIBUTED TO THIS OXIDASE 0REISIG ET AL SO OTHER OXIDASES ARE PROBABLY FUNCTIONING /NE CANDIDATE FOR THE REMAIN ING ACTIVITY IS #OX789: A MUTATION IN WHICH WAS : SHOWN TO ADVERSELY AFFECT THE SYMBIOSIS 3URPIN AND -AIER ! #YT C OXIDASE WITH TO SUBUNITS CONTAINING #/ REACTIVE HEME C WAS PURIlED FROM " JAPONICUM BACTEROID MEMBRANES +EEFE AND -AIER 4HIS COMPLEX UNDOUBTEDLY CORRESPONDS TO THE CBB TYPE OXIDASE ENCODED BY THE lX./10 0 GENE CLUSTER AS SOME OF THE IDENTIlED SUBUNITS AS WELL AS THE HEME CONTENT MATCH WITH THE CBB OXIDASE 4HE LATTER OXIDASE COMPLEX WAS PURIlED FROM ANAEROBI CALLY GROWN " JAPONICUM USING SOLUBILIZATION AND PURIlCATION PROCEDURES 0REISIG ET AL SIMILAR TO THOSE USED TO lRST PURIFY THE #YT B AND C CONTAINING OXIDASE COMPLEX FROM THE " JAPONICUM BACTEROIDS +EEFE AND -AIER 4HE COMPLEX CORRESPOND ING TO lX./10 0 THAT WAS PURIlED FROM " JAPONICUM BACTEROIDS +EEFE AND -AIER ALSO CONTAINED SOME OF THE BC COMPLEX SUBUNITS INDICATING A TIGHT ASSOCIATION OF THE TERMINAL OXIDASE COMPLEX WITH THE INTERMEDIARY ELECTRON TRANSFER COMPLEX 4HE +DERIVED FOR / OF THE CBB COMPLEX CORRESPONDING TO lX./10 WAS N- AND CONSISTENT WITH AN OXIDASE THAT FUNCTIONS IN THE SYMBIOSIS 4HEE lX./10 0 OPERONS OF 2 ETLII ARE HIGHLY REGULATED BY &NR FAMILY TRANSCRIPTIONAL ACTIVATORS AND ARE POISED FOR HIGH EXPRESSION IN MICROAEROBIC CONDITIONS AND THEREFORE FOR THE SYMBIOSIS ,OPEZ ET AL )N !ZOSPIRILLUM BRASILENSE A MICROAEROBICALLY INDUCED #YT CBB WAS IDENTIlED -ARCHAL ET AL )T MAY NOT BE RELATED TO . lXATION AS A #YT . MUTANT WAS
3PCFSU+.BJFS STILL CAPABLE OF . lXATION RATES ALMOST EQUAL TO THOSE OF THE WILD TYPE 2EGULATION EXPERIMENTS ARE BADLY NEEDED FOR ALL THE SYMBIONTS IN ORDER TO UNDERSTAND THE ENVIRONMENTAL REGULATORY FACTORS FOR EXPRESSION OF THE VARIOUS OXIDASES &OR EXAMPLE UNDERSTANDING THE AFFECT OF A SERIES OF CONCENTRATIONS OF VARIOUS REGULATORS LIKE / ( lXED . AND COPPER ON ALL THE OXIDASES EXPRESSION WOULD BE USEFUL $'BDUPSTBGGFDUJOHOPEVMFBOECBDUFSPJE SFTQJSBUJPO 3TUDIES OF 2 LEGUMINOSARUM BACTEROIDS (AAKER ET AL SHOWED THAT RESPIRATION RATES MAY NOT ONLY BE DETERMINED BY THE AFlNITIES OF THE TERMINAL OXIDASES BUT BY RESPIRATORY CONTROL IN EFFECT BY THE ADENYLATE POOLS )N THIS WAY BY GENERATING mUCTUATING ADENYL ATE POOLS AND REQUIRING !40 NITROGENASE IN EFFECT CAN REGULATE ITSELF 4HE ADDITION OF LEGHEMOGLOBIN TO BACTEROID SUSPENSIONS RESULTED IN LARGE INCREASES IN NITROGENASE ACTIVITY WITH ONLY MODEST INCREASES IN / CONSUMPTION 7ITTENBERG ET AL 4HIS INDICATED THAT !40 PRODUCTION IS GREATEST AT THE LOWEST / LEVELS BUT OTHER EXPLANATIONS ARE PLAUSIBLE SEE BELOW &ROM THE EARLY STUDIES ON BACTEROIDS IT WAS PRE DICTED THAT EFlCIENT RESPIRATION OCCURRED AT THE LOWEST / TENSIONS AND THAT !40 SYNTHESIS IS HIGHEST IN " JAPONICUM BACTEROIDS AT LEGHEMOGLOBIN BUFFERED / CONCENTRATIONS OF n - "ERGERSEN AND 4URNER )T APPEARED THAT RESPIRATION BECAME UNCOUPLED FROM !40 SYNTHESIS AT LEVELS OF OXYGEN ABOVE - "ERGERSEN AND 4URNER )T WAS WIDELY BELIEVED THAT UNCOUPLED ELECTRON TRANSPORT ACTED AS A @PROTECTIVE RESPIRATION MECHANISM TO MAINTAIN A LOW INTRACELLULAR / CONCENTRATION AT THE EXPENSE OF HIGH ENERGY ELECTRONS TO PREVENT INACTI VATION OF THE / LABILE NITROGENASE ENZYME -AIER ET AL (OWEVER THE MULTIPLE OXYGEN BINDING PHASES OBSERVED IN BACTEROIDS CAN BE DUE TO ARTIFACTS OF THE POLAROGRAPHIC METHOD (AAKER ET AL AND SIMPLY A CHANGE IN THE TYPE OF REDUCTANT USED BY BACTEROIDS COULD LOWER THE !40 LEVELS AT THE HIGH / CONCENTRATIONS 4HIS WOULD MAKE IT APPEAR AS IF HIGH RESPIRATORY RATES WERE LESS COUPLED TO ENERGY PRODUC TION &OR EXAMPLE IF RESPIRATION WAS ALTERED SO THAT IT IS DRIVEN BY ELECTRON DONORS ENTERING AT THE LEVEL OF 1 RATHER THAN USE OF LOWER POTENTIAL SUBSTRATES SUCH AS NITROGENASE PRODUCED ( A LOWER !40!$0 RATIO BUT HIGHER / USE WOULD BE OBSERVED 4HE BEST WAY TO DETERMINE THE FUNCTION OF THE DIFFERENT OXIDASES SEE 4ABLE WOULD BE TO RECONSTITUTE EACH INTO LIPOSOMES
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO AND MEASURE THE COUPLING EFlCIENCY 6^ AND 6P( OF EACH AS A FUNCTION OF / LEVEL 4HE EFlCIENCIES OF !40 PRODUCTION AND THUS NITRO GENASE ACTIVITIES BY BACTEROIDS WERE HYPOTHESIZED TO BE DETERMINED BY THE / AFlNITY OF THE EFlCIENT VERSUS INEFlCIENT TERMINAL OXIDASE PATHWAYS "ERGERSEN AND 4URNER (OWEVER SINCE NITROGENASE IN BACTE ROIDS CAN BE CONTROLLED BY mUCTUATING ADENYLATE POOLS AND OTHER FACTORS THAT ARE IN TURN CONTROLLED BY / CONCENTRATION SEE (AAKER ET AL THE EFlCIENT VERSUS INEFlCIENT HYPOTHESIS IS NOT NEEDED TO EXPLAIN THE EARLY RESULTS 4HE @UNCOUPLED OXIDASE HYPOTHESIS IS THEREFORE OPEN TO SCRUTINY (AAKER ET AL OBSERVED THAT NITROGENASE INHIBITION BY EXCESS / WAS ASSOCIATED WITH HIGH !40!$0 RATIOS AND THAT THE PROTON MOTIVE FORCE CONTROLS RESPIRATORY ACTIVITY AND THUS NITROGENASE TOO !40 HYDROLYSIS BY NITROGENASE MAY KEEP THE !40!$0 RATIO SUFlCIENTLY LOW TO RE LIEVE INHIBITION OF RESPIRATION BY RESPIRATORY CONTROL MECHANISMS /"RIAN AND -AIER (AAKER ET AL !T LOW N- LEVELS OF / RESPIRATION WOULD CONTINUE AND NITROGENASE WOULD BE ACTIVE REGARDLESS OF THE VARIABILITY IN COUPLING EFlCIENCY OF THE DIFFER ENT ELECTRON TRANSPORT PATHWAYS )N A RECENT STUDY " JAPONICUM BACTEROID RESPI RATION WAS INCREASED BY INCREASING THE / SUPPLY INCLUDING BY ADDING OXYLEGHEMOGLOBIN ,I ET AL )T WAS FOUND THAT MALATE TRANSPORT INTO THE CELLS WAS STIMULATED ALONG WITH RESPIRATION AND THAT THE FREE / LEVEL HAD LITTLE INmUENCE ON THE MALATE UPTAKE RATES 4HE STUDY UNDERSCORES THE IMPORTANCE OF PERFORMING / USE EXPERIMENTS VIA OXYLEGHEMOGLOBIN BY BACTEROIDS 3UCH CONDITIONS ARE NEEDED IN ORDER TO MIMIC THE NEAR ANAEROBIC NODULE SITUATION TO RELATE THE lNDINGS TO IN SITU BACTERIAROID METABOLISM *75IF'F4**1SPUFJOBOE$POGPSNBUJPOBM 1SPUFDUJPO "4IFUIOB1SPUFJOT ! PROBLEM INHERENT TO AEROBIC NITROGEN lXING OR GANISMS IS THAT / IS REQUIRED FOR !40 SYNTHESIS VIA RESPIRATORY METABOLISM YET THE NITROGENASE ENZYME IS / LABILE .ITROGEN lXING BACTERIA HAVE DEVEL OPED A NUMBER OF CREATIVE STRATEGIES TO DEAL WITH THIS PARADOX 4HE STRATEGIES MUST ENSURE A STEADY SUPPLY OF !40 DURING NITROGENASE FUNCTIONING YET PERMIT NITROGENASE TO BE PROTECTED FROM / INACTIVA TION 4HE ! VINELANDII &E3)) PROTEIN @IRON SULFUR ))
PROTEIN WAS ORIGINALLY ISOLATED AS A PINK PROTEIN PRESENT IN RELATIVELY LARGE AMOUNTS IN NITROGEN lXING CELLS 3HETHNA ET AL 3PECTROSCOPIC AS WELL AS DIRECT METAL ANALYSIS INDICATED IT CONTAINED TWO &E 3 CLUSTERS &EDIMER ,AANE ET AL 3HERINGS ET AL "ULEN AND ,E#OMPTE SHOWED THAT THE &E 3 PROTEIN WAS INTIMATELY ASSOCI ATED WITH NITROGENASE 4HE PURE NITROGENASE COMPLEX WAS COMPOSED OF !V) COMPONENT OF NITROGENASE !V)) IS COMPONENT OF NITROGENASE AND THE &E3)) PROTEIN AS SUCH NITROGENASE WAS RELATIVELY STABLE TO AIR EXPOSURE 4HE ASSOCIATION OF THE ! CHROOCOCCUM &E3)) PROTEIN WITH THE -O &E PROTEIN AND THE &E PROTEIN OF NITROGENASE IN THE FORM OF AN ISOLATABLE OXYGEN STABLE COMPLEX WAS RIGOROUSLY DEMONSTRATED BY 2OBSON 2OBSON 4HE &E3)) PROTEIN WAS FOUND TO CO PURIFY WITH THE !V) AND !V)) THROUGH A SUCROSE DENSITY GRADIENT STEP YIELDING A PREPARATION WHICH RETAINED A RELATIVELY HIGH TOLERANCE TOWARDS OXYGEN INACTIVATION 4HE &E3)) PROTEIN CANNOT PROTECT EITHER OF THE NITROGENASE COMPONENT PROTEINS IN THE ABSENCE OF THE OTHER COMPONENT PROTEIN 0OSSIBLE CLUES AS TO A REDOX BASED MECHANISM OF THE &E3)) PROTEIN HAVE COME FROM EXPERIMENTS WHICH INDICATE THAT THE &E3)) PROTEIN CAN ACCEPT ELECTRONS FROM THE &E PROTEIN ,AANE ET AL $ESPITE EVIDENCE OF AN IMPORTANT ROLE FOR THE ASSOCIATION OF THE &E3)) PROTEIN WITH AEROBIC NITROGEN lXATION IN !ZOTOBACTER THE ROLE OF THIS PROTEIN WAS ESSENTIALLY IGNORED FOR ALMOST TWO DECADES SEE -OSHIRI ET AL 4HE NITROGENASE PROTECTIVE &E3)) PROTEIN WAS lRST NAMED THE 3HETHNA PROTEIN BUT IT IS IMPORTANT TO NOTE THAT ANOTHER 3HETHNA PROTEIN RECENTLY TERMED 3HETHNA PROTEIN ) #HATELET AND -EYER IS ALSO A &E 3 PROTEIN BUT IT IS A DIFFERENT PROTEIN THAN THE PROTECTIVE 3HETHNA PROTEIN 4HE 3HETHNA PROTEIN ) USUALLY TERMED &E3) IS ENCODED WITHIN THE NIF GENE CLUSTER AND HAS HOMOLOGUES IN MANY . lXING BACTERIA #HATELET AND -EYER !ZOTOBACTER VINELANDII SYNTHESIZES AT LEAST DIFFERENT SMALL FERREDOXIN LIKE PROTEINS AND ONE RECENTLY SHOWN TO BE INVOLVED IN &E3 CLUSTER ASSEMBLY WAS CHARACTERIZED AND TERMED &E3 )6 *UNG ET AL 4HE STRUCTURAL GENE EN CODING THE ! VINELANDII &E3)) PROTEIN WAS USED FOR THE CONSTRUCTION OF STRAINS BEARING DElNED DELETIONS OF THE FES))) GENE -OSHIRI ET AL ! ROLE FOR &E3)) IN PROTECTION OF NITROGENASE WAS OBTAINED FROM EXPERIMENTS INVOLVING @ADDING BACK THE PURE &E3)) PROTEIN TO EXTRACTS FROM THE &E3)) MUTANT STRAIN AND ASSAYING THE LOSS OF NITROGENASE ACTIVITY IN / OVER A OR MIN PERIOD -OSHIRI ET AL
4HE ! VINELANDII &E3)) PROTEIN WAS OVER EXPRESSED IN % COLI AND CHARACTERIZED SPECTRALLY AND CRYSTAL LIZED -OSHIRI ET AL 4HE SPECTROSCOPIC STUD IES USING 56 VISIBLE ABSORPTION #$ AND VARIABLE TEMPERATURE -#$ %02 AND 2AMAN APPROACHES SHOWED THE &E 3 CLUSTER IS COORDINATED BY CYS TEINE RESIDUES ONLY 4HE DATA WAS UNAMBIGUOUS IN ASSIGNING ONE &E 3 CLUSTER TO EACH SUBUNIT OF THE HOMODIMER 4HE %02 PROPERTIES SHOWED THE CLUSTER IS LIKE THE PLANT TYPE CHLOROPLAST FERREDOXINS BUT THE OTHER SPECTRA JUSTIlED CLASSIlCATION OF THE &E3)) PROTEIN CLUSTER INTO THE HYDROXYLASE TYPE FERREDOXINS 4HE &E3)) PROTEIN IS THEREFORE UNUSUAL IN THAT IT HAS CHARACTERISTICS OF BOTH CLASSES OF &E 3 CLUSTER CONTAINING PROTEINS 4HE VIBRATIONAL PROPERTIES OF THE CLUSTER INDICATED IT IS NOT EXPOSED TO SOLVENT BUT IT MAY BE BURIED WITHIN THE STRUCTURE 4HE NATURE OF THE TRIGGER THAT FACILITATES ASSOCIA TIONDISSOCIATION OF THE &E3)) PROTEIN WITH NITROGENASE IS UNKNOWN BUT AN ATTRACTIVE HYPOTHESIS IS THAT THIS IS DETERMINED BY THE OXIDATION STATE OF THE CLUSTER SO THAT THE CLUSTER ACTS AS A SENSOR OF THE INTRACELLULAR REDOX POTENTIAL 4HEREFORE THE REDOX POTENTIAL OF THE CLUSTER IS A CRUCIAL DETERMINANT TO ITS FUNCTION 4HAT POTENTIAL WAS DETERMINED TO BE n M6 A VALUE SUBSTANTIALLY HIGHER THAN FOR THE CHLOROPLAST TYPES OF &E 3 CLUSTERS 4HE REDOX POTENTIAL IS IN THE RANGE OF VALUES ONE WOULD EXPECT FOR A SENSOR THAT RESPONDS TO THE OVERALL POTENTIAL WITHIN AN AEROBIC CELL -OSHIRI ET AL 3ITE DIRECTED MUTANTS OF THE &E3)) PROTEIN WERE CREATED TO TEST THE IMPORTANCE OF CERTAIN RESIDUES NEAR THE CLUSTER IN MAINTAINING THE INTEGRITY OF THE REDOX POTENTIAL ,OU ET AL 4HE MUTANT VERSIONS WERE PURIlED AND SHOWN TO RETAIN NORMAL LIKE WILD TYPE %02 AND .-2 SPECTROSCOPIC PROPERTIES -UTATION OF A HISTIDINE RESIDUE NEAR A CLUSTER LIGANDING CYSTEINE WAS SHOWN CREATE A &E3)) PROTEIN WITH LITTLE NITROGENASE PROTECTIVE ABILITY ! P+ INCREASE OF THAT HISTIDINE UPON &E 3 CLUSTER OXIDA TION IS INDICATIVE OF IONIZATION OF ANOTHER GROUP NEAR THE CLUSTER WHICH COULD MODULATE THE &E3)) PROTEINS AFlNITY FOR NITROGENASE IN A REDOX DEPENDENT MAN NER !LSO MUTATION REPLACEMENT WITH ALANINES OF TWO LYSINE RESIDUES OF &E3)) CREATED AN &E3)) PROTEIN THAT PERMITTED RAPID DEGRADATION OF NITROGENASE IN CARBON STARVED CELLS AND THIS DOUBLE LYSINE MUTANT VERSION PROVIDED ALMOST NO PROTECTIVE ABILITY WHEN ADDED TO NITROGENASE CONTAINING CELL EXTRACTS 4HESE LYSINE RESIDUES MAY BE ASSOCIATED WITH THE SURFACE OF &E3)) AND MAY PLAY A ROLE IN THE INITIAL STEPS OF
3PCFSU+.BJFS RECOGNIZING THE NITROGENASE COMPONENT PROTEINS ,OU ET AL #0YZHFOBOE/JUSPHFOBTF.FEJBUFE$FMM %FBUI "ASED ON ANALYSIS OF MUTANTS IT WAS CONCLUDED THAT THE &E3)) PROTEIN IS MOST IMPORTANT TO ! VINELANDII WHEN THE CELLS ARE CARBON SUBSTRATE DEPLETED IE WHEN RESPIRATORY PROTECTION IS INOPERABLE 7E CULTURED CELLS IN A LOW CARBON M- SUCROSE RATHER THAN THE NORMAL M- MEDIUM TO ADDRESS THE EFFECTS OF THE PRESENCE OF THE &E3)) PROTEIN ON THE SURVIVABILITY OF ! F VINELANDII -AIER AND -OSHIRI 4HIS EFFECT WAS ASSESSED BY PLATING FOR VIABLE CELL NUMBER AT HR INTERVALS AFTER THE CELLS TERMINATED THEIR EXPONENTIAL GROWTH PHASE &ROM THE RESULTS IT WAS CONCLUDED THE &E3)) PROTEIN IS IMPORTANT FOR AEROBIC SURVIVAL OF THE CELLS AND THAT MINIMIZING OXIDATIVE STRESS ASSOCIATED WITH NITROGENASE INACTIVATION MAY BE A NOVEL ROLE OF &E3)) -AIER AND -OSHIRI 4HAT AEROBIC RESPIRATORY METABOLISM IS RESPONSIBLE FOR THE PRODUCTION OF HARMFUL PARTIALLY REDUCED OXYGEN SPECIES IS WELL DOCUMENTED 4HAT METALLOEN ZYMES NOT INVOLVED IN RESPIRATORY ELECTRON TRANSPORT CAN BE THE SOURCE OF THESE OXYGEN RADICALS IN VIVO HAS ONLY RELATIVELY RECENTLY BEEN REALIZED 'AUDU ET AL )MLAY 3TORZ AND )MLAY 4HE BINDING OF OXYGEN TO OXYGEN SENSITIVE BACTERIAL RE DOX PROTEINS CAN LEAD TO SUPEROXIDE FORMATION WHEN ANAEROBICALLY GROWN CELLS ARE EXPOSED TO OXYGEN 3TORZ AND )MLAY 4HE ACCUMULATION OF HY DROGEN PEROXIDE AND SUPEROXIDE CAN LEAD TO HIGHLY DAMAGING HYDROXYL RADICALS VIA &ENTON CHEMISTRY IN THE PRESENCE OF TRANSITION METAL IONS 'ALLON 3TORZ AND )MLAY 3UCH RADICALS CAN DAMAGE A NUMBER OF CELLULAR COMPONENTS INCLUDING $.! AND THE COMPONENTS OF MEMBRANES AS %/( RADICALS WILL ATTACK VIRTUALLY EVERY MOLECULE WITHIN THE CELL !LTHOUGH AEROBIC . lXING BACTERIA HAVE INHERENT PHYSIOLOGICAL MECHANISMS TO REDUCE CONTACT OF NITRO GENASE WITH / NITROGENASE CAN REDUCE LOW LEVELS OF / TO (/ WITHOUT BECOMING INACTIVATED 4HORNELEY AND !SHBY 5PON EXPOSURE TO HIGH LEVELS OF / HOWEVER NITROGENASE WILL BE INACTIVATED DURING / REDUCTION THROUGH A PROPOSED REACTION INVOLVING THE &E PROTEIN AND SUPEROXIDE OR HYDROXYL RADICALS 4HORNELY AND !SHBY 'ALLON )T IS THOUGHT THAT NITROGENASE EVEN IN VIVO CAN GENERATE SUPEROXIDE %/( AND (/ BY REDUCING / 'ALLON
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO )T WAS DETERMINED THAT 3/$ LEVELS COULD BE VARIED BY VARYING THE SUPPLY OF IRON TO % COLI SO EXPERIMENTS WERE DONE TO MEASURE 3/$ LEVELS FOR ! VINELANDII AS A FUNCTION OF THE LEVEL OF IRON IN THE MEDIUM -AIER AND -OSHIRI )T WAS FOUND THAT 3/$ ACTIVITIES FOR BOTH THE WILD TYPE AND THE &E3)) I STRAIN WERE CONSIDERABLY LOWER IN ! VINELANDII GROWN IN &E LIMITING CONDITIONS THAN IN CELLS WITH SUFlCIENT IRON 4HE VIABILITY AS PERCENT OF THE T TIME POINT T IS THE POINT AT WHICH TIME THE CARBON SUPPLY IS LIMITED AND EXPONENTIAL GROWTH TERMINATES WAS DETERMINED AT TWO TIMES AFTER THE T TIME POINTS )N 3/$ LOWERED CULTURES THE SURVIVAL OF THE &E3)) MUTANT WAS AFFECTED MUCH MORE THAN THE WILD TYPE IN THE TIME POINTS AFTER CARBON EXHAUSTION 4HE RE SULTS RELATED THE LOSS OF VIABILITY BY LACK OF THE &E3)) PROTEIN TO LOWERED 3/$ LEVELS OR ABILITY OF THE CELLS TO DISSIPATE SUPEROXIDE -AIER AND -OSHIRI 4HUS THE CELL DEATH OBSERVED BY LACK OF &E3)) MAY BE RELATED TO INCREASES IN TOXIC SUPEROXIDE LEVELS . lXATION RELATED PROTEINS MAY GENERATE THE REACTIVE OXYGEN SPECIES /XIDATIVE STRESSES DUE TO / EXPOSURE IS A PROBLEM FOR OTHER . lXERS AS WELL SEE 'ALLON 4HE POWERFUL REDUCTANTS ASSOCIATED WITH NITROGENASE LIKE mAVODOXIN FERREDOXIN AND NITROGENASE ITSELF COULD GENERATE /n AND (/ BY PARTIALLY REDUCING / 2EACTIVE OXYGEN SPECIES ARE PRODUCED AS AN EARLY RESPONSE BY ALFALFA TO 3INORHIZOBIUM MELILOTI INFEC TION 3ANTOS ET AL IT SEEMS THE RHIZOBIA MUST COMBAT THE SAME BARRIERS PUT FORTH BY THE LEGUME TO DETER INFECTION BY OTHER MICROORGANISMS 2HIZOBIA IN MATURE ROOT NODULES ARE THOUGHT TO BE SUBJECT TO TOXIC OXIDATIVE PRODUCTS OF LEGHEMOGLOBIN REACTIONS 'ALLON )N LEGUME NODULES THE MAINTENANCE OF LEGHEMOGLOBIN IN AN @/ CARRYING STATE AS &E BY FERRIC LEGHEMOGLOBIN REDUCTASE MAY BE RESPONSIBLE FOR (/ GENERATION SEE 'ALLON (YDROGEN PEROXIDE IN TURN CAN DEGRADE LEGHEMOGLOBIN LIBER ATING FREE &E WHICH CAN GIVE RISE TO EVEN MORE DAMAGING %/( THROUGH &ENTON REACTIONS 0UPPO AND (ALLIWELL $)PNPMPHVFTPG'F4** )N COMPARING THE NUCLEIC ACID AND DEDUCED AMINO ACID SEQUENCE OF THE &E3)) PROTEIN AGAINST DATABASES THE SEQUENCES WITH THE GREATEST HOMOLOGY WITH THE &E3)) PROTEIN WAS FOUND TO BE THE PRODUCT OF THE 2 CAPSULATUS FDX$ GENE -OSHIRI ET AL ,ESS HOMOLOGY WAS SEEN BETWEEN THE &E3)) PROTEIN AND
THE &DX( PROTEINS ENCODING HETEROCYST SPECIlC FER REDOXINS FROM THE NITROGEN lXING BLUE GREEN ALGAE !NABAENA SP AND &REMYELLA DIPLOSIPHON -OSHIRI ET AL 4HE HOMOLOGOUS AREA OF THESE FERREDOXINS AND &E3)) WAS MAINLY IN THE AREAS AROUND THE CYSTEINE RESIDUES WHICH LIGATE THE &E3 CLUSTER 4HE FDX$ GENE 2 CAPSULATUS ENCODES A PROTEIN OF AMINO ACIDS INCLUDING THE INITIATOR METHIONINE AND THE DEDUCED AMINO ACID SEQUENCE OF ITS PRODUCT IS IDENTICAL WITH AN ADDITIONAL SIMILARITY TO THE &E3)) PRO TEIN WITHOUT ANY GAPS IN THEIR ALIGNMENT .OTABLY ALL FOUR OF THE CYSTEINE RESIDUES WHICH ARE PRESUMED TO BE THE LIGAND FOR THE ;&E 3= CLUSTER OF THE &E3)) PROTEIN ARE PRESENT IN THE SAME POSITION IN THE &DX$ SEQUENCE )N THE OTHER SEQUENCES WHICH WERE FOUND TO BE HOMOLOGOUS WITH &E3)) SUCH AS THE !NABAENA &DX( PROTEIN THE THREE CLOSELY SPACED CYSTEINES IE # 8 # 8 # COULD ALSO BE ALIGNED BUT THE FOURTH CYSTEINE WAS PRESENT IN RATHER VARIABLE POSITIONS IN THE REMAINING SEQUENCE )NTERESTINGLY THERE IS A DI LYSINE MOTIF WHICH IS ALSO PRESENT CLOSE TO THE . TERMINUS OF &DX$ AT POSITIONS 4HE 2 CAPSULATUS &DX$ PROTEIN WAS EXPRESSED IN % COLI AND THE PURIlED PROTEIN WAS PARTIALLY CHARACTER IZED !RMENGAUD ET AL 4HE EXPRESSED PROTEIN CALLED FERREDOXIN 6 WAS SHOWN TO BE A ;&E 3= PROTEIN WHICH WAS UNABLE TO FUNCTION AS AN ELECTRON DONOR TO NITROGENASE IN VITRO A PROPERTY CONSISTENT WITH ITS RELATIVELY HIGH MID POINT POTENTIAL OF n M6 5NAWARE OF THE SEQUENCE HOMOLOGIES BETWEEN &DX$ AND &E3)) THE &RENCH RESEARCHERS !RMENGAUD ET AL *OUANNEAU ET AL NOTED THE STRIK ING SIMILARITIES BETWEEN THE SPECTROSCOPIC PROPERTIES AND THE REDOX POTENTIAL OF &DX$ AND &E3)) BUT STILL A FUNCTION FOR &DX$ COULD NOT BE PROPOSED )T WAS NEV ERTHELESS CONCLUDED THAT &DX$ MAY PLAY A ROLE IN . lXATION IN 2 CAPSULATUS *OUANNEAU ET AL %.PEFMGPS1SPUFDUJWF1SPUFJO'VODUJPO .FDIBOJTN 4HE WORKING MODEL FOR THE OXYGEN PROTECTIVE FUNC TION OF THE &E3)) PROTEIN IN THE AEROBIC NITROGEN lXING PHYSIOLOGY OF !ZOTOBACTER IS AS FOLLOWS DURING PE R
RIODS OF OXYGEN STRESS THE &E3)) PROTEIN BY FORMING THE OXYGEN STABLE COMPLEX WITH NITROGENASE ALLOWS THE COMPLEX TO ASSUME A REVERSIBLY INACTIVE STATE 7E PREDICT THAT THE NITROGENASE COMPONENTS OF THE COMPLEX UNDERGO ONE lNAL REDOX TURNOVER WHEREBY THE -O&E AND &E PROTEIN ATTAIN AN OXIDIZED FORM AND THE &E3)) PROTEIN IS REDUCED 4HE REDUCED &E3))
PROTEIN IS AUTO OXIDIZED BY ITS REACTION WITH OXYGEN AND ALL THREE COMPONENTS REMAIN ASSOCIATED ! NUM BER OF REDOX TITRATION EXPERIMENTS PERFORMED USING DIFFERENT REDOX MEDIATORS AND VARYING OXYGEN LEVELS HAVE DEMONSTRATED THAT BOTH THE -O&E PROTEIN AS WELL AS THE &E PROTEIN CAN EXIST IN A NUMBER OF REVERSIBLE INTERMEDIATE REDUCED AND OXIDIZED STATES 7ANG ET AL )T IS LIKELY THAT IN THE INITIAL FORMATION OF THE OXYGEN STABLE COMPLEX THE &E3)) PROTEIN BY VIRTUE OF ITS INTERMEDIATE REDOX POTENTIAL CAN ACT AS A REDOX MEDIATOR BETWEEN THE -O&E AND &E PROTEINS AND OXYGEN AND MAINTAIN THESE COMPONENTS IN A STABLE OXIDIZED STATE )N THE ABSENCE OF THE &E3)) PROTEIN HOWEVER OXIDATION OF THE NITROGENASE COMPONENTS RAPIDLY PROCEEDS TO AN IRREVERSIBLY OXIDIZED INACTI VATED STATE 7ANG ET AL AND TOXIC / DERIVED PRODUCTS MAY BE GENERATED AS WELL 0ERHAPS WITH THE FORMATION OF THE TRI PARTITE COMPLEX PRODUCTION OF THESE TOXIC PRODUCTS IS ELIMINATED OR MINIMIZED (OW EVER OUR IN VITRO EXPERIMENTS USING SPIN TRAP REAGENTS IN CONJUNCTION WITH %02 HAVE BEEN UNSUCCESSFUL AT PROVIDING EVIDENCE OF GREATER NITROGENASE PRODUCED PARTIAL / REDUCTION PRODUCTS IN EXTRACTS LACKING THE &E3)) PROTEIN COMPARED TO EXTRACTS THAT CONTAINED THE &E3)) PROTEIN UNPUBLISHED OBSERVATION .EVERTHE LESS THE IN VIVO RESULTS OF CELL VIABILITY LOSS POINT TO A LINK BETWEEN A &E3)) NITROGENASE COMPLEX AND MINIMIZING / TOXICITY AND A PROBABLE ROLE FOR THE &E3)) PROTEIN IN PREVENTING SUCH TOXICITY PROBLEMS 70UIFS0 1SPUFDUJPO.FDIBOJTNT "#MPDLJOHUIF"DDFTTJCJMJUZPG.PMFDVMBS 0YZHFO ! RECENT STUDY IMPLICATED AN ALGINATE CAPSULE ON THE SURFACE OF ! VINELANDII F IN PLAYING A ROLE IN PROTECTION FROM / 3ABRA ET AL BY PREVENTING / DIFFU SION ACROSS THE CELL WALL 4HIS IS A MECHANISM THAT APPARENTLY IS UNIQUE TO PHOSPHATE LIMITED CULTURES .EVERTHELESS GROWTH IN THE PRESENCE OF AMMONIA IN HIBITS ALGINATE FORMATION AS EXPECTED FOR A PROTECTION SYSTEM FOR NITROGENASE )NTERESTINGLY GROWTH OF CELLS IN INCREASING / TENSION LED TO CONCOMITANT FORMATION OF HIGHER -7 ALGINATE AND WITH A HIGHER GLUCURONIC ACID CONTENT 4HIS MADE THE CAPSULE THICKER FURTHERING ITS FUNCTION AS AN EFFECTIVE / BARRIER #ARBON STARVATION CONDITIONS FOR EXAMPLE FOR ! VINELANDII CAUSE THE CELLS TO BE PARTICULARLY VULNERABLE TO / DAMAGE OF NITROGENASE -OSHIRI ET
3PCFSU+.BJFS AL 4HIS MAY BE DUE TO EITHER A DElCIENCY IN RESPIRATORY PROTECTION BLOCKING ACCESS OF / TO THE INTERIOR OF THE CELL OR VIA A LACK OF REDUCTANT mUX TO NITROGENASE !LSO AN ! VINELANDII MUTANT THAT HAS A LIMITED ABILITY TO ACCUMULATE POLY ` HYDROXY BUTYR ATE A 0TS0 0 MUTATION MAY BE CONCOMITANTLY LIMITED IN ITS ABILITY TO PROTECT NITROGENASE FROM / DAMAGE 3EGURA AND %SPIN 4HE INABILITY OF THE 0TS0 MUTANT TO GROW DIAZOTROPHICALLY WAS EXACERBATED BY CARBON STARVATION AND WAS RELIEVED BY SUPPLYING EITHER AMMONIUM OR GLUCOSE 4HE RESULTS INDICATE A RESPIRATORY PROTECTION PROBLEM FOR THE MUTANT BUT IT WAS ACKNOWLEDGED 3EGURA AND %SPIN THAT AN UNREALIZED INTERACTION BETWEEN &E3)) THE PROTECTIVE PROTEIN AND 0(" ACCUMULATION MAY EXIST TO ACCOUNT FOR THE RESULTS 4HE MARINE lLAMENTOUS . lXING CYANOBACTERIA 4RICHODESMIUM SPP A MAJOR PLAYER IN . lXED IN THE OCEANS DOES NOT FORM HETEROCYSTS YET lXES . WHILE CARRYING OUT / EVOLVING PHOTOSYNTHESIS :EHR ET AL #HEN ET AL 4HIS STRATEGY IS UNUSUAL AS MOST CYANOBACTERIA SEPARATE / SENSITIVE . lXATION FROM / EVOLVING PHOTOSYNTHESIS BY lXING . IN THE DARK OR IN THE SPECIALIZED CELLS WHERE PHOTOSYSTEM )) IS ABSENT 3TILL 4RICHODESMIUMS NITROGENASE PROTEINS ARE SENSITIVE TO / INACTIVATION AND THE PROTECTION MECHANISM PROPOSED INVOLVES BOTH SPATIAL AND TEM PORAL SEPARATION OF . lXATION AND PHOTOSYNTHESIS / EVOLVING TYPE 4HE PROTECTION MECHANISM USED BY 4RICHODESMIUM SPP TO lX . "ERMAN &RANK ET AL INVOLVES A BRIEF SEVERAL HR PERIOD WITHIN THE ENTIRE PHOTOPERIOD IN WHICH . lXATION CAN TAKE PLACE 4HE . lXATION RELATED EVENTS CAN TAKE PLACE ONLY WHEN 03)) IS DOWN REGULATED AND STORED CARBO HYDRATE IS USED SO THAT / CONSUMPTION VIA RESPIRA TION EXCEEDS THE RATE OF / EVOLUTION !T THIS BRIEF TIME INTERVAL THE ENVIRONMENT FOR NITROGENASE IS ESSENTIALLY ANAEROBIC AND NITROGENASE IS ACTIVE &ROM THIS INTERESTING WORK IT IS PROPOSED THAT NITROGENASE ONCE SERVED AS AN ELECTRON ACCEPTOR FOR ANAEROBIC HETEROTROPHIC METABOLISM DURING EARLY EVOLUTION OF OXYGENIC PHOTOSYNTHESIS !NOTHER ABUNDANT DIAZO TROPH IN THE OCEANS ARE THE CYANOBACTERIAL SYMBIONTS OF DIATOMS :EHR AND 7ARD 4HEY REPRESENT A SIGNIlCANT SOURCE OF lXED . IN MANY PARTS OF THE OCEAN 4HE DIATOMS CONTAINING THE HETEROCYST FORMING CYANOBACTERIA FORM LARGE AGGREGATES WHICH MAY BE ANOTHER WAY TO REDUCE DAMAGING / LEVELS 4HE OBSERVED BEHAVIOR OF !ZOSPIRILLUM BRASILENSE WHEN EXPOSED TO INCREASED / CONCENTRATIONS WHILE INCUBATED IN SOFT AGAR INDICATES THE BACTERIA NEEDS
$IBQUFS 3FTQJSBUJPOBOE/JUSPHFO'JYBUJPO TO BALANCE ITS / EXPOSURE :HULIN ET AL BE TWEEN THAT NEEDED FOR RESPIRATION AND THAT SO HIGH AS TO BE DETRIMENTAL 4HIS IS PROBABLY A REmECTION OF THE / LEVEL REQUIRED FOR EFlCIENT / RESPIRATION COUPLED WITH THE / LEVEL THAT WILL BE TOLERATED BY ITS NITROGENASE !GGREGATION IS APPARENTLY USED BY MANY CELLS INCLUDING !ZOTOBACTER VINELANDII TO LIMIT / TO THE INNER CELLS SO THAT . lXATION CAN OCCUR IN THE MICROENVIRONMENT IN THE CENTER OF THE AGGREGATE SEE 'ALLON $IAZOTROPHS CAN SEPARATE THE SITES OF . REDUCTION FROM HIGH / AREAS BY A SPATIAL SEPARA TION OF THE PROCESSES 4HE FORMATION OF HETEROCYSTS BY lLAMENTOUS CYANOBACTERIA AS THE DIFFERENTIATED AND SPECIALIZED . lXATION SITE TO AVOID / EXPOSURE PROBLEMS 0AERL OR FORMING ACTINORHIZAL VESICLES &RANKIA TO PERMIT AERO TOLERANT . lXATION WITHIN AN / BARRIER ,UNDQUIST ARE HIGHLY INTERESTING MECHANISMS 4HE &RANKIA !LNUS ROOT NODULES RESPONDED TO SUPRA ATMOSPHERIC / BY DECREASING NITROGENASE AC TIVITY AND A lXED / DIFFUSION BARRIER BY THE NODULES WAS PROPOSED ,UNDQUIST 4HAT VESICLES OF FREE LIVING &RANKIA PRODUCE THICKER CELL WALLS WHEN EXPOSED TO HIGHER / LEVELS (ARRIS AND 3ILVESTER INDICATE THE VESICLE STRUCTURE MAY BE REGULATED FOR . lXATION TOLERANCE FROM / ,IKE HETEROCYST FORMATION ANOTHER MECHANISM INVOLVING SPATIAL SEPARATION OF PROCESSES WOULD INCLUDE THE PRODUC TION OF POLYSACCHARIDE SHEATHES IN SLIME PRODUCING CONSORTIA 0HYSICAL VISCOUS BARRIERS TO / SUCH AS POLYSACCHARIDE CAPSULE PRODUCTION MAY BE A COM MON MECHANISM IN NATURE TO PREVENT / PENETRATION OR DIFFUSION INTO CELLS OR CELL AGGREGATES FOR MANY NITROGEN lXING ASSOCIATIONS SEE 'ALLON )T IS PROPOSED THAT THE / DEPLETED INTERNAL REGIONS DEEP WITHIN THE SLIME LAYER OF MARINE CONSORTIA SUPPORT THE HIGHEST . lXATION RATES 3TAL .ON HETEROCYST FORMING CYANOBACTERIA SUCH AS UNICELLULAR #LOEOTHECE AND lLAMENTOUS TYPES /SCIL LATORIA AND -ICROCOLEOUS CAN TEMPORALLY SEPARATE . lXATION OCCURS AT NIGHT FROM OXYGENIC PHOTOSYNTHE SIS OCCURS IN DAYLIGHT 4HEREFORE THE MECHANISMS FOR OCCLUDING / ACCESS TO THE NITROGENASE PEPTIDES ARE NUMEROUS )T WOULD BE MOST INTERESTING TO STUDY THE EXPRESSION OF EACH OF THESE MECHANISMS IN CONCERT WITH . lXATION GENES 4HE . lXING ENDOPHYTE OF SUGARCANE 'LUCONACE TOBACTER DIAZOTROPHICUS CAN ADAPT TO A VARIETY OF / TENSIONS APPARENTLY TO MAINTAIN NITROGENASE ACTIVITY 0AN AND 6ESSEY 2ESPIRATORY RATES OF COLONIES VARIED LITTLE IN RESPONSE TO / TENSION VARIATIONS AND
NITROGENASE ACTIVITY RETURNED TO NORMAL WITHIN MIN AFTER A HIGH / EXPOSURE 4HEREFORE A SWITCH ONOFF OF NITROGENASE IN RESPONSE TO / mUX WAS PROPOSED A MECHANISM THAT WILL PRESUMABLY INVOLVE OTHER PRO TEINS OR MOLECULES THAT INTERACT WITH NITROGENASE #3FTQJSBUPSZ1SPUFDUJPO 4HE ENERGETIC DEMANDS ASSOCIATED WITH NITROGEN lXATION REQUIRE THAT EFlCIENT ELECTRON TRANSPORT PATHWAYS FUNCTION IN AEROBIC DIAZOTROPHS (OWEVER RESPIRATORY PATHWAYS THAT PLAY PROTECTIVE ROLES BY SCAVENGING OXYGEN ARE ALSO PROPOSED TO EXIST 4HE RESPIRATORY CHAIN OF ! VINELANDII IS TERMINATED WITH EITHER A CYTOCHROME O TYPE OR A BDD TYPE OXIDASE (ADDOCK AND *ONES -AIER ET AL 4HE HEME MOIETY ASSOCIATED WITH THE CYTOCHROME O MAY BE EITHER A HEME " OR A HEME / 4HE BRANCH POINT AT THE QUINONE LEVEL THEREFORE YIELDS B A DD AND C A O TERMINAL PATHWAYS )NSTEAD OF THE COMBINED CYTOCHROME CC A O PATHWAY THAT WAS PROPOSED INITIALLY BASED ON INHIBITOR EXPERIMENTS (ADDOCK AND *ONES 9ATES SEPARATE C A O AND CYTOCHROME C A O PATHWAYS ARE CONSISTENT WITH THE SPECTRAL STUDIES ON TERMINAL OXIDASE MUTANTS OF ! VINELANDII .G ET AL 3INCE CYTOCHROME O WAS STILL REDUCED IN A CYTOCHROME C MUTANT SEPA RATE PATHWAYS TO CYTOCHROME O FROM THE INDIVIDUAL C TYPE CYTOCHROMES IS INDICATED 4HE TERMINAL OXIDASE PATHWAYS OF ! VINELANDII THEREFORE CONSIST OF THREE SEPARATE BRANCHES AND THIS mEXIBILITY UNDOUBTEDLY RELATES TO . lXATION PHYSIOLOGY 4HE COMPLEMENT OF TERMINAL OXIDASE IN !ZOTOBACTER VINELANDII AND !ZORHIZOBIUM CAULINODANS ARE INCLUDED IN 4ABLE 4HE CYTOCHROME BD D TERMINAL OXIDASE OF !ZOTO BACTER VINELANDII HAS BEEN IMPLICATED AS PLAYING ROLE IN USING OXYGEN SPECIlCALLY TO PROTECT NITROGENASE FROM / DAMAGE SEE +ELLY ET AL -OSHIRI ET AL 4HE / AFlNITY OF THIS OXIDASE WOULD BE SUFlCIENT FOR / BINDING WITHIN A LARGE RANGE OF / LEVELS +OLONAY ET AL AS WOULD BE EXPECTED FOR RESPIRATORY PROTECTION 4HE UP REGULATION OF THIS OXIDASE WITH NITROGEN lXATION GENES SUPPORTS SUCH A ROLE -OSHIRI ET AL !LSO THE CHARACTERISTICS OF A CYTOCHROME BD MUTANT IT CANNOT lX . D IN AIR IS CONSISTENT WITH A RESPIRATORY PROTECTION ROLE FOR THE BD OXIDASE PATHWAY +ELLY ET AL 4HE CYTOCHROME BDD OF ! VINELANDII IS NEVERTHELESS A COUPLED OXIDASE AS P( AND POTENTIAL GRADIENTS ARE WELL ESTABLISHED BY THE OXIDASE INCORPORATED INTO LIPOSOME +OLONAY AND -AIER !NALYSIS OF C TYPE CYTOCHROME MUTANTS
OF ! VINELANDII INDICATES THEY MAY ALSO PLAY ROLES IN RESPIRATORY PROTECTION OF NITROGEN lXATION ! GENE DIRECTED MUTANT IN BOTH CYTOCHROMES C AND C WAS UNABLE TO GROW AT / TENSIONS OF OR LESS ON . FREE MEDIUM 2EY AND -AIER 3INCE THE WILD TYPE GROWTH WAS UNAFFECTED BY THE VARIOUS / TEN SIONS THE CONCLUSION THAT ONE ROLE OF THE C DEPENDENT PATHWAYS IS TO PROVIDE FOR RESPIRATION TO SUPPORT . lXATION AT INTERMEDIATE n AND LOW BELOW / CONCENTRATIONS WAS REACHED .EVERTHELESS THESE PATHWAYS CYTOCHROMES C AO ARE STILL IMPORTANT FOR RESPIRATION IN THE ABSENCE OF NITROGEN lXATION AS THE CC DOUBLE MUTANT WAS GROWTH RETARDED IN . CONTAIN ING MEDIUM AS WELL &ROM MONITORING GROWTH OF CELLS IN EXPERIMENTAL CONDITIONS EMPLOYING BOTH LIQUID AND SOLID SUPPORT MEDIA IN CLOSED GAS SYSTEMS IT APPEARS THAT THE #YT C DEPENDENT TERMINAL PATHWAYS AUGMENT RESPIRATION TO PERMIT SURVIVAL )N THE INTERMEDIATE / LEVELS OF PARTIAL PRESSURE OF THE GAS EVENTUALLY A LOWER THRESHOLD LEVEL OF / CAN BE ACHIEVED BELOW PARTIAL PRESSURE WHERE THE #YT BD PATH FAILS TO D FUNCTION SO THAT THE WILD TYPE BACTERIUM RELIES ONLY ON THE C TYPE CYTOCHROME PATHWAYS 7ITHOUT THESE ALTERNATIVE C TYPE CYTOCHROME DEPENDENT PATHWAYS THE CELLS FAIL TO SURVIVE AND THIS IS MOST EVIDENT WHEN THE ENERGY AND RESPIRATORY DEMANDS ARE THE GREATEST IE . lXATION CONDITIONS /XYGEN LEVELS DID NOT INmUENCE THE TRANSCRIPTIONAL REGULATION OF THE TWO C TYPE CYTOCHROMES C AND C BUT THEY WERE REGULATED BY lXED . SUPPLEMENTA TION 4HE PROMOTER ACTIVITIES OF THE CYC! #YT C AND CYC" #YT C WERE UP REGULATED AND FOLD BY . STARVATION 4HE . STARVATION CONDITION USED ALSO CAUSED NITROGENASE TO BE EXPRESSED /THER GENES KNOWN TO PLAY ROLES IN !ZOTOBACTER OXYGEN PROTECTION MECHANISMS #YT DD AND THE &E3)) PRO TEIN ARE MODERATELY UPREGULATED BY CONVERSION OF THE CELLS TO NITROGEN lXATION CONDITIONS -OSHIRI ET AL (OWEVER THE C TYPE CYTOCHROMES THAT PASS ELECTRONS TO CYTOCHROME O APPEAR TO BE MORE HIGHLY . REGULATED IN CONCERT WITH NIF F GENES THAN THE OTHER ELECTRON TRANSPORT PATHWAYS OR PROTECTIVE &E3)) TYPE MECHANISMS -OSHIRI ET AL 4HE #YT C AND C TRANSCRIPT UPREGULATION THAT COINCIDES WITH lXED . STARVATION COULD BE DUE TO A FACTOR LIKE THE +LEBSIELLA NITROGEN ASSIMILATION CONTROL .!# PROTEIN THAT COUPLES TRANSCRIPTION OF m DEPENDENT PROMOTERS TO REGULATION BY THE . REGULATION SYSTEM -USE AND "ENDER 4HE SUPPORT FOR THE CONCEPT OR DOGMA OF RESPIRATORY PROTECTION OF NITROGENASE IN !ZOTOBACTER SPECIES HAS R
3PCFSU+.BJFS RECENTLY BEEN BROUGHT INTO QUESTION /ELZE 4HE HYPOTHESIS THAT HAS EXISTED FOR YEARS IS THAT / CONSUMPTION BY THE CELL MEMBRANE PROTECTS !ZO TOBACTERR NITROGENASE FROM / DAMAGE /NE OF THE TENETS OF THE CONCEPT IS THAT AS / LEVELS ARE INCREASED THE RESPIRATORY SYSTEM WILL BECOME MORE UNCOUPLED 4HIS HAS BEEN OBSERVED BUT IT IS NOT ALWAYS THE CASE /ELZE 4HE ARGUMENTS THAT CONTRADICT THE IMPORTANCE OF RESPIRATORY PROTECTION ARE BASED ON THE OBSERVATIONS THAT THE RESPIRATORY ACTIVITY IN !ZO TOBACTER VINELANDII IS CONTROLLED BY THE #. RATIO IE THE CONSUMPTION OF CARBON PER . COMPOUND USED )T WAS CONCLUDED /ELZE THAT RESPIRATORY PROTEC TION IS ONLY IMPORTANT AT THE LOWER END BELOW - OF PHYSIOLOGICAL / LEVELS !LTHOUGH THE #. RATIO MAY BE INVOLVED THERE IS CONSIDERABLE EVIDENCE THAT RESPIRATORY PROTECTION IS AN IMPORTANT MECHANISM ESPECIALLY FOR !ZOTOBACTERR SPECIES 4HIS HAS BEEN CORROBORATED BY STUDIES ON GENE DIRECTED MUTANTS R 4HAT !ZOTOBACTER RESPIRATORY ACTIVITY IS STIMULATED UNDER . lXATION CONDITIONS APPEARS TO BE A PHYSI OLOGICAL ADAPTATION TO / STRESS 2OBSON 4HE UP REGULATION OF TERMINAL OXIDASE PATHWAYS 2EY AND -AIER WITH ONSET OF NITROGENASE EXPRESSION IS ONE PIECE OF EVIDENCE THAT RESPIRATORY PATHWAYS ARE CLOSELY CORRELATED TO . lXATION IN AEROBES &ROM STUDIES ON CHEMOSTAT CULTURES OF ! VINELAN DII IT WAS CONCLUDED THAT RATHER THAN / CONSUMING RESPIRATION AS THE FACTOR TO MAINTAIN NITROGENASE ACTIVITY IN AEROBIC !ZOTOBACTER VINELANDII THE !40 SUPPLY TO NITROGENASE IS A KEY ELEMENT ,INKERHAG NER AND /ELZE )T IS IMPORTANT TO NOTE THAT . lXATION ACTIVITIES AND NOT NITROGENASE INACTIVATION WAS MEASURED SO THAT THE ABOVE STUDIES DO NOT DIRECTLY IMPACT CONCLUSIONS ON RESPIRATORY PROTECTION 4HAT !40 LEVELS ARE ALSO IMPORTANT IS INDISPUTABLE 4HE CONTROLLED GROWTH CONDITIONS TO INmUENCE NITROGENASE ACTIVITY ARE MORE LIKELY A FUNCTION OF !40 AND REDUC TANT SUPPLY TO THE ENZYME RATHER THAN PROTECTION OF NITROGENASE FROM / DAMAGE )N !NABAENA 0## RESPIRATORY PROTECTION OF NITROGENASE MAY FUNCTION IN THE HETEROCYST BY SPECIlC OXIDASES DESIGNED FOR THIS PURPOSE OF PROVIDING !40 FOR . lXATION *ONES AND (ASELKORN )N ADDITION TO TERMINAL OXIDASES OTHER ELECTRON TRANSPORT COMPONENTS ON THE REDUCING SIDE OF THE OXIDASES MAY PLAY PROTECTION ROLES &OR EXAMPLE AN ! VINELANDII MUTANT IN A NON COUPLED .!$( UBIQUI I
NONE REDUCTASE WAS SEVERELY IMPAIRED IN RESPIRATORY ACTIVITY "ERTSOVA ET AL 4HE MUTANT STRAIN WAS ABLE TO GROW lNE AT LOW AERATION OR IN THE PRESENCE
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4VNNBSZ 4HIS CHAPTER DEALS WITH THE OXIDATION OF AMMONIA .( / A (./ (/ AS A SOURCE OF REDUCING POWER IN THE CHEMOLITHOTROPHIC BACTERIUM .ITROSOMONAS EUROPAEA $IRECT KNOWLEDGE OF THE ENZYMES INVOLVED TOGETHER WITH THE SEQUENCE OF THE GENOME REVEAL CORE ELEMENTS OF A REDOX SYSTEM UNIQUE TO OXIDATION OF AMMONIA TO NITRITE WHICH FEEDS INTO A MORE TRADITIONAL BACTERIAL ELECTRON TRANSPORTTERMINAL OXIDASE SYSTEM 4HE APPARENTLY LOW STOICHIOMETRY OF PROTONS TRANSLOCATED PER AMMONIA OXIDIZED HINTS AT THE BASIS OF THE LOW GROWTH YIELDS OF THIS BACTERIUM 2EMARKABLY THE PUTATIVE COMPLEX OF HYDROXYLAMINE OXIDOREDUCTASE (!/ CYTOCHROME C #YT C AND THE MEMBRANE CYTOCHROME C- #YT C- WHICH CATALYZES THE OXIDATION OF A MOLECULE OF HYDROXYLAMINE AND TRANSFER OF FOUR ELECTRONS TO MEMBRANE UBIQUINONE WOULD INVOLVE C HEMES PER CATALYTIC SITE OR HEMES FOR THE HYPOTHETICAL AGGREGATE CONTAINING THE TRIMERIC (!/ 4HE DEHYDROGENATION CATALYZED AT THE NOVEL CATALYTIC HEME HEME 0 IS UNIQUE BY COMPARISON WITH OTHER KNOWN CATALYTIC HEMES WHICH BIND SUBSTRATE TO THE IRON IN ALL OTHERS ELECTRONS ENTER THE SYSTEM AND REDUCE THE SUBSTRATE WHEREAS THE REVERSE IS TRUE WITH (!/ 4HIS MODE OF CATALYSIS MAY BE FUNCTIONALLY RELATED TO A CROSS LINK WHICH IS FOUND ONLY IN (!/ A COVALENT BOND BETWEEN A METHYNE CARBON OF HEME 0 AND A RING CARBON OF A PEPTIDE TYRO SINE 4HE DRAMATIC CRYSTAL STRUCTURES OF (!/ AND #YT C HAVE PROVIDED INSIGHTS INTO CATALYSIS AND ELECTRON TRANSFER AS WELL AS ILLUSTRATING EVOLUTIONARY RELATIONSHIPS WHICH ARE NOT REmECTED IN HOMOLOGY OF AMINO ACID SEQUENCE #ONSIDERING THEIR RELATIVE SPATIAL ARRANGEMENT THE HEMES OF #YT C CAN BE PRECISELY SUPERIMPOSED WITH OF THE HEMES OF (!/ %VIDENCE SUGGESTS THAT THEY HAVE A COMMON ANCESTOR AND HAVE PRESERVED HEME CONlGURATIONS EVEN WHEN SEQUENCE HOMOLOGY HAD BEEN LOST 4HE NOVEL ANAEROBIC OXIDATION OF AMMONIA .( (./ A . (/ BY A PLANCTOMYCETE BACTERIUM AND THE OXIDATION OF AMMONIA TO NITRITE IN HETEROTROPHIC BACTERIA ARE DESCRIBED MORE BRIEmY !BBREVIATIONS !-/ n AMMONIA MONOOXYGENASE !.!--/8 n ANAEROBIC AMMONIA OXIDATION ##0 n CYTOCHROME C PEROXIDASE #U .I2 n COPPER CONTAINING NITRITE REDUCTASE #YT n CYTOCHROME #YT C- n MEMBRANE CYTOCHROME C- OF .ITROSOMONAS #YT AA COMPLEX n HEME AA CONTAINING TERMINAL OXIDASE #YT BC COM PLEX n UBIQUINOL CYTOCHROME C OXIDO REDUCTASE %02 n ELECTRON PARAMAGNETIC RESONANCE (!/ n HYDROXYLAMINE OXIDOREDUCTASE .(% n NORMAL HYDROGEN ELECTRODE .I2 n PENTA HEME CYTOCHROME C TYPE NITRITE REDUCTASE . SIDE n NEGATIVELY CHARGED REGION CYTOPLASMIC SIDE OF THE PLASMA MEMBRANE /2& n OPEN READING FRAME 0 SIDE n POSITIVELY CHARGED REGION PERIPLASMIC SIDE OF THE PLASMA MEMBRANE
**OUSPEVDUJPO.FUBCPMJTNPG /JUSPTPNPOBT 4HIS CHAPTER DEALS WITH THE BIOCHEMISTRY OF THE OXI DATION OF AMMONIA AS A SOURCE OF REDUCING POWER FOR ENERGY TRANSDUCTION AND BIOSYNTHESIS IN BACTERIA )T WILL FOCUS ON THE AEROBIC OXIDATION OF AMMONIA TO NITRITE .( / A (./ (/ IN THE CHEMOLITHOTROPHIC BACTERIUM .ITROSOMONAS EURO
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB PAEA SINCE THAT PROCESS HAS BEEN MOST EXTENSIVELY CHARACTERIZED 4HE BACTERIAL ANAEROBIC OXIDATION OF AMMONIA !.!--/8 PATHWAY .( (./ A . (/ AND THE OXIDATION OF AMMONIA TO NITRITE IN HETEROTROPHIC BACTERIA WILL BE REVIEWED BRIEmY 4HE OBLIGATE AEROBIC CHEMOLITHOTROPHIC AMMONIA OXIDIZING BACTERIA THOUGHT TO ACCOUNT FOR THE GREATER FRACTION OF AMMONIA OXIDATION IN NATURE ARE FOUND IN THE ` AND a SUBDIVISIONS OF THE PROTEOBACTERIA 4ESKE ET AL 4HE SPECIES IN THE ` SUBDIVISION INCLUD ING .ITROSOMONAS EUROPAEA ARE CLOSELY RELATED AND FOUND IN MARINE AND FRESH WATERS AND IN SOILS 4HE a SUBDIVISION IS REPRESENTED BY THE OBLIGATE MARINE FORM .ITROSOCOCCUS OCEANUS !LTHOUGH UNABLE TO REDUCE NITRATE TO NITRITE OR ./ TO . AT LOW CONCENTRATIONS OF / THESE ORGANISMS CATALYZE DISSIMILATORY REDUCTION OF NITRITE TO ./ AND ./ TO ./ AND MAY BE A MAJOR SOURCE OF THESE GASES IN NATURE 'OREAU ET AL !NDERSON AND ,EVINE #ONRAD 4HE OXIDATION OF AMMONIA TO NITRITE BY .ITROSOMO NAS IS THE SOLE SOURCE OF REDUCTANT FOR BIOSYNTHESIS AND FOR CREATION OF A PROTON GRADIENT FOR ENERGY TRANSDUC TION 'ROWTH OCCURS IN MEDIA CONTAINING ONLY .( AS REDUCTANT AND . SOURCE #/ AS # SOURCE 3/n AS 3 SOURCE 0/nn AND METAL IONS )N LABORATORY CULTURE THE DOUBLING TIME IS APPROXIMATELY HOURS $URING GROWTH THE MEDIA IS ACIDIlED BY NITROUS ACID 4HE POPULATION DENSITY CAN APPROACH CELLSML IN A CHEMOSTAT WITH P( CONTROL ,OGAN ET AL 4HE GENOME OF . EUROPAEA HAS BEEN SEQUENCED #HAIN ET AL AND REVEALS THAT THE ORGANISM IS HIGHLY SPECIALIZED FOR AN AUTOTROPHIC MODE OF LIFE REQUIRING GENES ENCODING THE . OXIDIZING SYSTEMS AS WELL AS BIOGENESIS OF ALL CELLULAR COMPONENTS FROM INORGANIC . # AND 3 BUT ALMOST NONE OF THE PATHWAYS FOR THE OXIDATION OF ORGANIC MOLECULES "ASED ON INFORMATION FROM THE GENOME lXATION OF #/ IS BY THE #ALVIN "ENSON CYCLE AND THE CITRIC ACID CYCLE IS PRESENT .( IS ASSIMILATED BY WAY OF GLUTAMATE DEHYDROGE NASE .ITROSOMONAS IS ABLE TO REDUCTIVELY ASSIMILATE NITRITE . BUT NOT NITRATE . INTO PROTEIN 7ALLACE AND .ICHOLAS 4HIS REVIEW DEALS WITH PATHWAYS FOR THE OXIDATION OF AMMONIA AND PRODUCTION OF REDUCING POWER .!$( AND !40 FOR BIOSYNTHESIS 0REVIOUS REVIEWS FOCUSING ON THE BIOCHEMISTRY OF THE OXIDATION OF AMMONIA AND RELATED ELECTRON TRANSFER AND ENERGY TRANSDUCTION IN .ITROSOMONAS HAVE APPEARED 7OOD 6ANNELLI ET AL (OOPER ET AL 7HIT TAKER ET AL
**4VNNBSZPGUIF0YJEBUJWF1BUIXBZT "NNPOJB.POPPYZHFOBTF )ZESPYZMBNJOF 0YJEPSFEVDUBTFBOEUIF1BTTBHFPG&MFD USPOTUP6CJRVJOPOF ""O0WFSWJFXPGUIF1SPDFTT !MMONIA IS OXIDIZED TO HYDROXYLAMINE .( En / ( A .(/( (OLLOCHER BY THE ENZYME AMMONIA MONOOXYGENASE !-/ (YDROXYL AMINE IS OXIDIZED TO NITRITE .(/( (/ A (./ En ( !NDERSSON AND (OOPER BY THE EN ZYME HYDROXYLAMINE OXIDOREDUCTASE (!/ &IG 4HE OXIDATION OF HYDROXYLAMINE MAY BE THOUGHT OF AS A DEHYDROGENATION OF SUBSTRATE SINCE ELECTRONS AND PROTONS ARE THE PRODUCT 4HE REACTION IS THE SOURCE OF ALL ELECTRONS REQUIRED FOR REDUCTIVE REACTIONS IN THE ORGANISM &OR EACH TURNOVER OF (!/ THE EQUIVALENT OF TWO ELECTRONS REMOVED FROM HYDROXYLAMINE MUST BE EXPENDED IN THE !-/ REACTION 4HE !-/ REACTION YIELDS NO ELECTRONS AND THERE IS NO EVIDENCE TO DATE THAT AS A MEMBRANE REDOX ENZYME IT CAN CONTRIBUTE TO A CHEMIOSMOTIC GRADIENT DURING TURNOVER 4HUS THE ROLE OF !-/ IS THE PRODUCTION OF THE PRIMARY ENERGY GENERATING MOLECULE HYDROXYLAMINE /NLY TWO OF THE FOUR ELECTRONS FROM THE TURNOVER OF A MOLECULE OF (!/ ARE THUS AVAILABLE FOR THE REDUCTIVE REACTIONS OF THE CELL THEY MIGHT BE CALLED @USABLE ELECTRONS /F THE TWO THEORETICAL @USABLE ELECTRONS AN ESTIMATED APPROXIMATELY TWO ELECTRONS EVERY SIX TURNOVERS PASS TO .!$ FOR USE IN THE REDUCTIVE REACTIONS OF BIOSYNTHESIS 7OOD )T IS ASSUMED THAT .!$( DEHYDROGENASE WHICH IS FOUND TO BE ENCODED IN THE GENOME #HAIN ET AL CATALYZES ENERGY DE PENDENT @REVERSE ELECTRON TRANSFER FROM UBIQUINOL TO .!$ 4HE REMAINING ELECTRON EQUIVALENTS THE USABLE PAIR OF ELECTRONS IN THE OTHER lVE TURNOVERS OF (!/ WILL ULTIMATELY PASS TO TERMINAL ELECTRON AC CEPTORS INCLUDING ONLY / NITRITE OR ./ CATALYZED BY A TERMINAL OXIDASE NITRITE REDUCTASE OR ./ REDUC TASE RESPECTIVELY UNLESS THEY SERVE AS REDUCTANT IN DETOXIlCATION OF POTENTIALLY TOXIC FORMS OF / SUCH AS (/ THE ASSIMILATORY REDUCTION OF SULFATE OR NITRITE OR THE PHYSIOLOGICALLY FUTILE OXYGENATION OF ORGANIC MOLECULES BY !-/ SEE BELOW 4HE ELECTRON TRANSFER REACTIONS RELATED TO . OXIDA TION AND ENERGY TRANSDUCTION IN .ITROSOMONAS CONSIST OF A PATHWAY UNIQUE TO AMMONIA OXIDIZING NITRIlERS IN WHICH ELECTRONS FROM HYDROXYLAMINE ARE FED INTO MEMBRANE UBIQUINONE AND THEN EITHER RETURNED TO
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!-/ OR CHANNELED INTO A SECOND @TRADITIONAL RE DOX AND ENERGY TRANSDUCING PATHWAY STARTING WITH UBIQUINOL #.FNCSBOF4ZTUFNTPG/JUSPTPNPOBT .ITROSOMONAS IS GRAM NEGATIVE AND HAS AN EXTENSIVE INTERNAL MEMBRANE SYSTEM PRODUCED BY INVAGINATION OF THE PLASMA MEMBRANE -URRAY AND 7ATSON GIVING RISE TO A LARGE SURFACE AREA OF MEMBRANE AND AN EXTENSIVE PERIPLASMIC SPACE )T IS LIKELY THAT THE ENZYMES OF ENERGY TRANSDUCTION IN .ITROSOMONAS ARE IN THE INTERNAL REGIONS OF THE MEMBRANE AND PERIPLASM (OOPER ET AL A $I3PIRITO ET AL A AS IS FOUND WITH THE ANALOGOUS ENZYMES OF METHANE OXIDATION IN -ETHYLOMICROBIUM ALBUM "RANTNER ET AL $5IF/PYJEBUJPO1BUIXBZ 4HE MAJORITY OF ENZYMES AND ELECTRON TRANSFER CARRI ERS UNIQUE TO . OXIDATION IN .ITROSOMONAS ARE NOW KNOWN AND MANY HAVE BEEN EXTENSIVELY CHARACTERIZED (!/ IS A TRIMER EACH SUBUNIT CONTAINS EIGHT C HEMES HEMES CROSSLINKED TO THE PROTEIN BY TWO THIOETHER LINKAGES TO PEPTIDE CYSTEINES /NE OF THE C HEMES THE CATALYTIC HEME 0 HAS A THIRD COVALENT BOND A CROSSLINK TO A TYROSINE OF THE PROTEIN %ACH (!/ SUBUNIT HAS A BINDING SITE FOR ITS ELECTRON ACCEPTOR THE TETRAHEME CYTOCHROME C #YT C %LECTRONS
MAY THEN PASS TO THE TETRAHEME MEMBRANE CYTO CHROME C- #YT C- AND THEN TO UBIQUINONE 4HE PUTATIVE (!/ TO UBIQUINONE ELECTRON TRANSFER PATHWAY STARTING WITH A MOLECULE OF HYDROXYLAMINE IN EACH OF THE THREE ACTIVE SITES OF (!/ INCLUDES C HEMES AND CAN IN THEORY REDUCE MOLECULES OF UBIQUINONE TO UBIQUINOL PER TURNOVER )T IS THOUGHT THAT UBIQUINOL IS THE ELECTRON DONOR TO !-/ "ECAUSE IT IS NOT ACTIVE IN EXTRACTS !-/ HAS NOT BEEN WELL CHARACTERIZED "ASED ON ANALYSIS OF THE SEQUENCE OF A GENE ENCODING A PROTEIN DERIVATIZED BY ACETYLENE A MECHANISM BASED INHIBITOR OF !-/ AND ADJOINING GENE CLUSTER !-/ APPEARS TO HAVE THREE SUBUNITS 4HE TRANSLATED PROTEINS ARE PREDICTED TO HAVE A LARGE NUMBER OF TRANSMEMBRANE DOMAINS 4HERE ARE SEVERAL COPIES OF SEVERAL OF THE GENES INVOLVED IN AMMONIA OXIDATION -C4AVISH ET AL B 4HE GENE CLUSTERS CONTAINING !-/ SUBUNITS (!/ CYTOCHROME C AND CYTOCHROME C- DO NOT SEEM TO HAVE ADDITIONAL /2&S #HAIN ET AL (ENCE ALL OR MOST OF THE PROTEINS INVOLVED WITH THE OXIDATION OF AMMONIA MAY HAVE BEEN IDENTIlED %5IFA5SBEJUJPOBM3FEPYBOE&OFSHZ5SBOT EVDJOH1BUIXBZTPG/JUSPTPNPOBT 4HE COMPONENTS OF THE @TRADITIONAL OR @UNDERLY ING 7OOD ENERGY TRANSDUCING PATHWAY IN .ITROSOMONAS HAVE PROPERTIES VERY SIMILAR TO THE EQUIVALENT ENZYMES OF HETEROTROPHIC BACTERIA !S IN
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB MANY BACTERIAL ELECTRON TRANSPORT PATHWAYS UBIQUINOL IS A MOBILE ELECTRON CARRIER AND A BRANCHING POINT TO THE !-/ SYSTEM TO THE .!$ REDUCTASE AND TO THE TERMINAL ELECTRON ACCEPTOR PATHWAYS $ZUPDISPNFCD 'ENES FOR ALL PROTEINS OF A PROTON DEPENDENT UBI QUINOL CYTOCHROME C REDUCTASE CYTOCHROME BC COMPLEX ARE FOUND IN A SINGLE GENE CLUSTER OF THE GENOME #HAIN ET AL 4HE AEROBIC OXIDATION OF EITHER HYDROXYLAMINE OR AMMONIA BY CELLS OF .I TROSOMONAS IS INHIBITED BY CLASSIC INHIBITORS OF THE UBIQUINOL CYTOCHROME C REDUCTASE SUCH AS ANTIMYCIN ! MYXOTHIAZOL OR STIGMATELLIN INDICATING THAT THE BC FUNCTIONS @DOWNSTREAM FROM (!/ IN THE ELECTRON TRANSPORT PATHWAY 7HITTAKER ET AL $ZUPDISPNFD #YTOCHROME C 9AMANAKA AND 3HINRA IS A PERIPLASMIC MONO HEME C CYTOCHROME WHICH IS HOMOLOGOUS TO THE C FAMILY OF BACTERIAL CYTO CHROMES AND MITOCHONDRIAL C )TS GENE IS PRESENT IN A SINGLE COPY WHICH IS NOT IN ANY OF THE THREE GENE CLUSTERS CONTAINING (!/ #YT C AND #YT C- #YT C IS THOUGHT TO MEDIATE ELECTRON TRANSFER BETWEEN THE #YT BC COMPLEX AND THE AA TERMINAL OXIDASE $I3PIRITO ET AL 'ENETIC OR KINETIC EVIDENCE HAS NOT ESTABLISHED THE ROLE FOR CYTOCHROME C AL THOUGH THE VALUE OF REDUCTION POTENTIAL M6 9AMANAKA AND 3HINRA IS IN KEEPING WITH THIS FUNCTION #YTOCHROME C IS ALSO ABLE TO RAPIDLY RE DUCE CYTOCHROME C PEROXIDASE !RCIERO AND (OOPER AND SLOWLY REDUCE THE COPPER CONTAINING NITRITE REDUCTASEOXIDASE $I3PIRITO ET AL B IN VITRO 4HUS DISTRIBUTION OF ELECTRONS TO ALTERNATE CARRIERS MAY ALSO BE A FUNCTION OF #YT C
AMMONIA TO NITRITE AND YET STIMULATE THE OXIDATION OF HYDROXYLAMINE TO NITRITE AS CATALYZED BY INTACT CELLS (OOPER AND 4ERRY 4HIS STRONGLY SUGGESTS THAT WHEN THE mUX OF ELECTRONS FROM UBIQUINONE THROUGH THE #YT BC COMPLEX AND THE #YT AA OXIDASE COMPLEX IS NOT RETARDED BY THE STEADY STATE CHEMIOSMOTIC GRADIENT ALL OR MOST OF ELECTRONS FROM UBIQUINOL GO TO THE TERMINAL OXIDASE PATHWAY RATHER THAN TO !-/ 7HITTAKER ET AL 4HE RESULTS INDICATE THAT IN THE ABSENCE OF UNCOUPLER !-/ CATALYSES THE RATE LIMITING STEP IN THE CONVERSION OF AMMONIA TO NITRITE 4HE PRESENCE OF ARTIlCIAL ELECTRON ACCEPTORS EG PHENAZINE METHOSULFATE OR METHYLENE BLUE (OOPER AND 4ERRY ALSO SELECTIVELY INHIBIT AMMONIA OXIDATION AND STIMULATE THE RATE OF OXIDA TION OF HYDROXYLAMINE IN WHOLE CELLS 4HIS EFFECT IS ALSO PROBABLY CAUSED BY DEPRIVING !-/ OF A mUX OF ELECTRONS )T WILL BE INTERESTING TO KNOW HOW THE CELL REGULATES .!$( DEHYDROGENASE ACTIVITY 'IVEN THE LARGE EX PENDITURE OF ENERGY REQUIRED TO REDUCE .!$ NOTED BELOW ITS RE OXIDATION BY THE ELECTRON TRANSPORT CHAIN AND TERMINAL OXIDASE WOULD RESULT IN A NET LOSS TO THE ORGANISM 'ENES ARE FOUND WHICH ENCODE ALL ENZYMES OF THE CITRIC ACID CYCLE #HAIN ET AL .O ACTIVITY OF _ KETOGLUTARATE DEHYDROGENASE WAS FOUND IN EXTRACTS (OOPER 4HIS APPARENT ABSENCE WAS ONCE RELATED TO A POSSIBLE BIOCHEMICAL BASIS OF OBLIGATE AUTOTROPHY WHICH STILL ALLOWED SYNTHESIS OF AMINO ACIDS DEPENDENT ON THE OTHER ENZYMES OF THE CYCLE )T REMAINS TO BE SEEN WHETHER ALL GENES ARE EXPRESSED )T IS LIKELY THAT MEMBRANES CONTAIN SUCCINATE DEHY DROGENASE &.JOPS&MFDUSPO5SBOTGFS1BUIXBZTUP5FSNJ OBM&MFDUSPO"DDFQUPST 0YJEBUJPOPG"MUFSOBUF4VCTUSBUFTCZ".0
$V"5FSNJOBM0YJEBTF ! #U! TYPE CYTOCHROME AA OXIDASE HAS BEEN ISO LATED $I3PIRITO ET AL AND GENES ENCODING ALL SUBUNITS HAVE BEEN IDENTIlED IN THE GENOME $POUSPMBOE*OUFHSBUJPOPG&MFDUSPO5SBOTQPSU 1BUIXBZT 0ROTONOPHORE UNCOUPLERS OF OXIDATIVE PHOSPHORYLA TION SUCH AS DINITROPHENOL OR M CHLOROCARBONYL CYANIDEPHENYLHYDRAZONE INHIBIT THE OXIDATION OF
)N VIVO AMMONIA MONOOXYGENASE OF .ITROSOMONAS IS ABLE TO OXIDIZE MANY ORGANIC COMPOUNDS (OOPER ET AL .OTE THAT TWO OF THE @USABLE ELECTRONS ARE USED PER TURNOVER OF !-/ 4HERE IS NO EXAMPLE WHERE THE PRODUCT OF THE REACTION CAN BE UTILIZED FOR ENERGY TRANSDUCTION HENCE THIS IS AN ENERGETICALLY UNPRODUCTIVE ACTIVITY :&IG "MUFSOBUF0YJEBTFT )T IS NOTABLE THAT .ITROSOMONAS HAS SO FEW OPTIONAL
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TERMINAL OXIDASES 'ENES ENCODING NO OTHER MEMBERS OF THE MEMBRANE BOUND SUPER FAMILY OF HEME COPPER RESPIRATORY OXIDASES ARE FOUND "Y (0,# ANALYSIS HEME O IS NOT PRESENT 4HE ABSENCE OF A QUINOL OXIDASE IN THIS FAMILY SUGGESTS THAT THE CONTRIBUTION TO THE PROTON GRADIENT OF THE TURNOVER OF THE #YT BC COM PLEX IS VERY IMPORTANT .ITROSOMONAS HAS A SOLUBLE PERIPLASMIC COPPER PROTEIN WHICH CATALYSES OXIDATION OF DYES BY OXYGEN OR NITRITE -ILLER AND 7OOD $I3PIRITO ET AL B )TS POSSIBLE ROLE IN REDUCTION OF DIOXYGEN AS A TERMINAL ELECTRON ACCEPTOR IS UNDER INVESTIGATION %FOJUSJàDBUJPO 4HE ./ AND ./ PRODUCED DURING OXIDATION OF AM MONIA BY .ITROSOMONAS AT LOW CONCENTRATIONS OF OXYGEN CAN ACCOUNT FOR OR MORE OF THE .( n. OXIDIZED ,IPSCHULTZ ET AL 0RODUCTION OF ./ OR ./ IN VIVO APPEARS TO OCCUR BY THE REDUCTION OF NITRITE PRODUCED BY (!/ RATHER THAN THE INCOMPLETE OXIDATION OF HYDROXYLAMINE BY (!/ 0OTH AND &O CHT (OOPER ET AL 2EMDE AND #ONRAD ! SOLUBLE ENZYME WITH NITRITE REDUCTASE AND DYE OXIDASE ACTIVITY HAS BEEN ISOLATED $I3PIRITO ET AL B )NTERESTINGLY THE GENE FOR THIS BLUE COPPER OXIDASENITRITE REDUCTASE IS IMMEDIATELY UPSTREAM OF GENES FOR A MONOHEME C CYTOCHROME AND A DIHEME C CYTOCHROME BOTH OF WHICH ARE ALSO EXPRESSED AND SOLUBLE AND THEN A NIR+ TYPE GENE FOR A COPPER CONTAINING NITRITE REDUCTASE 4HE ROLE OF THE PROTEINS ENCODED BY THIS @NIR+GENE CLUSTER IS
UNDER INVESTIGATION "EAUMONT ET AL CURRENT SPECULATION IS THAT BOTH ENZYMES CAN CONTRIBUTE TO DENITRIlCATION )N KEEPING WITH PHYSIOLOGICAL OBSER VATIONS GENES ENCODING A ./ REDUCTASE ARE IN THE GENOME WHEREAS GENES FOR NITRATE OR ./ REDUCTASE ARE NOT #HAIN ET AL 1PTTJCMF#ZQBTTPGUIF$ZUCD $PNQMFY )N VIVO INHIBITION OF THE OXIDATION OF AMMONIA OR HYDROXYLAMINE BY ANTIMYCIN ! MYXOTHIAZOL OR STIGMATELLIN IS NOT COMPLETE (ENCE UNDER CERTAIN CIRCUMSTANCES A SMALL FRACTION OF THE mUX OF ELECTRONS FROM (!/ CAN POSSIBLY BYPASS THE #YT BC COMPLEX !LTHOUGH THE 8 RAY STRUCTURE OF CYTOCHROME CSHOWS A DOCKING SITE FOR (!/ BUT NOT FOR CYTOCHROME C )VERSON ET AL IN VITRO ELECTRON mOW FROM HYDROXYLAMINE TO CYTOCHROME C IS EXTREMELY RAPID IN THE PRESENCE OF CATALYTIC AMOUNTS OF (!/ AND CYTOCHROME C 9AMANAKA AND 3HINRA 4HIS COULD IN THEORY PROVIDE A BYPASS OF THE BC COMPLEX (OWEVER THE RESPONSE OF CELLS TO INHIBITORS OF THE BC COMPLEX INDICATES THAT THIS ACTIVITY IS NOT NEARLY AS RAPID IN VIVO AS IN VITRO ! SECOND CANDIDATE FOR A BYPASS OF THE #YT BC COMPLEX WOULD BE ELECTRON TRANSFER THROUGH A CYTOCHROME C LIKE PROTEIN WHICH IS PRESENT IN MEMBRANES OF .ITROSOMONAS AT ON A MOLAR BASIS RELATIVE TO THE CORE PROTEINS OF . OXIDATION 7HITTAKER ET AL %XAMPLES OF CYTOCHROME C IN OTHER ORGANISMS ARE THOUGHT TO PARTICIPATE IN ELECTRON TRANSFER FROM UBIQUINOL TO CYTOCHROMES AS PART OF A PATH LEADING TO A TERMINAL OXIDASE
&IG !LTERNATE FATES OF THE HYPOTHETICAL @USABLE TWO ELECTRONS FROM THE (!/ REACTION @5SABLE REFERS TO THE OF ELECTRON EQUIVALENTS PER CATALYTIC CYCLE OF (!/ WHICH ARE NOT OBLIGATORILY CONSUMED BY THE !-/ REACTION $ASHED LINES INDICATE ELECTRON TRANSFER STEPS WHICH HAVE NOT BEEN CONCLUSIVELY ESTABLISHED BY EXPERIMENT
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB .PMBS3BUJPPG3FEPY$PNQPOFOUT 4HE RELATIVE AMOUNTS OF ELECTRON TRANSFER COMPONENTS ARE APPROXIMATELY (!/ SUBUNIT CYTOCHROME C CYTOCHROME C- UBIQUINONE #YT BC COMPLEX TOGETHER WITH ./ REDUCTASE CYTOCHROME C CYTOCHROME AA 7HITTAKER ET AL )T IS INTRIGUING THAT THERE ARE ROUGHLY EQUAL AMOUNTS OF CYTOCHROME C CYTOCHROME C- #YT BC COMPLEX AND CYTOCHROME AA PER SUBUNIT OF (!/ )N CONTRAST THE MOBILE ELECTRON CARRIERS MEMBRANE UBIQUINONE AND THE SOLUBLE PERIPLASMIC CYTOCHROME C ARE IN CONSIDERABLE EXCESS 4HE EQUAL AMOUNTS OF CYTOCHROME C AND CYTOCHROME C- PER SUBUNIT OF (!/ PROMPT SPECULATION THAT THESE COMPONENTS MIGHT FORM A STOICHIOMETRIC AGGREGATE RADIATING FROM THE (!/ TRIMER '0UIFSSFEPYQSPUFJOT /JUSPTPDZBOJO .ITROSOCYANIN IS A NOVEL SOLUBLE RED COPPER PROTEIN LOCATED IN THE PERIPLASM OF .ITROSOMONAS !RCIERO ET AL )T IS FOUND IN QUANTITIES EQUIVALENT TO (!/ AND SO IS THOUGHT TO POSSIBLY HAVE A ROLE IN A CENTRAL . OXIDATION PATHWAYS "ASED ON ITS PRIMARY AND TER TIARY STRUCTURE IT BELONGS TO THE CUPREDOXIN FAMILY OF REDOX PROTEINS ,IEBERMAN ET AL (OWEVER IT DIFFERS FROM OTHER CUPREDOXINS IN BINDING COPPER IN A @TYPE SITE HAVING AN OPEN COPPER COORDINATION SITE 4HUS THE POSSIBILITY OF EITHER AN ELECTRON TRANSPORT OR CATALYTIC ROLE IS BEING CONSIDERED $ZUPDISPNF1 #YTOCHROME 0 A SOLUBLE PERIPLASMIC MONO HEME C CYTOCHROME WITH HYDROXYLAMINE OXIDIZING ACTIVITY IS ALSO FOUND IN SMALL QUANTITY %RICKSON AND (OOPER .UMATA ET AL "ERGMANN AND (OOPER B )T IS CHARACTERIZED BY AN OPTICAL SPECTRUM WITH A BROAD 3ORET BAND AT NM IN THE FERRIC FORM AND NM IN THE FERROUS FORM _ AND ` ABSORPTION BANDS OF TYPICAL C HEMES ARE NOT SEEN WHEREAS THE HEME 0 OF (!/ IS A C HEME ALSO COVALENTLY BOUND TO A PEPTIDE TYROSINE THE C HEME OF CYTOCHROME 0 IS CROSS LINKED TO A PEPTIDE LYSINE !RCIERO AND (OOPER )TS PHYSIOLOGICAL ROLE IS NOT KNOWN 2ECENT WORK SUGGESTS THAT ITS ROLE IS IN THE DETOXIlCATION OF ./ OR RELATED COMPOUNDS )TS SEQUENCE IS HOMOLOGOUS TO THAT OF A CYTOCHROME 0
WITH SIMILAR OPTICAL PROPERTIES AND REACTIVITY WITH . OXIDES FOUND IN THE METHANE OXIDIZING BACTERIA "ERG MANN ET AL 3IGNIlCANTLY THE LATTER ALSO HAS GREAT SEQUENCE HOMOLOGY WITH CYTOCHROME C| FROM THE SAME ORGANISM A PROTEIN WHOSE ROLE IS THOUGHT TO BE SEQUESTRATION OF ./ &URTHER REPLACEMENT OF THE CROSSLINKED LYSINE WITH !RG !LA OR 4YR RESULTS IN HETEROLOGOUSLY EXPRESSED MUTANT PROTEIN WITH LIGAND BINDING ABILITY WITHOUT CATALYTIC ACTIVITY AND WITH OPTICAL ABSORPTION PROPERTIES DISSIMILAR TO THE WILD TYPE PROTEIN BUT RESEMBLING THOSE OF CYTOCHROME C| "ERGMANN AND (OOPER %JIFNF$ZUPDISPNFD1FSPYJEBTF .ITROSOMONAS CONTAINS A PERIPLASMIC DIHEME CYTO CHROME C PEROXIDASE WHICH MAY SCAVENGE HYDROGEN PEROXIDE !RCIERO AND (OOPER #YT C SERVES AS AN EFFECTIVE ELECTRON DONOR 4HE DI FERRIC FORM OF THE DI HEME CYTOCHROME C PEROXIDASE OF .ITRO SOMONAS HAS THE NOVEL ABILITY TO REACT WITH (/ TO FORM A STABLE FERRYL OXYGEN DERIVATIVE COMPOUND ) AND (/ 4HIS CONTRASTS WITH THE MORE COMMONLY OBSERVED TYPE OF DIHEME CYTOCHROME C PEROXIDASE FROM 0 AERUGINOSA WHICH REQUIRES REDUCTION OF THE HIGH POTENTIAL ELECTRON TRANSFER HEME BEFORE THE CATA LYTIC HEME BECOMES REACTIVE WITH (/ AND CAN THEN FORM COMPOUND ) #OMPARISON OF THE CRYSTAL DERIVED STRUCTURES OF THE TWO PROTEINS &IG INDICATES THAT A HISTIDINE IS THE SIXTH AXIAL LIGAND OF THE CATALYTIC HEME OF THE ENZYME FROM 0 AERUGINOSA WHEREAS IN THE ENZYME FROM .ITROSOMONAS THE LOOP CONTAINING THE HOMOLOGOUS HISTIDINE IS HELD AWAY FROM THE HEME THUS PROVIDING ACCESS OF PEROXIDE TO THE REACTIVE IRON 3HIMIZU ET AL 4HIS HISTIDINE CONTAINING LOOP OF THE ENZYME FROM 0 AERUGINOSA IS PRESUMED TO FOLD AWAY UPON REDUCTION OF THE ELECTRON TRANSFER HEME TO ALLOW CATALYSIS TO COMMENCE )T IS POSSIBLE THAT THIS PROPERTY OF THE ENZYME FROM .ITROSOMONAS ALLOWS SEQUESTRATION OF (/ AT RELATIVELY HIGH STEADY STATE REDOX POTENTIALS WITHIN THE CELL &ULL REDUCTION OF THE SECOND OXYGEN AND COMPLETION OF TURNOVER OF ##0 OF .ITROSOMONAS STILL REQUIRES THE ENTRY OF TWO ADDITIONAL ELECTRONS (1SPUPO(SBEJFOU (FOFSBUJPOPGUIF(SBEJFOU 4HE STOICHIOMETRY OF PROTON GRADIENT GENERATED PER ELECTRON PAIR FROM HYDROXYLAMINE IS HARD TO PREDICT
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&IG #OMPARISON OF THE STRUCTURES OF THE RESTING OXIDIZED DIHEME CYTOCHROME C PEROXIDASES ##0 OF .ITROSOMONAS EUROPAEA AND 0SEUDOMONAS AERUGINOSA 4HE TWO ENZYMES HAVE SIMILAR DOMAINS ENCLOSING A LOW POTENTIAL ,0 CATALYTIC C HEME AND HIGH POTENTIAL (0 ELECTRON TRANSFERING C HEME (IS OF THE 0SEUDOMONAS ENZYME IS THE SIXTH AXIAL LIGAND OF THE FERRIC ,0 HEME WHEREAS THE LOOP CONTAINING THE HOMOLOGOUS (IS OF THE .ITROSOMONAS ENZYME IS RELATIVELY DISPLACED RESULTING IN AN OPEN CATALYTIC SITE AND PENTA COORDINATE CATALYTIC C HEME
)T IS FAIR TO ASSUME THAT OXIDATION OF UBIQUINOL BY THE CYTOCHROME BC COMPLEX AND CYTOCHROME OXIDASE WOULD YIELD THE NET TRANSLOCATION OF SIX PROTONS FROM . TO 0 SIDE &IG 4HE REACTION OF (!/ RELEASES FOUR PROTONS ON THE 0 SIDE TWO PER UBIQUINONE REDUCED 4HE IONIZA TION OF NITRITE MIGHT BE CONSIDERED AS CONTRIBUTING A lFTH PROTON (OWEVER THE ACIDIlCATION OF THE MEDIA DURING GROWTH IS APPROXIMATELY STOICHIOMETRIC WITH NITRITE PRODUCTION INDICATING THAT THIS SHOULD NOT BE CONSIDERED AN ENERGETICALLY USEFUL PROTON IE ONE WHICH COULD BE TRANSLOCATED TO . SIDE DURING TURNOVER OF AN ENZYME SUCH AS !40 SYNTHASE 4HE NET TRANS LOCATION OF PROTONS IN THE REDUCTION OF UBIQUINONE BY ELECTRONS FROM HYDROXYLAMINE WOULD DEPEND ON THE MEMBRANE LOCATION OF THE SITE OF REDUCTION OF AND UPTAKE OF PROTONS BY UBIQUINONE #YTOCHROME C- IS ATTACHED TO THE 0 SIDE OF THE MEMBRANE BY AN . TERMINAL TRANSMEMBRANE _ HELIX )F THE GLOBULAR TETRAHEME REGION LIES ON THE MEMBRANE SURFACE AND REDUCES UBIQUINONE DURING CATALYSIS PROTONS WOULD BE ABSORBED INTO MEMBRANE QUINOL ON THE 0 SIDE )N THIS CASE THE NET PROTON TRANSLOCATION DURING THE REDUC TION OF UBIQUINONE BY ELECTRONS FROM HYDROXYLAMINE WOULD BE )NTERESTINGLY THIS ORIENTATION OF #YT C- COULD ALLOW THE REDUCTION OF UBIQUINONE n M6 BY THE M6 HEMES OF CYTOCHROME C !RCIERO ET AL B TO BE FACILITATED BY THE STEADY STATE TRANSMEMBRANE CHARGE DIFFERENTIAL )F ON THE OTHER
HAND CYTOCHROME C- PROJECTS INTO THE MEMBRANE FAR ENOUGH TO REDUCE UBIQUINONE ON THE . SIDE TWO . SIDE PROTONS COULD BE TAKEN UP )N THAT CASE THE REDUCTION OF ONE MOLECULE OF UBIQUINONE BY TWO ELEC TRONS FROM (!/ WOULD RESULT IN THE EQUIVALENT OF THE TRANSLOCATION OF TWO PROTONS FROM . TO 0 SIDE )T IS NOT KNOWN WHETHER NET TRANSLOCATION OF PROTONS OCCURS IN THE !-/ REACTION )T DEPENDS ON WHETHER THE RELEASE OF TWO PROTONS PRODUCED IN THE OXIDATION OF UBIQUINOL UBIQUINOL A UBIQUINONE En ( AND THE UPTAKE OF THE SUBSTRATE PROTONS OF THE MONOOXY GENASE REACTION .( / En ( TAKE PLACE ON THE 0 OR . SIDE 4HE LARGE NUMBER OF TRANSMEMBRANE _ HELICES PREDICTED IN !-/ SEE !-/ BELOW HAVE THE POTENTIAL TO PROVIDE THE SCAFFOLDING TO SUPPORT A SYSTEM FOR MOVING EITHER PROTONS OR ELECTRONS ACROSS THE MEMBRANE AND COULD THEORETICALLY SUPPORT SEVERAL SCENARIOS 2ELEASE OF TWO PROTONS ON THE 0 SIDE AND UPTAKE OF TWO PROTONS ON THE . SIDE WOULD RESULT IN THE EQUIVALENT OF THE TRANSLOCATION OF TWO PROTONS TO THE 0 SIDE WHEREAS THE REVERSE ORIENTATION OF THE REACTIONS WOULD RESULT IN A NET TRANSLOCATION OF TWO PROTONS TO THE . SIDE )NTERESTINGLY THE LATTER ORIENTA TION OF REACTIONS WOULD ALLOW THE !-/ REACTION TO BE DRIVEN BY THE STEADY STATE TRANSMEMBRANE CHARGE DIFFERENTIAL )F THIS WERE SO IT COULD ACCOUNT FOR THE GREAT SENSITIVITY OF THE !-/ REACTION TO UNCOUPLERS )F BOTH THE PROTON YIELDING AND PROTON UTILIZING REAC TIONS OF !-/ ARE ON THE SAME SIDE OF THE MEMBRANE
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB THE NET TRANSLOCATION OF PROTONS WOULD BE ZERO 5NTIL MORE IS KNOWN REGARDING THE HYDROXYLAMINE TO UBIQUINONE STEPS AND THE !-/ REACTION ONE CAN ONLY GUESS THE NUMBER OF PROTONS WHICH ARE TRANSLO CATED TO THE 0 SIDE PER TRANSFER OF FOUR ELECTRONS FROM .(/( TWO OF WHICH THEN REDUCE / AND TWO OF WHICH REDUCE !-/ TO REPLACE THE OXIDIZED .(/( 4HE EQUIVALENT OF SIX PROTONS ARE TRANSLOCATED BY #YT BC AND #YT AA IN THE REOXIDATION OF A MOLECULE OF UBIQUINOL 4HE HIGH AND LOW VALUES OF THE POSSIBLE CONTRIBUTION OF THE ELECTRON HYDROXYLAMINE TO UBIQUINONE STEP ARE AND PROTONS 4HE HIGH AND LOW VALUES OF THE CONTRIBUTION OF THE TWO ELECTRON !-/ REACTION ARE AND n (ENCE THE THEORETICAL OVERALL YIELD RANGES FROM BY (!/QUINONE BY #YT BC#YT AA AND BY !-/ TO BY (!/QUINONE #YT BC AND #YT AA AND n FROM !-/
TASE AND .A ( ANTIPORTER WHICH MIGHT HAVE ROLES IN SODIUM SYMPORT OR ANTIPORT ***)ZESPYZMBNJOF0YJEPSFEVDUBTF "0WFSWJFXPG3FBDUJPO 4HE REACTION CATALYZED BY (!/ CAN BE THOUGHT OF AS A DEHYDROGENATION OF HYDROXYLAMINE TO NITROSONIUM .(/( A ./ En (
FOLLOWED BY HYDROLYSIS TO NITROUS ACID ./ (/ A (./ (
(!/ WILL ALSO RAPIDLY OXIDIZE HYDRAZINE TO DINI TROGEN
6UJMJ[BUJPOPGUIF(SBEJFOU B"514ZOUIBTF !40 SYNTHASE IS DRIVEN BY A PROTON GRADIENT WHICH CAN BE EXPERIMENTALLY DEMONSTRATED DURING OXIDATION OF HYDROXYLAMINE +UMAR AND .ICHOLAS ! STEADY STATE VALUE OF 6P OF M6 INSIDE NEGATIVE HAS BEEN ESTIMATED FOR .ITROSOMONAS (OLLOCHER ET AL !LL GENES FOR A PROTON DEPENDENT !40 SYNTHASE ARE PRESENT #HAIN ET AL )T WOULD PRESUMABLY TRANSLOCATE APPROXIMATELY FOUR PROTONS TO THE . SIDE PER !40 FORMED .OTE THAT THIS MAY CORRESPOND TO ALL OR MORE THAN THE PROTON GRADIENT YIELD OF THE OXIDA TION OF ONE MOLECULE OF AMMONIA C3FEVDUJPOPG1ZSJEJOF/VDMFPUJEFT ! GENE CLUSTER ENCODES ALL SUBUNITS OF A PROTON DEPENDENT .!$ DEHYDROGENASE COMPLEX ) WHICH IS PRESUMED TO FUNCTION PREDOMINANTLY OR ONLY AS A .!$ REDUCTASE IN THIS ORGANISM )NTERESTINGLY EACH TURNOVER OF THIS ENZYME MAY REQUIRE !-/ AND (!/ TO TURNOVER TWICE ONCE TO REDUCE UBIQUINONE AND THEN .!$ AND ONCE TO REPLENISH THE ( GRADIENT EQUIVALENTS UTILIZED IN THE REDUCTION OF .!$ BY UBIQUINOL !NOTHER GENE CLUSTER ENCODES A PROTON GRADIENT DEPENDENT .!$.!$0 TRANSHYDROGENASE PRESUMABLY FOR THE GENERATION OF .!$0( (ENCE .!$0( IS VERY ENERGETICALLY EXPENSIVE POSSIBLY REQUIRING THREE TURNOVERS OF THE !-/(!/ SYSTEM 4HE GENOME ENCODES A .A DEPENDENT .!$ REDUC
.( .( A . E (
4HE OXIDATION OF HYDRAZINE BY (!/ HAS BEEN EXPERIMENTALLY USEFUL BUT HAS NO KNOWN FUNCTION IN .ITROSOMONAS 4HE REACTION AS CATALYZED BY AN (!/ WITH SIMILAR CATALYTIC AND MOLECULAR PROPER TIES HAS AN IMPORTANT ROLE IN THE BACTERIAL ANAEROBIC TRANSFORMATION OF AMMONIUM AND NITRITE TO DINITROGEN DISCUSSED LATER 3CHALK ET AL 4HE TURNOVER OF THE ENZYME IS COMMONLY MEASURED IN THE PRESENCE OF AN ARTIlCIAL ELECTRON ACCEPTOR SUCH AS PHENAZINE METHOSULFATE CATALYTIC IN THE PRESENCE OF / DICHLO ROPHENOLINDOPHENOL MAMMALIAN CYTOCHROME C OR CYTOCHROMES OF .ITROSOMONAS EG CYTOCHROME C IN THE PRESENCE OF CATALYTIC AMOUNTS OF CYTOCHROME C 9AMANAKA AND 3HINRA 3EVERAL ELEMENTS OF THE POSSIBLE CATALYTIC MECHA NISM MAY BE SUGGESTED #ATALYSIS MUST INVOLVE SELECTIVE BINDING OF SUBSTRATE . INTERMEDIATES MUST BE STABILIZED TO PREVENT THE RELEASE OF SUCH POSSIBLE COMPOUNDS AS (./ OR ./ !CTIVATION OF SUBSTRATE AND . ( INTERMEDIATES MAY INVOLVE CONVERSION TO ELECTRON RICH FORMS BY DEPROTONATION 7ATER MAY ALSO BE ACTIVATED BY DEPROTONATION %LECTRONS MAY BE REMOVED IN SINGLE STEPS OR PAIRS POSSIBLY AS A HYDRIDE OR A COMBINATION EG AS A HYDRIDE IN ONE STEP FOLLOWED BY TWO ONE ELECTRON STEPS %LECTRONS MUST BE WITHDRAWN AND ALSO EFFECTIVELY REMOVED FROM THE SITE OF CATALYSIS TO HIGH POTENTIAL REDOX CENTERS SO AS TO PREVENT @BACK mOW OF ELECTRONS TO POSSIBLE OXIDANTS SUCH AS DIOXYGEN OR HYDROGEN PEROXIDE OR TO
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PREVENT REVERSAL OF THE . OXIDATION REACTION 2EMOVAL OF PROTONS COULD OCCUR BY WAY OF AN INTRA PROTEIN RELAY OR A CHANNEL 4HE PRODUCT NITROUS ACID MAY EXIT THROUGH A CHANNEL OR THROUGH SEPARATE NITRITE CHANNEL AND PROTON PATH 3UBSTRATE BINDS WITH HIGH AFlNITY (!/ HAS A +M FOR HYDROXYLAMINE OR HYDRAZINE IN THE LOW +- RANGE (OOPER AND .ASON 4HE MEASURED RATE OF TRANSFER OF ELECTRONS FROM SUBSTRATE TO HEMES OF THE ENZYME IS AT LEAST 3n (OOPER ET AL B !RCIERO ET AL A 5NDER SOME REACTION CONDITIONS THE ISOLATED ENZYME PRODUCES SOME ./ POSSIBLY THE PRODUCT OF THE DIMERIZATION AND HYDROLYSIS OF TWO NITROXYLS (./ HENCE THE lRST STEP MAY BE REMOVAL OF TWO ELECTRONS OR A HYDRIDE (OOPER ./ CAN ALSO BE A PRODUCT INDICATING THAT THE SECOND STAGE CAN SOMETIMES OCCUR IN VITRO IN SINGLE ELECTRON STEPS (OOPER ET AL (OOPER AND 4ERRY ./ WILL NOT SERVE AS SUBSTRATE FOR THE PRODUCTION OF NITRITE (ENDRICH ET AL HOWEVER ./ WILL RE OXIDIZE FERROUS C HEMES 2ELEASE OF INTERMEDIATES DOES NOT OCCUR IN VIVO BUT CAN OCCUR IN VITRO DEPENDING ON THE NATURE OF THE ARTIlCIAL ELECTRON ACCEPTOR AND THE ASSAY CONDITIONS #0WFSWJFXPGUIF4USVDUVSFPG)"0 $IFNJDBM$IBSBDUFSJ[BUJPO 0URIlED (!/ IS A RICH RED IN COLOR DUE TO ITS HIGH CON TENT OF HEME (OOPER ET AL )N FACT CELLS HAVE A STRIKING BROWNISH RED COLOR DUE TO THE PRESENCE OF LARGE AMOUNTS OF (!/ AND OTHER C TYPE CYTOCHROMES 3EVERAL YEARS OF RIGOROUS PROTEIN CHEMISTRY REVEALED (!/ TO BE AN OLIGOMER IN WHICH EACH MONOMER CON TAINED SEVEN C HEMES EACH COVALENTLY ATTACHED BY TWO THIOETHER BONDS FROM VINYL GROUPS TO CYSTEINES AND AN UNUSUAL C HEME DERIVATIVE HEME 0 4HIS WAS DETERMINED BY PROTEOLYSIS AND ISOLATION OF ALL HEME CONTAINING PEPTIDES !RCIERO AND (OOPER !S REVEALED BY ANALYSIS OF AN UNUSUAL CROSSLINKED DIPEP TIDE RECOGNIZED BY HAVING TWO . TERMINAL AMINO ACID RESIDUES HEME 0 HAS AN ADDITIONAL COVALENT BOND FROM A RING CARBON OF A PEPTIDE TYROSINE TO A METHYNE CARBON OF A HEME !RCIERO ET AL 4REATMENT OF FERRIC (!/ WITH HYDROGEN PEROXIDE SPECIlCALLY AND IRREVERSIBLY ALTERS THE ENZYME SO THAT THE NM OPTICAL BAND IS NO LONGER SEEN IN THE FERROUS ENZYME )N ADDITION THE TURNOVER OF THE ENZYME AND THE REDUC IBILITY OF C HEMES BY SUBSTRATE IS LOST (ENCE HEME
0 IS CRITICAL TO CATALYSIS (OOPER AND 4ERRY !LL EVIDENCE INDICATES THAT IN FACT HEME 0 BINDS TO SUBSTRATE 4HE SEQUENCE DEDUCED FROM THE CLONED GENE RESULTED IN NUMBERING OF HEMES n FROM THE . TERMINUS 3AYAVEDRA 3OTO ET AL 4HE OPTICAL SPECTRUM OF FERROUS (!/ CONSISTS OF A LARGE 3ORET BAND A SHOULDER AT NM OF HEME 0 AND _ AND ` BANDS AT AND NM DUE TO C HEMES &IG 4HE FERRIC SPECTRUM CONTAINS ONLY THE BROAD 3ORET BAND AND _ AND ` BANDS OF THE C HEMES AND A WEAK BROAD BAND AT NM DUE TO HEME 0 #OLLINS ET AL /PTICAL SPECTRO ELECTROCHEMISTRY &IG INSET REVEALED THE REMARKABLE RANGE OF MIDPOINT POTENTIALS 4ABLE TO BE M6 TO n M6 4HE REDUCTION POTENTIAL OF HEME 0 DECREASED BY M6 PER P( UNIT IE PROTONATION OF A MOIETY ON THE ENZYME PROMOTES UPTAKE OF ELECTRONS BY THE CATALYTIC IRON 4HE HEMES WITH POTENTIALS AT AND n M6 ARE REDUCED EITHER AT EQUILIBRIUM IN THE PRESENCE OF HYDROXYLAMINE OR IN THE STEADY STATE IN THE PRESENCE OF HYDROXYLAMINE AND ELECTRON ACCEPTORS 5IF$SZTUBM4USVDUVSFPG)"0 4HE DETERMINATION OF THE CRYSTAL STRUCTURE OF (!/ HAS ADVANCED THE lELD REMARKABLY )GARASHI ! SIDE VIEW OF A MONOMER &IG ! REVEALS C HEMES CONCENTRATED IN THE LOWER PORTION OF THE MOLECULE 4HE MOLECULE IS WIDE AT THE BASE AND A SPINE OF LONG _ HELICAL DOMAINS RISE AT THE BACK &IG " MORE CLEARLY SHOWS THE ARRANGEMENT IN SPACE OF THE HEMES OF A MONOMER )N THIS VIEW HEME HEME 0 AS IDENTIlED BY ITS CROSS LINKING TO TYROSINE IS THE HIGHEST HEME "Y THEIR SPACING THE HEMES OF EACH SUBUNIT APPEAR TO GROUP INTO FOUR CLUSTERS A TRIMER OF HEMES AND DIMERS OF HEMES AND AND AND AND A MONOMERIC HEME )GARASHI ET AL 4HE TRIMER OF (!/ HAS A THREE CLOVE @GARLIC SHAPE !S SEEN FROM THE SIDE VIEW &IG THE HEMES ARE CONCENTRATED IN THE WIDE BOTTOM HALF OF THE MOLECULE AROUND THE TOWER OF LONG _ HELICES ! SHALLOW CAVITY PROJECTS INTO THE BASE OF THE TRIMER 4HE THREE CATALYTIC HEME 0S ARE AT THE TOP OF THE CLOUD OF C HEMES IN THE (!/ TRIMER 2EMARKABLY THE HEME CROSSLINK OF HEME 0 IS TO A TYROSINE IN THE ADJACENT SUBUNIT (ENCE (!/ IS A COVALENTLY LINKED ENTITY CONTAINING HEMES &ROM THE TOP VIEW &IG THE HEMES APPEAR IN A RING WITH A HEME PROJECT ING RADIALLY FROM EACH SUBUNIT
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&IG /PTICAL ABSORPTION SPECTRUM OF (!/ OF .ITROSOMONAS EUROPAEA !BSORBANCE AS A FUNCTION OF WAVELENGTH $ARKER LINE OXIDIZED ENZYME ,IGHTER LINE REDUCED ENZYME )NSET REDOX TITRATION OF THE SEVEN C HEMES OF (!/ DATA FOR HEME 0 NOT SHOWN !BSORBANCE AS A FUNCTION OF POTENTIAL H $ATA BASED ON #OLLINS ET AL 4ABLE 2EDUCTION POTENTIALS FOR THE HEMES OF (!/ #OLLINS ET AL %|M .(%
!BSORPTION "AND
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&IG 3TRUCTURE OF A SUBUNIT OF (!/ ;BASED ON )GARASHI ET AL = 3IDE VIEW (EME NUMBERS ARE BASED ON THE PRIMARY STRUCTURE AND BEGIN AT THE . TERMINAL ! HEMES AND RIBBON VIEW OF PROTEIN " (EMES ALONE
1PTTJCMF1BUIPG&MFDUSPOTGSPN)FNF1 !CCESS TO SOLVENT BY THE HEMES IS LIMITED TO A SUB STRATE ACCESSIBLE CHANNEL LEADING TO AN OPEN AXIAL POSITION OF THE CATALYTIC HEME 0 HEME TO THE PORPHYRIN EDGE OF HEME MOST DISTAL FROM THE
CENTER OF THE HEME CLUSTER AND TO A REGION IN THE BASE CAVITY NEAR HEMES AND /N THIS BASIS HEME WAS PREDICTED TO BE THE POINT OF EXIT OF ELECTRONS FROM (!/ )GARASHI ET AL 4HE IRONS OF ALL HEMES ARE WITHIN ¯ FROM THE NEAREST HEME IRON (ENCE THERE IS NO APPARENT KINETIC BARRIER TO ELECTRON TRANSFER
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&IG 3TRUCTURE OF TRIMER OF (!/ WITH ONE #YT C DOCKED ;BASED ON )AGARASHI ET AL AND )VERSON ET AL = 3IDE VIEW
(EMES ARE ALSO CLOSE ENOUGH TO HEMES OF THE ADJA CENT SUBUNIT TO ALLOW RAPID ELECTRON TRANSFER (ENCE A LOW RESISTANCE ELECTRON PATH COULD BEGIN AT HEME 0 OF A GIVEN SUBUNIT AND AT HEME BIFURCATE TO A PATH THROUGH HEME OF THAT SUBUNIT AND A SECOND PATH THROUGH HEME AND AND HEMES AND OF THE ADJACENT SUBUNIT INTERESTINGLY THE SUBUNIT TO WHICH HEME 0 IS COVALENTLY BOUND &IG &MFDUSPOJDBOE3FEPY1SPQFSUJFTPGUIF )FNFT 4HE FERRIC ENZYME ANALYZED BY PERPENDICULAR MODE %02 IS SEEN TO CONTAIN A VARIETY OF LOW SPIN AXIAL BIS (IS HEME IRONS WITH G VALUES OF APPROXIMATELY AND PAIRS OF HEMES EXHIBITING ELECTRONIC EXCHANGE &IG /NE OF THE ELECTRONICALLY COUPLED PAIRS EXHIBITS SIGNALS AT G AND !N %02 SIGNAL FOR THE SECOND PAIR IS NOT SEEN EXCEPT BY PARALLEL MODE
%02 BY WHICH AN INTEGER SPIN SIGNAL AT G IS SEEN (ENDRICH ET AL 2EDUCTION OF THE ENZYME WITH SUBSTRATE ,IPSCOMB AND (OOPER OR REDOX TITRATION OF THE ENZYME 0RINCE AND (OOPER RESULTS IN AN ARRAY OF SHIFTING %02 SIGNALS REmECTING INTERACTION BETWEEN HEMES !N EXHAUSTIVE SERIES OF STUDIES HAVE BEEN CARRIED IN WHICH %02 SPECTRA WERE MEASURED ON POISED SAMPLES OF (!/ -EASUREMENT OF %02 SPECTRA AT SEVERAL FREQUENCIES AND ANALYSIS OF THE HEME GEOMETRIES FROM THE CRYSTAL STRUCTURE ALLOWED ASSIGNMENT OF %02 PROPERTIES AND REDOX POTENTIALS TO SEVERAL HEMES (ENDRICH ET AL 4HE CATALYTIC HEME 0 n M6 IS EXCHANGE COUPLED TO THE NEIGHBORING HEME 4HE VALUES OF MIDPOINT REDUCTION POTENTIAL OF n M6 AND n M6 WERE ASSIGNED TO HEME 0 AND HEME RESPEC TIVELY 4HESE LOW VALUES OF MIDPOINT POTENTIAL ARE IN KEEPING WITH THE INABILITY TO EXPERIMENTALLY OBSERVE REDUCTION OF HEME 0 BY SUBSTRATE IN THE STEADY
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&IG 3PATIAL ARRANGEMENT OF HEMES OF TRIMER OF (!/ ;BASED ON )GARASHI ET AL = 4OP VIEW (EMES 0 OF THE THREE SUBUNITS ARE NUMBERED | AND || AND ARE DARKER
STATE OR BY STOPPED mOW MEASUREMENTS IN THE MSEC RANGE (OOPER ET AL B )N THE FERRIC ENZYME THE COUPLING BETWEEN THESE TWO HEMES CAN BE DISRUPTED BY BINDING OF SUBSTRATE OR CYANIDE TO HEME 0 (ENDRICH ET AL #YANIDE IS A LIGAND TO HEME 0 AND AN INHIBITOR OF THE ENZYME ACTIVITY 4ABLE ,OGAN ET AL 4HE TWO HEMES WITH MIDPOINT POTENTIALS NEAR ARE ALSO EXCHANGE COUPLED AND HAVE BEEN IDENTIlED AS HEMES AND .OTABLY THESE ARE TWO OF THE HEMES THAT ARE REDUCED DURING REACTION OF (!/ WITH HY DROXYLAMINE OR HYDRAZINE 4HE ELECTRONIC COUPLING BETWEEN HEMES IN THESE TWO PAIRINGS IMPLIES THAT ELECTRONS EQUILIBRATE RAPIDLY BETWEEN THE TWO HEME IRONS 4HIS PROPERTY MIGHT MAKE THEM ESPECIALLY SUIT ABLE AS ELECTRON REDOX MEDIATORS OR SINKS (EME HAS A MIDPOINT POTENTIAL OF n M6 4HE OTHER THREE HEMES AND HAVE NOT YET BEEN MATCHED WITH THE UNASSIGNED VALUES OF MIDPOINT POTENTIAL n AND n )NTERESTINGLY BECAUSE THE ELECTRON PATHS IN THE ENZYME DIVIDE AND REJOIN &IG THE HIGHEST PO TENTIAL HEMES OF THE (!/ TRIMER SIX HEMES OF ^ M6 AND THREE OF M6 CAN IN THEORY PROVIDE A RESERVOIR FOR AT LEAST ELECTRONS AND THUS ALLOW COM PLETE TURNOVER AND RELEASE OF NITRITE ONCE A SUBSTRATE MOLECULE IS BOUND
&IG $IAGRAM OF POSSIBLE ELECTRON PATHS FROM THE THREE ACTIVE SITES OF THE (!/ TRIMER THROUGH HEMES OF (!/ #YT C AND #YT C- (EMES ARE SHOWN AS CIRCLES
&IG %02 SPECTRUM OF (!/ OF .ITROSOMONAS #ONDITIONS ARE AS DESCRIBED IN (ENDRICH ET AL $ARKER LINE PERPEN DICULAR MODE %02 ,IGHTER LINE PARALLEL MODE %02
4QFDVMBUJPO3FHBSEJOHUIF.FDIBOJTNPG $BUBMZTJT B#JOEJOHBOE%FQSPUPOBUJPOPG4VCTUSBUF &IGURE SHOWS THE ORIENTATION OF AMINO ACID RESIDUES OF THE ACTIVE SITE AS DETERMINED BY CRYSTALLOGRAPHY )GARASHI ET AL MODELED WITH A MOLECULE OF HYDROXYLAMINE BOUND BY ITS NITROGEN TO THE IRON !TOMS OF SEVERAL RESIDUES PROJECT INTO THE ACTIVE
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SITE WITHIN HYDROGEN BONDING DISTANCES n¯ OF HYDROGENS OF HYDROXYLAMINE AND ARE THUS PO TENTIALLY CAPABLE OF FACILITATING SELECTIVE BINDING OF SUBSTRATE STABILIZATION OF INTERMEDIATES AND ACTIVATION OF THE SUBSTRATE BY DEPROTONATION 4HEY INCLUDE THE CROSSLINKED TYROSINE AS WELL AS HISTIDINE ASPARTATE AND TYROSINE WHICH ARE ABOVE THE SUBSTRATE ( BONDS COULD EXTEND FROM THE HYDROXYL ( OF SUBSTRATE TO THE EPSILON . OF HISTIDINE FROM ONE SUBSTRATE AMINO ( TO THE RING OXYGEN OF TYROSINE FROM THE OTHER SUBSTRATE AMINO ( TO THE CARBOXYLATE / OF ASPARTATE (YDROXYLAMINE AND HYDRAZINE REDUCE THE C HEMES OF THE ENZYME . METHYLHYDROXYLAMINE WILL ALSO REDUCE C HEMES OF (!/ BUT / METHYL HYDROXYLAMINE IS NOT REACTIVE 2ITCHIE AND .ICHOLAS EMPHASIZING THE POS SIBLE IMPORTANCE OF BEING ABLE TO FORM AN OXYANION DURING OXIDATION OF HYDROXYLAMINE
&IG (EME 0 OF (!/ SHOWING NEARBY PEPTIDE RESIDUES AND PUTATIVE MODE OF BINDING OF SUBSTRATE $ATA BASED ON )GARASHI ET AL 3HOWN ARE THE CROSSLINKED TYROSINE AND THREE RESIDUES THAT PROJECT INTO THE ACTIVE SITE ASPARTATE TYROSINE AND HISTIDINE (YDROXYLAMINE IS MODELED AS HAVING AN . &E BOND TO THE HEME 4HE AXIAL HISTIDINE APPEARS BELOW THE HEME
C&MFDUSPO3FNPWBM 4HE ELECTRONIC COUPLING OF THE CATALYTIC HEME PAIR AND MAKE IT A REASONABLE HYPOTHESIS THAT THE REACTION OCCURS IN TWO ELECTRON STEPS )N THE lRST STEP DEPROTONATION FOLLOWED EITHER BY REMOVAL OF A HYDRIDE OR A SECOND DEPROTONATION AND REMOVAL OF TWO ELECTRONS WOULD PRODUCE IRON (./ )N THE SECOND STEP HYDRIDE TRANSFER OR DEPROTONATION AND REMOVAL OF TWO ELECTRONS WOULD PRODUCE IRON NITROSONIUM ./
4HE LOW VALUES OF REDOX POTENTIAL FOR THE CATALYTIC HEME PAIR MAKE IT LIKELY THAT ELECTRONS QUICKLY PASS TO THE M6 PAIR HEMES AND IN KEEPING WITH THE INABILITY TO EXPERIMENTALLY DEMONSTRATE REDUCTION OF HEME 0 DURING CATALYSIS
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB D5IF3FBDUJPOPG)"0JT6OJRVFGPSB$BUB MZUJD)FNF )N THE REACTION CATALYZED BY (!/ SUBSTRATE lRST BINDS TO THE IRON OF A HEME )N THE COURSE OF THE REACTION ELECTRONS ARE REMOVED FROM SUBSTRATE AND ARE MOVED OUT OF THE SYSTEM THROUGH THE OTHER C HEMES OF THE PROTEIN 4HIS IS THE ONLY REACTION OF THIS TYPE IN NA TURE !LL OTHER REDOX REACTIONS OF SUBSTRATES BOUND TO THE IRON OF A HEME INVOLVE TRANSFER OF ELECTRONS INTO THE CATALYTIC SYSTEM AND THEN TO THE IRON BOUND SUBSTRATE 4ABLE 4HIS DESCRIBES THE REACTIONS OF THE SIROHEME CONTAINING NITRITE OR SULlTE REDUCTASE THE CYTOCHROME CD NITRITE REDUCTASE THE PENTA HEME NITRITE REDUCTASE .I2 AND THE NITRIC OXIDE REDUC TASE AS WELL AS REDUCTION OF OXYGEN BY CYTOCHROME OXIDASE OR CYTOCHROME C PEROXIDASE )N THE CASE OF CYTOCHROME OXIDASE ELECTRONS MAY BE THOUGHT OF AS ENTERING THE CATALYTIC SYSTEM AND REDUCING ONE OF THE OXYGEN ATOMS TO WATER !T THE SAME TIME THE IRON AND PORPHYRIN RING DONATE ELECTRONS AS THE FERRYL OXYGEN INTERMEDIATE IS FORMED 4HE LATTER IS REDUCED TO WATER AS TWO ADDITIONAL ELECTRONS ENTER THE SYSTEM )N THE CASE OF CYTOCHROME 0 MONOOXYGENASE ELECTRONS MAY ALSO BE THOUGHT OF AS ENTERING THE CATALYTIC SYS TEM AND REDUCING ONE OXYGEN TO WATER ELECTRONS ARE ALSO WITHDRAWN FROM THE IRON AND PORPHYRIN RING AS THE FERRYL OXYGEN INTERMEDIATE IS FORMED 4HE LATTER DECAYS AS SUBSTRATE IS HYDROXYLATED )N THE REACTION OF CATALASE WITH A MOLECULE OF HYDROGEN PEROXIDE THE PORPHYRIN AND IRON DONATE ELECTRONS AS THE FER RYL OXYGEN INTERMEDIATE AND WATER ARE FORMED 4HIS IS FOLLOWED BY REACTION OF THE OXYGEN WITH A SECOND MOLECULE OF HYDROGEN PEROXIDE 3INCE THE TRAIT OF ELECTRON WITHDRAWAL FROM SUBSTRATE WHICH IS BOUND TO THE IRON OF HEME 0 IS SO NOVEL WE STRONGLY SUSPECT THAT IT IS RELATED TO THE MOST NOVEL ASPECT OF THE STRUCTURE OF (!/ THE TYROSINE CROSS LINKED TO THE CATALYTIC HEME 4HIS IS REINFORCED BY A COMPARISON OF (!/ WITH THE PENTA C HEME NITRITE REDUCTASE )N BOTH ENZYMES CATALYSIS TAKES PLACE AT THE IRON OF A C HEME AND ELECTRONS ARE EITHER WITHDRAWN TO (!/ OR DONATED BY .I2 AUXILIARY C HEMES OF THE ENZYME 2EMARKABLY ALTHOUGH SIGNIlCANT HOMOLOGY OF AMINO ACID SEQUENCE IS NOT OBSERVED THE TERTIARY STRUCTURE OF SUBUNITS OF (!/ AND .I2 ARE VERY SIMILAR AND THE HEMES OF .I2 HAVE EXACTLY THE SAME ORIENTATION IN SPACE RELATIVE TO ONE ANOTHER AS HEMES n OF (!/ %INSLE ET AL 4HE CATALYTIC HEME IRON OF .I2 DIFFERS FROM HEME 0 IN HAVING A LYSINE RATHER THAN HISTIDINE AS THE
AXIAL LIGAND -OST NOTABLY HOWEVER THE CATALYTIC HEME OF .I2 LACKS THE TYROSINE CROSS LINK FOUND IN (!/ 7ITH THIS IN MIND DETERMINATION OF THE ROLE OF THE TYROSINE CROSS LINK IN (!/ IS A HIGH PRIORITY FOR FUTURE WORK "Y SHOWING THAT THE PLANE OF THE TYROSINE RING IS PERPENDICULAR TO THE PORPHYRIN PLANE &IG RATHER THAN IN THE SAME PLANE THE CRYSTAL STRUCTURE OF (!/ ELIMINATED MECHANISMS DEPENDENT ON A CONTINUOUS CONJUGATED / ELECTRON SYSTEM BETWEEN PORPHYRIN AND TYROSINE RINGS &OR EXAMPLE ONE HYPOTHETICAL MECHANISM HAD INVOLVED THE OXIDATION OF A 3CHIFFS BASE WHICH WULD HAVE BEEN PREVIOUSLY FORMED BY REACTION OF HYDROXYLAMINE WITH A CARBONYL OXYGEN OF TYROSINE CONJUGATED WITH THE HEME RING !RCIERO AND (OOPER )N FACT THE ORIENTATION OF THE TYROSINE TO PORPHYRIN COVALENT BOND INDICATES THAT THE RELEVANT PORPHYRIN CARBON IS A TETRAHEDRAL CENTER BONDED TO THE TWO ADJACENT PYRROLE CARBONS THE TYROSYL CARBON AND PRESUMABLY HYDROGEN IE THE PORPHYRIN RING COULD NOT BE UNSATURATED AT THAT POINT 4HIS STRUCTURE A DIHYDROPORPHYRIN COULD THEORETICALLY PARTICIPATE IN HYDRIDE REMOVAL FROM IRON BOUND HYDROXYLAMINE !RCIERO AND (OOPER )N THAT MODEL THE ROLE OF IRON WAS THE STABILIZATION AND ACTIVATION OF SUBSTRATE RATHER THAN ELECTRON TRANSFER /THER POSSIBLE MODELS WHICH ARE UNDER CONSIDERATION CALL FOR A CLEARER DETERMINATION OF THE NATURE OF THE TYROSYL PORPHYRIN STRUCTURE !RCIERO AND (OOPER E3FBDUJWJUZPGUIF"DUJWF4JUFPG)"0 /RGANOHYDRAZINES ARE SUICIDE SUBSTRATES OF (!/ ,OGAN AND (OOPER )T IS HYPOTHESIZED THAT ORGANOHYDRAZINE IS OXIDIZED TO DINITROGEN AND A RADICAL CATION WHICH SUBSEQUENTLY REACTS WITH HEME 0 !S NOTED ABOVE HYDROGEN PEROXIDE REACTS WITH AND DESTROYS HEME 0 OF (!/ (YDROGEN PEROXIDE IS HYPOTHESIZED TO BIND AT THE IRON UNDERGO DEPROTONATION AND REACT WITH FERRIC HEME 0 TO FORM A FERRYL OXYGEN INTERMEDIATE WHICH MAY DECAY OR DISMUTATE TO REACTIVE FORMS OF OXYGEN THAT OPEN THE PORPHYRIN RING 4HIS INACTIVATION IS APPARENTLY PREVENTED IN VIVO %ITHER / OR (/ ARE CATALYTICALLY REDUCED TO (/ OR (/ RESPECTIVELY AT HEME 0 OF FULLY FERROUS (!/ OR IN THE PRESENCE OF AN EXCESS OF REDUCTANT IE THE ENZYME CAN ACT AS AN OXIDASE OR PEROXIDASE (OOPER AND "ALNY (OOPER ET AL 4ABLE 4HE REACTIONS ARE RAPID ONLY DURING THE OXIDATION OF THE LOW POTENTIAL HEMES OF THE ENZYME 4HESE REAC
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HAVE REDOX POTENTIALS OF %M| VS .(% n AND n M6 !RCIERO ET AL B $URING TURNOVER WITH (!/ THE TWO HEMES WITH MIDPOINT POTENTIALS OF M6 ARE REDUCED !RCIERO ET AL B 4HE MAGNETIC PROPERTIES OF THE C HEMES HAVE BEEN PARTIALLY CHARACTERIZED AND EXHIBIT{ ELEC TRONIC INTERACTIONS SIMILAR TO THOSE OBSERVED IN (!/ !NDERSSON ET AL -OST OF #YT C IS SOLUBLE IN EXTRACTS (OWEVER A SIGNIlCANT FRACTION OF #YT C IS MEMBRANE ASSOCIATED AND CAN BE WASHED OFF WITH HIGH CONCENTRATIONS OF .A#L 4HE DISSOCIATED #YT C RE BINDS IN APPROXIMATELY STOICHIOMETRIC AMOUNTS -C4AVISH ET AL 4HE BINDING CAPABILITIES OF #YT C IN THE INITIAL SUPERNATANT THE SALT WASH AND HIGHEST LEVEL OF PURIlCATION ARE THE SAME 4HE AMINO ACID SEQUENCE "ERGMANN ET AL (OM MES ET AL AND THE 8 RAY STRUCTURE HAVE BEEN COMPLETED )VERSON ET AL A ! SIDE VIEW OF THE 8 RAY STRUCTURE IS SHOWN IN &IG (EMES ARE NUMBERED )n)6 STARTING FROM THE . TERMINUS 2EDUCTION POTENTIALS AND SPECTROSCOPIC PROPERTIES AND HEME HEME INTERACTIONS HAVE BEEN ASSIGNED TO THE HEMES OF THE PROTEIN 5PADHYAY ET AL 4HE DISTRIBUTION OF SURFACE CHARGE AND THE SURFACE GEOMETRY OF (!/ AND CYC REVEAL A REASONABLE DOCKING SITE WHICH BRINGS THE EXPOSED EDGE OF HEME OF (!/ WITHIN ¯ OF THE SOLVENT EXPOSED EDGE OF HEME ) OF #YT C &IG )VERSON ET AL 4HE
TIONS APPARENTLY DO NOT OCCUR IN VIVO AND IN FACT MUST BE AVOIDED !S EXPECTED FOR A FERROUS HEME WHICH REACTS WITH DIOXYGEN FERROUS HEME 0 BINDS #/ CAUSING THE 3ORET BAND TO SHIFT TO NM !S DETER MINED BY OPTICAL SPECTROSCOPY FERRIC (!/ BINDS CYANIDE BUT DOES NOT BIND THE ANIONS AZIDE CYANATE OR THIOCYANATE mUORIDE CHLORIDE OR "ROMIDE ,OGAN ET AL #YANIDE INHIBITS ENZYME ACTIVITY ./ FORMS A DERIVATIVE WITH FERRIC HEME 0 IN THE RESTING ENZYME AND CAUSES THE REOXIDATION OF FERROUS HEMES OF THE PARTIALLY REDUCED ENZYME (ENDRICH ET AL 3EVERAL LINES OF EVIDENCE INDICATE THAT FERROUS (!/ REDUCES NITRITE 4HE STOICHIOMETRIC CONVERSION OF HYDROXYLAMINE AND NITRITE TO NITROUS OXIDE IN THE PRESENCE OF (!/ POSSIBLY INVOLVES THE INTERMEDI ATE PRODUCTION OF (./ FROM NITRITE (OOPER &URTHER THE REDUCED ENZYME IS REOXIDIZED IN THE PRESENCE OF NITRITE 9AMANAKA ET AL 3CHALK ET AL 4ABLE *75SBOTGFSPG&MFDUSPOTGSPN )"0UP6CJ RVJOPOF "$ZUPDISPNFD %LECTRONS FROM (!/ PASS TO #YT C 9AMANAKA AND 3HINRA 4HE C HEMES OF THIS CYTOCHROME
4ABLE 2EDOX CATALYSIS BY HEMES 2EDUCTION OF &E BOUND SUBSTRATE #YT CDD NITRITE REDUCTASE 3IROHEME SULlTENITRITE REDUCTASE
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IRON IRON DISTANCE BETWEEN THE TWO HEMES IS ¯ 7ITHIN #YT C A POSSIBLE ELECTRON TRANSPORT CHAIN THROUGH HEMES ) ))) )6 AND )) WOULD HAVE IRON IRON DISTANCES OF n ¯ AND ALLOW RAPID EQUILIBRIUM OF ELECTRONS AMONG THE HEMES OF #YT C AND BOUND (!/ )N THE STEADY STATE OR AT EQUILIBRIUM ELECTRONS COULD ACCUMULATE IN THE TWO M6 HEMES OF #YT C AS WELL AS THE ^ M6 AND M6 HEMES OF (!/ 2ECALLING THE APPARENT CAPACITY FOR ELECTRON TRANSPORT BETWEEN SUBUNITS A SINGLE ACTIVE SITE OF (!/ IN A FULLY OCCUPIED ;(!/#YT C=COMPLEX THEORETICALLY HAS AN ELECTRON RESERVOIR OF HIGH POTENTIAL C HEMES ON (!/ AND ON #YT C &IG 4HE UTILITY OF THIS ARRANGEMENT MAY BE THE ASSURANCE OF COMPLETE TURN OVER ONCE A SUBSTRATE MOLECULE HAS BOUND #$ZUPDISPNFD. "1VUBUJWF$ZUPDISPNF D6CJRVJOPOF3FEVDUBTF )N A GENE CLUSTER CONTAINING (!/ 3AYAVEDRA 3OTO ET AL THE GENE FOR #YT C SHARES AN OPERON AND IS CO TRANSCRIBED WITH THE GENE ENCODING THE MEMBRANE TETRAHEME #YT C- "ERGMANN AND (OOPER 4HE . TERMINAL SEQUENCE OF THE PURIlED DETERGENT SOLUBILIZED PROTEIN IS THE ONLY TRANSMEMBRANE DOMAIN 6ANNELLI ET AL "ASED ON ITS GENE SEQUENCE
THIS PROTEIN IS A MEMBER OF THE @.IR4.AP# FAM ILY OF TETRAHEME MEMBRANE ANCHORED CYTOCHROMES WHICH ARE IMPLICATED IN ELECTRON TRANSFER FROM MEMBRANE QUINOLS TO PERIPLASMIC TERMINAL ELECTRON ACCEPTORS 4HIS FAMILY IS EXEMPLIlED BY .IR4 .AP# AND .RF( WHICH DIRECT ELECTRONS TO THE #U CONTAINING NITRITE REDUCTASE *UNGST ET AL THE NITRATE RE DUCTASE 2OLDAN ET AL OR THE PENTA HEME NITRITE REDUCTASE 3IMON ET AL RESPECTIVELY %LECTRON TRANSFER FROM QUINOL TO THE APPROPRIATE PERIPLASMIC CYTOCHROME HAS BEEN EXPERIMENTALLY DEMONSTRATED WITH .RF( )N ALL THE MEMBERS OF THIS FAMILY THE SUBSEQUENT PERIPLASMIC ELECTRON TRANSFER STEP IS MEDIATED BY A SOLUBLE PERIPLASMIC C CYTOCHROME OR A lFTH C HEME IN AN ADDITIONAL VERY LARGE # TERMINAL DOMAIN OF THE QUINOL DEHYDROGENASE )T IS PROPOSED THAT #YT C- OF .ITROSOMONAS TRANSFERS ELECTRONS FROM THE PERIPLASMIC OXIDATION OF HYDROXYLAMINE BY (!/C TO UBIQUINONE IN THE MEMBRANE !LTHOUGH MEMBERS OF THE .IR4.AP# FAMILY HAVE SIMILAR LARGE CENTRAL AMINO ACID SEQUENCE DOMAINS CONTAINING THE FOUR C HEME BINDING MOTIFS THEY DIFFER VERY SIGNIlCANTLY IN THE SEQUENCE OF # TERMINAL DOMAINS 3INCE THE FAMILY OF PROTEINS ARE ALL THOUGHT TO REACT WITH MEMBRANE QUINONES THE VARIABLE
&IG 3TRUCTURE OF #YT C OF .ITROSOMONAS ;BASED ON )VERSON ET AL = 3IDE VIEW 4HE PROTEIN IS ARRANGED AS IF PREPARED TO DOCK AT THE LEFT SIDE OF THE (!/ TRIMER OF &IG (EME NUMBERS ARE BASED ON THE PRIMARY STRUCTURE AND BEGIN AT THE . TERMINAL
# TERMINAL DOMAINS MAY PLAY A ROLE IN THE SPECIlCITY OF PERIPLASMIC REDOX PARTNER 4HE # TERMINAL DOMAIN OF THE .ITROSOMONAS #YT C- IS REMARKABLY ACIDIC OF THE AMINO ACIDS ARE ASPARTATE OR GLUTAMATE )F THE ENZYMATIC ROLE OF #YT C- IS CONlRMED THE TRANSFER OF ELECTRONS FROM AN (!/ BOUND .(/( TO UBIQUINONE WILL CONTAIN HEMES PER (!/ SUBUNIT OR IN THE HYPOTHETICAL FULLY OCCUPIED ;(!/#YT C#YT C-= COMPLEX AS IN &IG /BVIOUSLY ONE ASKS WHY SO MANY HEMES ARE REQUIRED BY THIS PROCESS 4HE ANSWER MIGHT INCLUDE RAPID AND IRREVERSIBLE REMOVAL OF ELECTRONS TO A SAFE DISTANCE 74QFDVMBUJPOPOUIF&WPMVUJPOPGUIF )ZESPYZMBNJOF0YJEJ[JOH4ZTUFN $URING ANALYSIS OF THE CRYSTAL STRUCTURE OF #YT C PAIRS OF HEMES WITHIN (!/ AND #YT C WERE SEEN TO HAVE THE SAME SPATIAL ORIENTATION RELATIVE TO ONE ANOTHER IE THE PAIRS FROM THE TWO PROTEINS COULD BE SUPERIMPOSED WITH HIGH PRECISION AND WERE THUS RECOGNIZED AS DIHEME DIMENSIONAL MOTIFS )VERSON ET AL B 4HE MOTIFS WERE SEEN TO OCCUR IN SEVERAL OTHER MULTIHEME CYTOCHROMES WHICH HAD
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AN ELECTRON TRANSFER ROLE IN THE OXIDATION OR REDUCTION OF INORGANIC NITROGEN OR SULFUR COMPOUNDS -OST DRAMATICALLY THE ENTIRE TETRAHEME MOTIF OF #YT C HEMES )) )6 ))) ) WAS PRECISELY SUPERIMPOSABLE ON HEMES OF (!/ 4HIS CAN BE VISUALIZED BY LIFTING THE COLLECTIVE HEMES OF #YT C FROM THE PROTEIN IN ITS DOCKING SITE WITH (!/ TURNING THEM END TO END AND SETTING THEM ON THE HEMES OF (!/ &IG 4HE SIMILARITY EXTENDS TO MUCH OF THE SECONDARY STRUCTURE SURROUNDING THE HEMES IN SPITE OF AN ALMOST COMPLETE LACK OF HOMOLOGY IN PRIMARY STRUCTURE )VERSON ET AL LB 'IVEN THE FUNCTIONAL INTERRELATIONSHIP BETWEEN THESE PROTEINS AND THEIR PRESENCE IN THE SAME GENOME THE HYPOTHESIS OF COMMON ANCESTRY IS SIMPLER AND PERHAPS MORE LIKELY THAN CONVERGENT EVOLUTION )NTERESTINGLY HEME )) OF #YT C WHICH IS HOMOLOGOUS TO THE CATALYTIC HEME 0 HEME OF (!/ AS REVEALED IN THE ALIGNMENT OF THE TETRAHEME MOTIF HAS THE UNUSUAL PROPERTY OF BEING PENTACOORDINATE AND YET BEING PROTECTED FROM ACCESS TO SOLVENT BY A PROTEIN FOLD 4HIS PROPERTY IS CORRELATED WITH ITS LACK OF REACTIVITY WITH LIGANDS AND THE SEEMINGLY PARADOXICAL POSSESSION OF A 3ORET OPTICAL BAND CHARACTERISTIC OF A HIGH SPIN %02 CEN TER #YT C COULD IN THEORY HAVE EVOLVED FROM A PRIMITIVE (!/ BY GENE DUPLICATION FOLLOWED BY THE LOSS OF HEME BINDING #89#( MOTIFS AND SOME OF THE PROTEIN DOMAINS FOR HEMES AND 4HIS WOULD HAVE BEEN ACCOMPANIED BY ACQUISITION OF A PROTEIN FOLD COVERING HEME )) AND LOSS OF SURFACE
PROPERTIES RELATED TO THE TRIMER STRUCTURE AND THE TYROSINE CROSSLINK !LTERNATIVELY THE MORE PRIMITIVE MOLECULE MAY HAVE BEEN AN ENZYME RESEMBLING #YT C WHICH POSSESSED A CATALYTIC SITE OPEN TO SOLVENT 4HIS ANCESTRAL ENZYME COULD HAVE EVOLVED TO (!/ AND THE ELECTRON TRANSPORT MEDIATOR #YT C FOLLOW ING GENE DUPLICATION 4HE COMPLETION OF THE CRYSTAL STRUCTURE OF THE PENTAHEME CYTOCHROME C NITRITE REDUCTASE FROM SEVERAL SPECIES ADDED ANOTHER COMPLEX MULTIHEME C CYTOCHROME TO THE FAMILY )T SHARES ITS HEME MOTIF WITH HEMES n OF (!/ AND HEMES ))n)6 OF #YT C %INSLE ET AL 4HESE RELATION SHIPS ARE DIAGRAMED IN &IG 2EMARKABLY THE THREE CATALYTIC OR PENTACOORDINATE HEMES HEME 0 OF (!/ HEME OF .I2 AND HEME )) OF #YT C ARE HOMOLOGOUS BY THIS CRITERION OF ORIENTATION IN SPACE 4HE RELATIONSHIP BETWEEN (!/ AND .I2 IS ESPECIALLY INTERESTING SINCE THE REACTIONS INVOLVE THE SIX ELECTRON REDUCTION OF NITRITE BY .I2 OR THE PRODUCTION OF NITRITE BY THE EXTRACTION OF FOUR ELECTRONS FROM SUBSTRATE BY (!/ REPECTIVELY 4HE SUBUNIT SIZE AMINO ACID COMPOSITION (OOPER ET AL AND MUCH OF THE SECONDARY STRUCTURE IS SIMILAR IN THE TWO ENZYMES THOUGH THEY HAVE LITTLE HOMOLOGOUS SEQUENCE &URTHER .I2 IS A DIMER AND LACKS THE INTER MOLECULAR TYROSINE CROSSLINK TO THE CATALYTIC HEME )N FACT STERIC CONSID ERATIONS MIGHT MAKE FORMATION OF TWO SUCH CROSSLINKS IN A DIMER OF (!/ SUBUNITS IMPOSSIBLE )F SO THE TRIMERIC (!/ MIGHT BE THE SMALLEST OLIGOMER ALLOW
&IG !LIGNMENT OF HEMES OF A SUBUNIT OF (!/ AS IN &IG " AND THE HEMES OF #YT C AS ROTATED IN THE PLANE OF THE PAGE FROM THEIR ARRANGEMENT IN &IG $ATA OF )VERSON ET AL A B
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ING EQUIVALENT CROSSLINKS TO ALL THREE CATALYTIC HEMES (EME OF .I2 IS THE lRST EXAMPLE OF A C HEME WITH A n#89#+ MOTIF WITH THE LYSINE AS AXIAL LIGAND TO THE IRON )N THE DIMER OF .I2 SUBUNITS ASSOCIATE AT THE BASE NEAR HEME EQUIVALENT TO HEME OF (!/ (EME OF .I2 WHICH IS PROPOSED TO BE THE POINT OF ENTRY OF ELECTRONS INTO THE ENZYME %INSLE ET AL CORRESPONDS TO HEME OF (!/ )NTERESTINGLY HEME IN (!/ IS THE APPARENT ELECTRON DONOR TO HEMES AND WHOSE HOMOLOGUES ARE NOT FOUND IN .I2 OF THE EXITT PATH FROM (!/ !S WITH (!/ AND #YT C (!/ AND .I2 COULD RELATIVELY EASILY HAVE ARISEN FROM A COMMON ANCESTOR 4HIS WOULD REQUIRE EITHER ACQUISITION OR LOSS OF THE BINDING SITES FOR HEMES AND OF (!/ AND THE HEME CROSSLINK AND TUNING OF THE CATALYTIC SITE FOR DEPROTONATION AND OXIDATION OR PROTONATION AND REDUCTION RESPECTIVELY OF . OXIDES 4HE EVOLUTIONARY RELATIONSHIPS BETWEEN GENES FOR THESE PROTEINS ARE SUBJECT FOR FUTURE SPECULATION ,OOKING AT EXAMPLES OF MEMBERS OF THIS FAMILY IT IS INTERESTING TO NOTE THAT BECAUSE THE ORDER OF SPATIALLY
HOMOLOGOUS HEMES ARE CO LINEAR WITH THE AMINO ACID SEQUENCE 4ABLE MOST EVOLUTIONARY TRANSFORMA TIONS SUGGESTED CAN OCCUR WITH SIMPLE MUTATION OF A HEME LIGATION SITE ANDOR DELETIONADDITION OF TERMINAL DOMAINS AND DO NOT REQUIRE EXCISION OR INSERTION OF DOMAINS INTERNAL TO THE GENE )MPORTANTLY THE LACK OF SEQUENCE HOMOLOGY INDICATES THAT THESE ARE OLD ENZYMES WHICH DIVERGED FROM COMMON ANCESTRY LONG AGO #AUTION MUST ACCOMPANY THE HYPOTHESIS THAT THESE PARTICULAR HEME MOTIFS ARE EVOLUTIONARILY RELATED )T HAS BEEN POINTED OUT THAT GIVEN THE TWO THIOETHER HEME TO PEPTIDE LINKAGES THE HISTIDINE AXIAL IRON LIGATION FROM THE n#89#( PRIMARY SEQUENCE MOTIF AND AXIAL LIGATION OF EACH HEME WITH A SECOND (IS STERIC HINDRANCE ALLOWS FEW ORIENTATIONS OF TWO HEMES IF PACKED WITHIN A HYDROPHOBIC ENVIRONMENT SUCH THAT THE HEMES ARE LESS THAN ¯ APART "RIGE ET AL &URTHER THE OBSERVED ORIENTATIONS WITHIN THE DIHEME MOTIFS OF THE CHARACTERIZED CYTOCHROMES ARE THE ONES PREDICTED FROM THESE STERIC CONSIDERATIONS
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"4USVDUVSFPG".04VCVOJUT 4HE REACTION BASED INACTIVATOR ACETYLENE LABELS A K$A MEMBRANE PROTEIN SEEN IN DENATURING GELS (YMAN AND 7OOD !N APPARENT COMPLEX CON SISTING OF THE ACETYLENE DERIVATIZED PROTEIN !-/ ! AND A K$A PROTEIN !-/ " WAS ISOLATED "ASED ON THE . TERMINI AN OPERON CONTAINING THE GENES AMO! AND AMO" FOR THE PUTATIVE SUBUNITS !-/ ! AND n" WAS CLONED AND SEQUENCED -C4AVISH ET AL A "ERGMANN AND (OOPER B (OMOLO GOUS GENES FOR SUBUNITS OF THE PARTICULATE METHANE MONOOXYGENASE P--/ WERE SUBSEQUENTLY FOUND IN -ETHYLOCOCCUS CAPSULATUS "ATH .GUYEN ET AL TOGETHER WITH A THIRD UPSTREAM GENE PMMO# ! HOMOLOGOUS GENE AMO# WAS THEN FOUND IN THE AMMONIA OXIDIZING NITRIlERS B UPSTREAM FROM AMO" +LOTZ ET AL 4HERE ARE NO OTHER OPEN READING FRAMES IN THE AMO GENE CLUSTER OF .ITRO SOMONAS #HAIN ET AL "ASED ON AMINO ACID SEQUENCE THE SUBUNITS OF !-/ AND P--/ ARE CLEARLY IN THE SAME EVOLUTIONARY FAMILY OF ENZYMES AND DISTINCT FROM THE SOLUBLE METHANE MONOOXYGEN ASE OF THE METHYLOTROPHIC BACTERIA (OLMES ET AL (YDROPHOBICITY PLOTS OF DEDUCED AMINO ACID SEQUENCES OF THE THREE SUBUNITS PREDICT A LARGE NUMBER OF TRANSMEMBRANE SEGMENTS IN BOTH !-/ ! AND
# AND IN !-/ " &IG 2EGIONS WITH SIGNIlCANT IDENTITY FOR !-/ AND P--/ ARE FOUND IN THE LARGE . AND # TERMINAL PERIPLASMIC DOMAINS OF SUBUNIT " AS WELL AS THE MOST # TERMINAL PERIPLASMIC LOOP OF SUBUNIT ! OF !-/ 4HE LATTER LOOP IS THOUGHT TO BE CYTOPLASMIC IN A THEORETICAL SEQUENCE BASED ANALYSIS OF THE HOMOLOGOUS SUBUNIT IN P--/ OF - CAPSULATUS WHERE SEVEN TRANSMEMBRANE SEGMENTS WERE PREDICTED 4UKHVATULLIN ET AL
4HE ELECTRON DONOR TO !-/ HAS NOT BEEN IDENTIlED $UROQUINOL SERVES AS ELECTRON DONOR IN CRUDE !-/ OR PURIlED P--/ SYSTEMS 3HEARS AND 7OOD :AHN AND $I3PIRITO 4HUS THE DONOR IS HYPOTH ESIZED TO BE UBIQUINOL -ANY OBSERVATIONS SUGGEST A ROLE FOR #U ION IN !-/ 7ITH CELLS THE COPPER BINDING AGENTS ALLYLTHIOUREA +#. OR DIETHYLDITHIOCARBAMATE INHIBIT AMMONIA OXIDATION (OOPER AND 4ERRY #OPPER IONS STIMULATE AMMONIA OXIDATION IN VITRO %NSIGN ET AL 3PECIlC PHOTO INACTIVATION OF AMMONIA OXIDIZING ACTIVITY BUT NOT HYDROXYLAMINE OXIDIZING ACTIVITY IN CELLS OF .ITROSOMONAS IS CAUSED BY NM LIGHT (OOPER AND 4ERRY %NZYME ACTIVITY IS LOW IN EXTRACTS (ENCE THE PRESENT LIMITED UNDERSTANDING OF THE MECHANISM OF !-/ HAS COME FROM STUDIES WITH INTACT CELLS )N CELLS !-/ IS REACTIVE WITH A REMARKABLE VARIETY OF NON POLAR SUBSTRATES WITH A SUSCEPTIBLE # ( BOND OR / ELECTRONS 4HE LIST INCLUDES .( #/ METHANE METHANOL PROPYLENE CYCLOHEXANE BENZENE PHENOL ETHYLENE HALOGENATED ALIPHATIC COMPOUNDS HALOBEN ZENES NAPHTHALENE AND CHLORO TRICHLOROMETHYL PYRIDINE (YMAN AND 7OOD 6ANNELLI AND (OOPER +EENER AND !RP REVIEWED IN (OOPER ET AL "ASED ON THE P( OPTIMUM FOR OXIDATION AMMONIA RATHER THAN AMMONIUM MAY BE THE PREFERRED SUBSTRATE 3UZUKI ET AL 0OLAR COMPOUNDS IN GENERAL ARE NOT GOOD SUBSTRATES SUG GESTING THAT THE ACTIVE SITE IS NON POLAR 4HE BROAD RANGE OF SUITABLE SUBSTRATES SUGGESTS THAT TURNOVER IS INITIATED BY THE ACTIVATION OF OXYGEN RATHER THAN THE ACTIVATION OF SUBSTRATE )N THEORY / ACTIVATION COULD INVOLVE BINDING AND ELECTRON REDUCTION OF / BY A METAL CENTER 4HE HYDROXYLATING AGENT COULD BE THE RESULTING PEROXYL DERIVATIVE OR A METAL / DERIVATIVE PRODUCED BY HETEROLYTIC CLEAVAGE
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OF PEROXYL WITH RELEASE OF WATER - E ( / A - / (/
! HYDROGEN MAY BE EXTRACTED FROM SUBSTRATE FORM ING A PLANAR TRICOORDINATE INTERMEDIATE BY EITHER THE PEROXIDE RELEASING WATER OR THE METAL / SPECIES TO FORM A METAL HYDROXIDE AND A SUBSTRATE RADICAL 4HE @REBOUND REACTION OF THE METAL HYDROXYL AND SUBSTRATE RADICAL WOULD GENERATE METAL AND PRODUCT .( - / A ;.(= - /(
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!-/ IN CELLS OF .ITROSOMONAS CATALYZES TWO REAC TIONS IN ADDITION TO MONOOXYGENATION WHICH CAN BE RATIONALIZED IN TERMS OF THE THESE MECHANISMS 4HE DEHYDROGENATION OF ETHYLBENZENE TO FORM STYRENE +EENER AND !RP COULD INVOLVE FORMATION OF THE - / INTERMEDIATE FOLLOWED BY TWO CYCLES OF DEHYDROGENATION OF SUBSTRATE AND RELEASE OF WATER 4HE ANAEROBIC REDUCTIVE DECHLORINATION OF CHLORO TRICHLOROMETHYL PYRIDINE TO CHLORO DICHLORO METHYL PYRIDINE 6ANNELLI AND (OOPER MIGHT HAVE RESULTED FROM ORIENTATION OF THE PYRIDINE IN A WAY THAT THE RING . WAS REDUCED INSTEAD OF DIOXYGEN 4QFDVMBUJPOPOUIF(FPNFUSZPGUIF"DUJWF 4JUF #OMPARISON OF THE REACTIVITY OF VARIOUS SUBSTRATES
ALSO SUGGESTS THAT THE OXYGENATING SITE IS DEEP IN A SOMEWHAT mATTENED TUBULAR POCKET &OR EXAMPLE ALKANES OF TO CARBONS IN LENGTH ARE PREFERENTIALLY HYDROXYLATED AT THE OR POSTION (YMAN ET AL &URTHER THE SUBSTITUTED AROMATIC COMPOUND ANISOLE IS OXIDIZED PREFERENTIALLY AT THE SUBSTITUENT GROUP AND WHEN THE RING IS HYDROXYLATED DURING THE REACTION THE PROCESS OCCURS PREFERENTIALLY AT THE P RATHER THAN O OR M POSITIONS +EENER AND !RP IE THE SUBSTITUTEnD AROMATIC COMPOUNDS ENTERS THE ACTIVE SITE LEADING WITH EITHER ONE END OR THE OTHER ! SERIES OF OBSERVATIONS INDICATE THAT THE AROMATIC RING OF THE SUBSTRATE IS IMMOBILIZED IN THE ACTIVE SITE SO AS TO LIMIT ROTATION OF THE BENZYL MOIETY AND TO LIMIT ROTATION AROUND THE # BENZYL BOND &URTHER THE DATA SUGGEST THAT THE ACTIVATED OXYGEN OF THE ACTIVE SITE IS POSITIONED ASYMMETRICALLY RELATIVE TO THE TWO # #ARBONS 6ANNELLI ET AL !-/ EXHIBITS STRONGLY REGIOSELECTIVE HYDROXYLATION OF # IN PREFERENCE TO # OF ETHYLBENZENE IE THE SUBSTRATE IS ORIENTED SO THAT THE ACTIVE OXYGEN IS CLOSER TO # &IG 3UBSTITUTION OF DEUTERIUM FOR HYDROGEN ON # CHANGED THE REGIOSELECTIVITY # RATHER THAN # WAS PREFERENTIALLY HYDROXYLATED 4HIS DEUTERIUM EFFECT INDICATED THAT A # ( BOND IS BROKEN DURING CATALYSIS )N TERMS OF THE CATALYTIC MODEL THIS WOULD BE THE ABSTRACTION OF AN ( OR $ BY THE REACTIVE OXYGEN 4HE RESULT SUGGESTS THAT ALTHOUGH THE ORIENTATION OF THE SUBSTRATE BY THE ACTIVE SITE PLACED # DEUTERIUM CLOSEST TO THE REACTIVE OXYGEN AS INDICATED BY THE REGIOSELECTIVITY OF THE NON DEUTERATED SUBSTRATE THE # HYDROGEN WAS CLOSE ENOUGH THAT THE LATTER WAS
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SELECTED DUE TO THE GREATER STABILITY OF THE # $ BOND AS COMPARED WITH THE # ( BOND !DDITIONAL DETAILS OF THE GEOMETRY OF THE ACTIVE SITE IN THE VICINITY OF THE OXYGEN ACTIVATING CENTER WERE SUGGESTED BY THE RELATIVE REACTIVITY OF THE PRO2 OR PRO3 CARBONS OF THE CHIRAL SUBSTRATES 2 OR 3 ; (= ETHYLBENZENE 7ITH 3 ; (= ETHYLBENZENE THE PRO 2 POSITION WAS HYDROX YLATED APPROXIMATELY SIX FOLD MORE FREQUENTLY THAN THE PRO 3 POSITION !SPECTS OF THE MODEL ARE ILLUSTRATED IN &IG !S SUGGESTED BY THE REGIOSELECTIVITY THE HYDROGENS OF THE METHYL GROUP ARE POSITIONED FURTHER FROM THE ACTIVE OXYGEN THAN EITHER HYDROGEN ON # !S SUGGESTED BY THE ENANTIOMERIC SELECTIVITY THE 2 HYDROGEN IS POSITIONED CLOSER TO THE ACTIVE OXYGEN THAN THE 3 HYDROGEN &WJEFODFGPSB3BEJDBM3FCPVOE.FDIB OJTN 4HE HYPOTHETICAL MECHANISM INVOLVES FORMATION OF A SUBSTRATE RADICAL WHICH REBOUNDS TO REACTION WITH THE METAL HYDROXYL 4HE OBSERVATION THAT INVERSION OF HYDROGENS OCCURS DURING HYDROXYLATION OF # OF ETHYLBENZENE BY !-/ IS CONSISTENT WITH THE IN VOLVEMENT OF A TRI COORDINATE RADICAL INTERMEDIATE 6ANNELLI ET AL )N OF THE CASES WHERE THE PRO2 HYDROGEN WAS EXTRACTED INVERSION AROUND THE # BENZYL BOND WAS FOUND TO OCCUR &OLLOWING PRO3 ABSTRACTION INVERSION OCCURRED IN OR OF THE CASES RESPECTIVELY DEPENDING ON WHETHER THE SUBSTRATE WAS $ OR ( IN THE PRO3 POSITION ! SIMPLE INTERPRETATION OF THIS RESULT IS THAT SUBSTRATE OXIDA TION WAS INITIATED WHEN ( OR $ WAS ABSTRACTED BY THE ACTIVATED OXYGEN AND THAT THE RESULTING SUBSTRATE RADICAL @REBOUNDED TO THE METAL /( CENTER WITH FORMATION OF PRODUCT !CCORDING TO THIS HYPOTHESIS THE RATE OF THE REBOUND PROCESS WAS SLOW ENOUGH TO ALLOW ROTATION OF SOME FRACTION OF THE SUBSTRATE RADICALS BEFORE ATTACK BY THE HYDROXYL $PNQBSJTPOPGUIF3FBDUJWJUZPGQ..0 BOE ".0XJUI"NNPOJBBOE.FUIBOF 4HE RATE OF OXIDATION OF AMMONIA BY !-/ IS ROUGHLY TEN FOLD GREATER THAN THE RATE OF OXIDATION OF METHANE 4HIS RATIO OF RELATIVE ACTIVITY WITH THE SUBSTRATES IS REVERSED WITH P--/ )NTERESTINGLY THE !-/ OF .ITROSOCOCCUS OCEANUS WHICH LIES BETWEEN THE !-/ OF ` NITRIlERS AND P--/ OF - CAPSULATUS IN SEQUENCE HOMOLOGY HAS SIMILAR ACTIVITY WITH EITHER AMMONIA OR METHANE 7ARD .ITROSOMONAS
&IG (YPOTHETICAL ORIENTATION OF ETHYLBENZENE RELATIVE TO THE PUTATIVE ACTIVATED OXYGEN IN THE ACTIVE SITE OF !-/ /PENING OF THE LONG ACTIVE SITE IS TO THE RIGHT 4HE ACTIVE SITE BINDS SUBSTRATE SO AS TO IMMOBILIZE THE AROMATIC RING RELATIVE TO THE ACTIVATED OXYGEN TO THE LEFT 4HE PUTATIVE / IS CLOSER TO THE 2 THAN THE 3 HYDROGEN
AND .ITROSOCOCCUS OXIDIZE METHANE TO #/ AND THE ENTIRE PROCESS IS LIKELY TO BE CATALYZED BY !-/ *ONES AND -ORITA 6OYSEY AND 7OOD )T MAY BE NUTRITIONALLY SIGNIlCANT THAT THE ORGANISM ALSO INCORPORATES A SIGNIlCANT FRACTION OF THE METHANE # INTO BIOMASS *ONES AND -ORITA 7ARD !-/ OF .ITROSOMONAS IS ALSO VERY MUCH MORE REAC TIVE WITH AROMATIC COMPOUNDS THAN P--/ ,ONTOH ET AL 7**"OBFSPCJD3FTQJSBUJPOCZ"VUPUSPQIJD /JUSJàFST 6ERY SLOW GROWTH OF .ITROSOMONAS EUROPAEA UNDER ANAEROBIC CONDITIONS HAS BEEN REPORTED WITH PYRUVATE AS REDUCTANT AND NITRITE AS TERMINAL ELECTRON ACCEP TOR !BIELOVICH AND 6ONSHAK 4HE DATA IN THE GENOME REVEAL THE PRESENCE OF GENES ENCODING MANY OF THE ENZYMES NECESSARY FOR THIS PROCESS AS MEDIATED BY PYRUVATE DEHYDROGENASE THE CITRIC ACID CYCLE AND NITRITE REDUCTASE #HAIN ET AL !NAEROBICALLY .ITROSOMONAS EUTROPHA AND .ITROSOMONA EUROPAEA ARE REPORTED TO OXIDIZE HYDROGEN WHILE REDUCING NITRITE "OCK ET AL ! GENE FOR HYDROGENASE IS NOT SEEN IN THE CHROMOSOME OF THE STRAIN OF . EUROPAEA WHICH HAS BEEN SEQUENCED .ITROSOMONAS EUTROPHA IS ALSO ABLE TO GROW AN AEROBICALLY WHILE CONSUMING EQUAL AMOUNTS OF .( AND ./ AND PRODUCING A MIXTURE OF ./ ./n AND . 3CHMIDT ET AL 4HE INITIATING REACTION IS THOUGHT TO INVOLVE ./ AS OXYGEN DONOR TO !-/ RESULTING IN THE PRODUCTION OF .(/( AND ./
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!N AUTOTROPHIC BACTERIUM #ANDIDATUS "ROCADIA ANAMMOXIDANS OF THE ORDER PLANCTOMYCETES GROWS ANAEROBICALLY BASED ON THE OVERALL ENERGY GENERAT ING REACTION .( (./ A . (/
VAN DE 'RAF ET AL 3TROUS ET AL 5SE OF .( WITH (./ RESULTED IN THE PRODUC TION OF ONLY . 4HIS ELEGANT OUTCOME INDICATED THAT A SINGLE ACTIVE SITE MUST HAVE SIMULTANEOUSLY BOUND AMMONIA AND NITRITE OR THEIR RESPECTIVE INTERMEDIATE PRODUCTS -EASURABLE AMOUNTS OF HY DRAZINE AND HYDROXYLAMINE ACCUMULATED DURING THE PROCESS 4HE HYPOTHESIS WHICH IS FAVORED BY THESE INVESTIGATORS INVOLVES THREE REACTIONS REDUCTION OF NITRITE TO HYDROXYAMINE REACTION OF AMMONIA WITH HYDROXYLAMINE TO FORM HYDRAZINE AND lNALLY THE DEHYDROGENATION OF HYDRAZINE (./ En ( A .(/( (/
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4HE REACTIONS ARE PROPOSED TO BE ARRANGED IN SEPARATE MEMBRANE COMPARTMENTS SO AS TO DRIVE FORMATION OF A CHEMIOSMOTIC GRADIENT 4HE ORGANISM HAS AN UNUSUAL INTERNAL MEMBRANE SYSTEM WHICH IS EX TREMELY DIFlCULT TO ANALYZE ,INDSAY ET AL )T IS DESCRIBED AS CONSISTING OF A PERIPHERAL CYTO PLASMIC MEMBRANE INSIDE THE CELL WALL 7ITHIN THE ENCLOSED @CYTOPLASM IS A REGION CALLED THE @RIBOPLASM WHICH IS ENCLOSED BY A SECOND INTERNAL MEMBRANE THE INTRACYTOPLASMIC MEMBRANE 4HE RIBOPLASM CONTAINS RIBOSOMES AND A NUCLEOID 4HE EXTENSIVE SPACE BETWEEN THE INTRACYTOPLASMIC MEMBRANE AND CYTOPLASMIC MEMBRANE IS TERMED @PARYPHOPLASM 4HE LATTER IS SEEN TO FORM A BALLOON LIKE INTRUSION INTO THE RIBOPLASM AND PERHAPS PINCH OFF PRODUCING YET A THIRD LAYER OF CONCENTRIC VACUOLES 4HE CELLS ARE RED IN COLOR DUE TO THE PRESENCE OF AN (!/ WHICH IS ABLE TO OXIDIZE HYDROXYLAMINE OR HYDRAZINE 3CHALK ET AL -OST OF THE MO LECULAR PROPERTIES ARE VERY SIMILAR TO THOSE OF (!/ OF .ITROSOMONAS AND .ITROSOCOCCUS 4HE REDUCED AND OPTICAL SPECTRA OF THE (!/ HAVE MAJOR 3ORET
AND _ AND ` BANDS ATTRIBUTABLE TO THE APPROXIMATELY C HEMES IN A TRIMER OF K$A 4HE FERROUS SPECTRUM HAS A WEAK 3ORET BAND AT NM WHICH IS NOT SEEN IF THE FERRIC ENZYME HAD BEEN TREATED WITH HYDROGEN PEROXIDE RESULTING IN LOSS OF ENZYME ACTIV ITY 4HIS APPEARS TO CORRESPOND TO HEME 0 OF THE .ITROSOMONAS ENZYME 4HE %02 SPECTRUM CONTAINS SOME SIGNALS FROM IRON OF LOW SPIN C HEMES AS WELL AS ATYPICAL SIGNALS POSSIBLY REPRESENTING ELECTRONI CALLY COUPLED HEME IRONS 4HE ENZYME IS THOUGHT TO AT LEAST ACCOUNT FOR THE OXIDATION OF HYDRAZINE TO DINITROGEN IN THE ORGANISM AND POSSIBLY CATALYZE THE REDUCTION OF NITRITE TO ./ 4HE HEME COMPOSITION AND CATALYTIC AND %02 PROPERTIES OF THE (!/S OF !.!--/8 AND OF .I TROSOMONAS ARE SIMILAR ENOUGH TO SUGGEST THAT THE HEMES OF THE TWO PROTEINS MIGHT ULTIMATELY BE FOUND TO ALIGN IN SIMILAR SPATIAL CONlGURATIONS *9)FUFSPUSPQIJD/JUSJàDBUJPO 4HE OXIDATION OF AMMONIA TO NITRITE ALSO OCCURS IN THE HETEROTROPHIC BACTERIA 4HE RATES OF OXIDATION OF AMMONIA PER CELL ARE LOWER THAN WITH THE AUTOTROPHIC NITRIlERS "IOCHEMICAL CHARACTERIZATION OF THE REAC TIONS INVOLVED HAS NOT BEEN EXTENSIVE !N AMMONIA MONOOXYGENASE FROM MEMBRANES OF 0ARACOCCUS DENITRIlCANS HAS BEEN SOLUBILIZED AND PURIlED TO A FORM CONTAINING SUBUNITS OF AND K$A -OIR ET AL 4HE ASSAY EMPLOYED DUROQUINOL AS REDUCTANT 4HE ENZYME RESEMBLED !-/ FROM .ITRO SOMONAS IN SENSITIVITY TO LIGHT AND COPPER BINDING AGENTS 4HE OXIDATION OF AMMONIA TO HYDROXYLAMINE NITRITE AND NITRATE BY 0SEUDOMONAS PUTIDA WAS SEEN TO BE INACTIVATED BY ACETYLENE $AUM ET AL !N OPEN READING FRAME IDENTIlED BY ITS HYBRIDIZATION WITH A PROBE FOR AMO! CONTAINED SEQUENCE SIMILARITY TO THE GENE IN AUTOTROPHIC NITRIlERS 4HUS IT APPEARS THAT !-/ OF THE !-/P--/ FAMILY IS UTILIZED BY MANY ORGANISMS )N CONTRAST THE HYDROXYLAMINE OXIDIZING ENZYMES THAT HAVE BEEN ISOLATED THUS FAR FROM HETEROTROPHIC BACTERIA DO NOT RESEMBLE (!/ OF THE AUTOTROPHIC NITRIlERS !N ENZYME FROM !R R THROBACTER GLOBIFORMIS +UROKAWA ET AL HAS HYDROXYLAMINE DYE REDUCTASE ACTIVITY OR HYDROXYL AMINE CYTOCHROME C REDUCTASE ACTIVITY BUT DOES NOT CONTAIN HEME ! SIMILAR ACTIVITY FROM 0ARACOCCUS DENITRIlCANS APPEARS TO BE CATALYZED BY AN IRON SULFUR PROTEIN -OIR ET AL
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"DLOPXMFEHNFOUT 4HE AUTHORS THANK -ARK 7HITTAKER 4OM 0OULUS 4ODD 6ANNELLI AND 0AUL +LUGE FOR DISCUSSIONS AND HELP IN PREPARATION OF THE MANUSCRIPT 4HIS WORK WAS SUPPORTED BY FUNDS FROM THE 53 .ATIONAL 3CIENCE &OUNDATION .3&-#" AND 53 $EPART MENT OF %NERGY $% &' %2 TO !"( AND FUNDS FROM .)( TO -0( 3FGFSFODFT !BIELOVICH ! AND 6ONSHAK ! !NAEROBIC METABOLISM OF .ITROSOMONAS EUROPAEA !RCH -ICROBIOL n !NDERSON )# AND ,EVINE *3 L 2ELATIVE RATES OF NITRIC OXIDE AND NITROUS OXIDE PRODUCTION BY NITRIlERS DENITRIlERS AND NITRATE RESPIRERS !PPL AND %NVIRON -ICROBIOL n !NDERSSON ++ AND (OOPER !" / AND (/ ARE EACH THE SOURCE OF ONE / OF (./ PRODUCED FROM .( BY .ITROSOMONAS . .-2 EVIDENCE &%"3 ,ETT n !NDERSSON ++ ,IPSCOMB *$ 6ELENTINE - -UNCK % AND (OOPER !" 4ETRAHEME CYTOCHROME C FROM .ITROSOMONAS EUROPAEA HEME HEME INTERACTIONS AND LIGAND BINDING * "IOL #HEM n !RCIERO $- AND (OOPER !" A (YDROXYLAMINE OXIDO REDUCTASE FROM .ITROSOMONAS EUROPAEA IS A MULTIMER OF AN OCTA HEME SUBUNIT * "IOL #HEM n !RCIERO $- AND (OOPER !" ! DI HEME CYTOCHROME C PEROXIDASE FROM .ITROSOMONAS EUROPAEA CATALYTICALLY ACTIVE IN BOTH THE OXIDIZED AND HALF REDUCED STATES * "IOL #HEM n !RCIERO $- AND (OOPER !" %VIDENCE FOR A PEPTIDYL LY SINE TO C HEME CROSSLINK IN CYTOCHROME 0 OF .ITROSOMONAS EUROPAEA &%"3 ,ETT n !RCIERO $- AND (OOPER !" #ONSIDERATION OF A PHLORIN STRUCTURE FOR HAEM 0 OF HYDROXYLAMINE OXIDOREDUCTASE AND ITS IMPLICATIONS REGARDING REACTION MECHANISM "IOCHEM 3OC 4RANS n !RCIERO $- AND (OOPER !" (EME 0 OF HYDROXYLAMINE OXIDOREDUCTASE -ODELS FOR CATALYSIS BASED ON DIHYDROPORPHYRIN AND ISOPORPHYRIN RING SYSTEMS * )NORG "IOCHEM !RCIERO $ "ALNY # AND (OOPER !" A 3PECTROSCOPIC AND RAPID KINETIC STUDIES OF REDUCTION OF CYTOCHROME C BY HYDROXYLAMINE OXIDOREDUCTASE FROM .ITROSOMONAS EUROPAEA "IOCHEMISTRY n !RCIERO $- #OLLINS - (ALADJIAN * "IANCO 0 AND (OOPER !" B 2ESOLUTION OF THE FOUR HEMES OF CYTOCHROME C FROM .ITROSOMONAS EUROPAEA BY REDOX POTENTIOMETRY AND OPTICAL SPECTROSCOPY "IOCHEMISTRY n !RCIERO $- (OOPER !" #AI - AND 4IMKOVICH 2 %VIDENCE FOR THE STRUCTURE OF THE ACTIVE SITE HEME 0 IN HYDROXYLAMINE OXIDOREDUCTASE OF .ITROSOMONAS "IOCHEMISTRY n !RCIERO $- 0IERCE "3 (ENDRICH -0 AND (OOPER !" .ITROSOCYANIN A RED CUPREDOXIN LIKE PROTEIN FROM M .ITROSOMONAS EUROPAEA "IOCHEMISTRY n "EAUMONT (%" (OMMES .' 3AYAVEDRA 3OTO ,! !RP $* !RCIERO $* (OOPER !" 7ESTERHOFF (6 AND 6AN 3PANNING
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4VNNBSZ 4HIS CHAPTER DEALS WITH THE OXIDATION OF AMMONIA .( / A (./ (/ AS A SOURCE OF REDUCING POWER IN THE CHEMOLITHOTROPHIC BACTERIUM .ITROSOMONAS EUROPAEA $IRECT KNOWLEDGE OF THE ENZYMES INVOLVED TOGETHER WITH THE SEQUENCE OF THE GENOME REVEAL CORE ELEMENTS OF A REDOX SYSTEM UNIQUE TO OXIDATION OF AMMONIA TO NITRITE WHICH FEEDS INTO A MORE TRADITIONAL BACTERIAL ELECTRON TRANSPORTTERMINAL OXIDASE SYSTEM 4HE APPARENTLY LOW STOICHIOMETRY OF PROTONS TRANSLOCATED PER AMMONIA OXIDIZED HINTS AT THE BASIS OF THE LOW GROWTH YIELDS OF THIS BACTERIUM 2EMARKABLY THE PUTATIVE COMPLEX OF HYDROXYLAMINE OXIDOREDUCTASE (!/ CYTOCHROME C #YT C AND THE MEMBRANE CYTOCHROME C- #YT C- WHICH CATALYZES THE OXIDATION OF A MOLECULE OF HYDROXYLAMINE AND TRANSFER OF FOUR ELECTRONS TO MEMBRANE UBIQUINONE WOULD INVOLVE C HEMES PER CATALYTIC SITE OR HEMES FOR THE HYPOTHETICAL AGGREGATE CONTAINING THE TRIMERIC (!/ 4HE DEHYDROGENATION CATALYZED AT THE NOVEL CATALYTIC HEME HEME 0 IS UNIQUE BY COMPARISON WITH OTHER KNOWN CATALYTIC HEMES WHICH BIND SUBSTRATE TO THE IRON IN ALL OTHERS ELECTRONS ENTER THE SYSTEM AND REDUCE THE SUBSTRATE WHEREAS THE REVERSE IS TRUE WITH (!/ 4HIS MODE OF CATALYSIS MAY BE FUNCTIONALLY RELATED TO A CROSS LINK WHICH IS FOUND ONLY IN (!/ A COVALENT BOND BETWEEN A METHYNE CARBON OF HEME 0 AND A RING CARBON OF A PEPTIDE TYRO SINE 4HE DRAMATIC CRYSTAL STRUCTURES OF (!/ AND #YT C HAVE PROVIDED INSIGHTS INTO CATALYSIS AND ELECTRON TRANSFER AS WELL AS ILLUSTRATING EVOLUTIONARY RELATIONSHIPS WHICH ARE NOT REmECTED IN HOMOLOGY OF AMINO ACID SEQUENCE #ONSIDERING THEIR RELATIVE SPATIAL ARRANGEMENT THE HEMES OF #YT C CAN BE PRECISELY SUPERIMPOSED WITH OF THE HEMES OF (!/ %VIDENCE SUGGESTS THAT THEY HAVE A COMMON ANCESTOR AND HAVE PRESERVED HEME CONlGURATIONS EVEN WHEN SEQUENCE HOMOLOGY HAD BEEN LOST 4HE NOVEL ANAEROBIC OXIDATION OF AMMONIA .( (./ A . (/ BY A PLANCTOMYCETE BACTERIUM AND THE OXIDATION OF AMMONIA TO NITRITE IN HETEROTROPHIC BACTERIA ARE DESCRIBED MORE BRIEmY !BBREVIATIONS !-/ n AMMONIA MONOOXYGENASE !.!--/8 n ANAEROBIC AMMONIA OXIDATION ##0 n CYTOCHROME C PEROXIDASE #U .I2 n COPPER CONTAINING NITRITE REDUCTASE #YT n CYTOCHROME #YT C- n MEMBRANE CYTOCHROME C- OF .ITROSOMONAS #YT AA COMPLEX n HEME AA CONTAINING TERMINAL OXIDASE #YT BC COM PLEX n UBIQUINOL CYTOCHROME C OXIDO REDUCTASE %02 n ELECTRON PARAMAGNETIC RESONANCE (!/ n HYDROXYLAMINE OXIDOREDUCTASE .(% n NORMAL HYDROGEN ELECTRODE .I2 n PENTA HEME CYTOCHROME C TYPE NITRITE REDUCTASE . SIDE n NEGATIVELY CHARGED REGION CYTOPLASMIC SIDE OF THE PLASMA MEMBRANE /2& n OPEN READING FRAME 0 SIDE n POSITIVELY CHARGED REGION PERIPLASMIC SIDE OF THE PLASMA MEMBRANE
**OUSPEVDUJPO.FUBCPMJTNPG /JUSPTPNPOBT 4HIS CHAPTER DEALS WITH THE BIOCHEMISTRY OF THE OXI DATION OF AMMONIA AS A SOURCE OF REDUCING POWER FOR ENERGY TRANSDUCTION AND BIOSYNTHESIS IN BACTERIA )T WILL FOCUS ON THE AEROBIC OXIDATION OF AMMONIA TO NITRITE .( / A (./ (/ IN THE CHEMOLITHOTROPHIC BACTERIUM .ITROSOMONAS EURO
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB PAEA SINCE THAT PROCESS HAS BEEN MOST EXTENSIVELY CHARACTERIZED 4HE BACTERIAL ANAEROBIC OXIDATION OF AMMONIA !.!--/8 PATHWAY .( (./ A . (/ AND THE OXIDATION OF AMMONIA TO NITRITE IN HETEROTROPHIC BACTERIA WILL BE REVIEWED BRIEmY 4HE OBLIGATE AEROBIC CHEMOLITHOTROPHIC AMMONIA OXIDIZING BACTERIA THOUGHT TO ACCOUNT FOR THE GREATER FRACTION OF AMMONIA OXIDATION IN NATURE ARE FOUND IN THE ` AND a SUBDIVISIONS OF THE PROTEOBACTERIA 4ESKE ET AL 4HE SPECIES IN THE ` SUBDIVISION INCLUD ING .ITROSOMONAS EUROPAEA ARE CLOSELY RELATED AND FOUND IN MARINE AND FRESH WATERS AND IN SOILS 4HE a SUBDIVISION IS REPRESENTED BY THE OBLIGATE MARINE FORM .ITROSOCOCCUS OCEANUS !LTHOUGH UNABLE TO REDUCE NITRATE TO NITRITE OR ./ TO . AT LOW CONCENTRATIONS OF / THESE ORGANISMS CATALYZE DISSIMILATORY REDUCTION OF NITRITE TO ./ AND ./ TO ./ AND MAY BE A MAJOR SOURCE OF THESE GASES IN NATURE 'OREAU ET AL !NDERSON AND ,EVINE #ONRAD 4HE OXIDATION OF AMMONIA TO NITRITE BY .ITROSOMO NAS IS THE SOLE SOURCE OF REDUCTANT FOR BIOSYNTHESIS AND FOR CREATION OF A PROTON GRADIENT FOR ENERGY TRANSDUC TION 'ROWTH OCCURS IN MEDIA CONTAINING ONLY .( AS REDUCTANT AND . SOURCE #/ AS # SOURCE 3/n AS 3 SOURCE 0/nn AND METAL IONS )N LABORATORY CULTURE THE DOUBLING TIME IS APPROXIMATELY HOURS $URING GROWTH THE MEDIA IS ACIDIlED BY NITROUS ACID 4HE POPULATION DENSITY CAN APPROACH CELLSML IN A CHEMOSTAT WITH P( CONTROL ,OGAN ET AL 4HE GENOME OF . EUROPAEA HAS BEEN SEQUENCED #HAIN ET AL AND REVEALS THAT THE ORGANISM IS HIGHLY SPECIALIZED FOR AN AUTOTROPHIC MODE OF LIFE REQUIRING GENES ENCODING THE . OXIDIZING SYSTEMS AS WELL AS BIOGENESIS OF ALL CELLULAR COMPONENTS FROM INORGANIC . # AND 3 BUT ALMOST NONE OF THE PATHWAYS FOR THE OXIDATION OF ORGANIC MOLECULES "ASED ON INFORMATION FROM THE GENOME lXATION OF #/ IS BY THE #ALVIN "ENSON CYCLE AND THE CITRIC ACID CYCLE IS PRESENT .( IS ASSIMILATED BY WAY OF GLUTAMATE DEHYDROGE NASE .ITROSOMONAS IS ABLE TO REDUCTIVELY ASSIMILATE NITRITE . BUT NOT NITRATE . INTO PROTEIN 7ALLACE AND .ICHOLAS 4HIS REVIEW DEALS WITH PATHWAYS FOR THE OXIDATION OF AMMONIA AND PRODUCTION OF REDUCING POWER .!$( AND !40 FOR BIOSYNTHESIS 0REVIOUS REVIEWS FOCUSING ON THE BIOCHEMISTRY OF THE OXIDATION OF AMMONIA AND RELATED ELECTRON TRANSFER AND ENERGY TRANSDUCTION IN .ITROSOMONAS HAVE APPEARED 7OOD 6ANNELLI ET AL (OOPER ET AL 7HIT TAKER ET AL
**4VNNBSZPGUIF0YJEBUJWF1BUIXBZT "NNPOJB.POPPYZHFOBTF )ZESPYZMBNJOF 0YJEPSFEVDUBTFBOEUIF1BTTBHFPG&MFD USPOTUP6CJRVJOPOF ""O0WFSWJFXPGUIF1SPDFTT !MMONIA IS OXIDIZED TO HYDROXYLAMINE .( En / ( A .(/( (OLLOCHER BY THE ENZYME AMMONIA MONOOXYGENASE !-/ (YDROXYL AMINE IS OXIDIZED TO NITRITE .(/( (/ A (./ En ( !NDERSSON AND (OOPER BY THE EN ZYME HYDROXYLAMINE OXIDOREDUCTASE (!/ &IG 4HE OXIDATION OF HYDROXYLAMINE MAY BE THOUGHT OF AS A DEHYDROGENATION OF SUBSTRATE SINCE ELECTRONS AND PROTONS ARE THE PRODUCT 4HE REACTION IS THE SOURCE OF ALL ELECTRONS REQUIRED FOR REDUCTIVE REACTIONS IN THE ORGANISM &OR EACH TURNOVER OF (!/ THE EQUIVALENT OF TWO ELECTRONS REMOVED FROM HYDROXYLAMINE MUST BE EXPENDED IN THE !-/ REACTION 4HE !-/ REACTION YIELDS NO ELECTRONS AND THERE IS NO EVIDENCE TO DATE THAT AS A MEMBRANE REDOX ENZYME IT CAN CONTRIBUTE TO A CHEMIOSMOTIC GRADIENT DURING TURNOVER 4HUS THE ROLE OF !-/ IS THE PRODUCTION OF THE PRIMARY ENERGY GENERATING MOLECULE HYDROXYLAMINE /NLY TWO OF THE FOUR ELECTRONS FROM THE TURNOVER OF A MOLECULE OF (!/ ARE THUS AVAILABLE FOR THE REDUCTIVE REACTIONS OF THE CELL THEY MIGHT BE CALLED @USABLE ELECTRONS /F THE TWO THEORETICAL @USABLE ELECTRONS AN ESTIMATED APPROXIMATELY TWO ELECTRONS EVERY SIX TURNOVERS PASS TO .!$ FOR USE IN THE REDUCTIVE REACTIONS OF BIOSYNTHESIS 7OOD )T IS ASSUMED THAT .!$( DEHYDROGENASE WHICH IS FOUND TO BE ENCODED IN THE GENOME #HAIN ET AL CATALYZES ENERGY DE PENDENT @REVERSE ELECTRON TRANSFER FROM UBIQUINOL TO .!$ 4HE REMAINING ELECTRON EQUIVALENTS THE USABLE PAIR OF ELECTRONS IN THE OTHER lVE TURNOVERS OF (!/ WILL ULTIMATELY PASS TO TERMINAL ELECTRON AC CEPTORS INCLUDING ONLY / NITRITE OR ./ CATALYZED BY A TERMINAL OXIDASE NITRITE REDUCTASE OR ./ REDUC TASE RESPECTIVELY UNLESS THEY SERVE AS REDUCTANT IN DETOXIlCATION OF POTENTIALLY TOXIC FORMS OF / SUCH AS (/ THE ASSIMILATORY REDUCTION OF SULFATE OR NITRITE OR THE PHYSIOLOGICALLY FUTILE OXYGENATION OF ORGANIC MOLECULES BY !-/ SEE BELOW 4HE ELECTRON TRANSFER REACTIONS RELATED TO . OXIDA TION AND ENERGY TRANSDUCTION IN .ITROSOMONAS CONSIST OF A PATHWAY UNIQUE TO AMMONIA OXIDIZING NITRIlERS IN WHICH ELECTRONS FROM HYDROXYLAMINE ARE FED INTO MEMBRANE UBIQUINONE AND THEN EITHER RETURNED TO
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!-/ OR CHANNELED INTO A SECOND @TRADITIONAL RE DOX AND ENERGY TRANSDUCING PATHWAY STARTING WITH UBIQUINOL #.FNCSBOF4ZTUFNTPG/JUSPTPNPOBT .ITROSOMONAS IS GRAM NEGATIVE AND HAS AN EXTENSIVE INTERNAL MEMBRANE SYSTEM PRODUCED BY INVAGINATION OF THE PLASMA MEMBRANE -URRAY AND 7ATSON GIVING RISE TO A LARGE SURFACE AREA OF MEMBRANE AND AN EXTENSIVE PERIPLASMIC SPACE )T IS LIKELY THAT THE ENZYMES OF ENERGY TRANSDUCTION IN .ITROSOMONAS ARE IN THE INTERNAL REGIONS OF THE MEMBRANE AND PERIPLASM (OOPER ET AL A $I3PIRITO ET AL A AS IS FOUND WITH THE ANALOGOUS ENZYMES OF METHANE OXIDATION IN -ETHYLOMICROBIUM ALBUM "RANTNER ET AL $5IF/PYJEBUJPO1BUIXBZ 4HE MAJORITY OF ENZYMES AND ELECTRON TRANSFER CARRI ERS UNIQUE TO . OXIDATION IN .ITROSOMONAS ARE NOW KNOWN AND MANY HAVE BEEN EXTENSIVELY CHARACTERIZED (!/ IS A TRIMER EACH SUBUNIT CONTAINS EIGHT C HEMES HEMES CROSSLINKED TO THE PROTEIN BY TWO THIOETHER LINKAGES TO PEPTIDE CYSTEINES /NE OF THE C HEMES THE CATALYTIC HEME 0 HAS A THIRD COVALENT BOND A CROSSLINK TO A TYROSINE OF THE PROTEIN %ACH (!/ SUBUNIT HAS A BINDING SITE FOR ITS ELECTRON ACCEPTOR THE TETRAHEME CYTOCHROME C #YT C %LECTRONS
MAY THEN PASS TO THE TETRAHEME MEMBRANE CYTO CHROME C- #YT C- AND THEN TO UBIQUINONE 4HE PUTATIVE (!/ TO UBIQUINONE ELECTRON TRANSFER PATHWAY STARTING WITH A MOLECULE OF HYDROXYLAMINE IN EACH OF THE THREE ACTIVE SITES OF (!/ INCLUDES C HEMES AND CAN IN THEORY REDUCE MOLECULES OF UBIQUINONE TO UBIQUINOL PER TURNOVER )T IS THOUGHT THAT UBIQUINOL IS THE ELECTRON DONOR TO !-/ "ECAUSE IT IS NOT ACTIVE IN EXTRACTS !-/ HAS NOT BEEN WELL CHARACTERIZED "ASED ON ANALYSIS OF THE SEQUENCE OF A GENE ENCODING A PROTEIN DERIVATIZED BY ACETYLENE A MECHANISM BASED INHIBITOR OF !-/ AND ADJOINING GENE CLUSTER !-/ APPEARS TO HAVE THREE SUBUNITS 4HE TRANSLATED PROTEINS ARE PREDICTED TO HAVE A LARGE NUMBER OF TRANSMEMBRANE DOMAINS 4HERE ARE SEVERAL COPIES OF SEVERAL OF THE GENES INVOLVED IN AMMONIA OXIDATION -C4AVISH ET AL B 4HE GENE CLUSTERS CONTAINING !-/ SUBUNITS (!/ CYTOCHROME C AND CYTOCHROME C- DO NOT SEEM TO HAVE ADDITIONAL /2&S #HAIN ET AL (ENCE ALL OR MOST OF THE PROTEINS INVOLVED WITH THE OXIDATION OF AMMONIA MAY HAVE BEEN IDENTIlED %5IFA5SBEJUJPOBM3FEPYBOE&OFSHZ5SBOT EVDJOH1BUIXBZTPG/JUSPTPNPOBT 4HE COMPONENTS OF THE @TRADITIONAL OR @UNDERLY ING 7OOD ENERGY TRANSDUCING PATHWAY IN .ITROSOMONAS HAVE PROPERTIES VERY SIMILAR TO THE EQUIVALENT ENZYMES OF HETEROTROPHIC BACTERIA !S IN
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB MANY BACTERIAL ELECTRON TRANSPORT PATHWAYS UBIQUINOL IS A MOBILE ELECTRON CARRIER AND A BRANCHING POINT TO THE !-/ SYSTEM TO THE .!$ REDUCTASE AND TO THE TERMINAL ELECTRON ACCEPTOR PATHWAYS $ZUPDISPNFCD 'ENES FOR ALL PROTEINS OF A PROTON DEPENDENT UBI QUINOL CYTOCHROME C REDUCTASE CYTOCHROME BC COMPLEX ARE FOUND IN A SINGLE GENE CLUSTER OF THE GENOME #HAIN ET AL 4HE AEROBIC OXIDATION OF EITHER HYDROXYLAMINE OR AMMONIA BY CELLS OF .I TROSOMONAS IS INHIBITED BY CLASSIC INHIBITORS OF THE UBIQUINOL CYTOCHROME C REDUCTASE SUCH AS ANTIMYCIN ! MYXOTHIAZOL OR STIGMATELLIN INDICATING THAT THE BC FUNCTIONS @DOWNSTREAM FROM (!/ IN THE ELECTRON TRANSPORT PATHWAY 7HITTAKER ET AL $ZUPDISPNFD #YTOCHROME C 9AMANAKA AND 3HINRA IS A PERIPLASMIC MONO HEME C CYTOCHROME WHICH IS HOMOLOGOUS TO THE C FAMILY OF BACTERIAL CYTO CHROMES AND MITOCHONDRIAL C )TS GENE IS PRESENT IN A SINGLE COPY WHICH IS NOT IN ANY OF THE THREE GENE CLUSTERS CONTAINING (!/ #YT C AND #YT C- #YT C IS THOUGHT TO MEDIATE ELECTRON TRANSFER BETWEEN THE #YT BC COMPLEX AND THE AA TERMINAL OXIDASE $I3PIRITO ET AL 'ENETIC OR KINETIC EVIDENCE HAS NOT ESTABLISHED THE ROLE FOR CYTOCHROME C AL THOUGH THE VALUE OF REDUCTION POTENTIAL M6 9AMANAKA AND 3HINRA IS IN KEEPING WITH THIS FUNCTION #YTOCHROME C IS ALSO ABLE TO RAPIDLY RE DUCE CYTOCHROME C PEROXIDASE !RCIERO AND (OOPER AND SLOWLY REDUCE THE COPPER CONTAINING NITRITE REDUCTASEOXIDASE $I3PIRITO ET AL B IN VITRO 4HUS DISTRIBUTION OF ELECTRONS TO ALTERNATE CARRIERS MAY ALSO BE A FUNCTION OF #YT C
AMMONIA TO NITRITE AND YET STIMULATE THE OXIDATION OF HYDROXYLAMINE TO NITRITE AS CATALYZED BY INTACT CELLS (OOPER AND 4ERRY 4HIS STRONGLY SUGGESTS THAT WHEN THE mUX OF ELECTRONS FROM UBIQUINONE THROUGH THE #YT BC COMPLEX AND THE #YT AA OXIDASE COMPLEX IS NOT RETARDED BY THE STEADY STATE CHEMIOSMOTIC GRADIENT ALL OR MOST OF ELECTRONS FROM UBIQUINOL GO TO THE TERMINAL OXIDASE PATHWAY RATHER THAN TO !-/ 7HITTAKER ET AL 4HE RESULTS INDICATE THAT IN THE ABSENCE OF UNCOUPLER !-/ CATALYSES THE RATE LIMITING STEP IN THE CONVERSION OF AMMONIA TO NITRITE 4HE PRESENCE OF ARTIlCIAL ELECTRON ACCEPTORS EG PHENAZINE METHOSULFATE OR METHYLENE BLUE (OOPER AND 4ERRY ALSO SELECTIVELY INHIBIT AMMONIA OXIDATION AND STIMULATE THE RATE OF OXIDA TION OF HYDROXYLAMINE IN WHOLE CELLS 4HIS EFFECT IS ALSO PROBABLY CAUSED BY DEPRIVING !-/ OF A mUX OF ELECTRONS )T WILL BE INTERESTING TO KNOW HOW THE CELL REGULATES .!$( DEHYDROGENASE ACTIVITY 'IVEN THE LARGE EX PENDITURE OF ENERGY REQUIRED TO REDUCE .!$ NOTED BELOW ITS RE OXIDATION BY THE ELECTRON TRANSPORT CHAIN AND TERMINAL OXIDASE WOULD RESULT IN A NET LOSS TO THE ORGANISM 'ENES ARE FOUND WHICH ENCODE ALL ENZYMES OF THE CITRIC ACID CYCLE #HAIN ET AL .O ACTIVITY OF _ KETOGLUTARATE DEHYDROGENASE WAS FOUND IN EXTRACTS (OOPER 4HIS APPARENT ABSENCE WAS ONCE RELATED TO A POSSIBLE BIOCHEMICAL BASIS OF OBLIGATE AUTOTROPHY WHICH STILL ALLOWED SYNTHESIS OF AMINO ACIDS DEPENDENT ON THE OTHER ENZYMES OF THE CYCLE )T REMAINS TO BE SEEN WHETHER ALL GENES ARE EXPRESSED )T IS LIKELY THAT MEMBRANES CONTAIN SUCCINATE DEHY DROGENASE &.JOPS&MFDUSPO5SBOTGFS1BUIXBZTUP5FSNJ OBM&MFDUSPO"DDFQUPST 0YJEBUJPOPG"MUFSOBUF4VCTUSBUFTCZ".0
$V"5FSNJOBM0YJEBTF ! #U! TYPE CYTOCHROME AA OXIDASE HAS BEEN ISO LATED $I3PIRITO ET AL AND GENES ENCODING ALL SUBUNITS HAVE BEEN IDENTIlED IN THE GENOME $POUSPMBOE*OUFHSBUJPOPG&MFDUSPO5SBOTQPSU 1BUIXBZT 0ROTONOPHORE UNCOUPLERS OF OXIDATIVE PHOSPHORYLA TION SUCH AS DINITROPHENOL OR M CHLOROCARBONYL CYANIDEPHENYLHYDRAZONE INHIBIT THE OXIDATION OF
)N VIVO AMMONIA MONOOXYGENASE OF .ITROSOMONAS IS ABLE TO OXIDIZE MANY ORGANIC COMPOUNDS (OOPER ET AL .OTE THAT TWO OF THE @USABLE ELECTRONS ARE USED PER TURNOVER OF !-/ 4HERE IS NO EXAMPLE WHERE THE PRODUCT OF THE REACTION CAN BE UTILIZED FOR ENERGY TRANSDUCTION HENCE THIS IS AN ENERGETICALLY UNPRODUCTIVE ACTIVITY :&IG "MUFSOBUF0YJEBTFT )T IS NOTABLE THAT .ITROSOMONAS HAS SO FEW OPTIONAL
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TERMINAL OXIDASES 'ENES ENCODING NO OTHER MEMBERS OF THE MEMBRANE BOUND SUPER FAMILY OF HEME COPPER RESPIRATORY OXIDASES ARE FOUND "Y (0,# ANALYSIS HEME O IS NOT PRESENT 4HE ABSENCE OF A QUINOL OXIDASE IN THIS FAMILY SUGGESTS THAT THE CONTRIBUTION TO THE PROTON GRADIENT OF THE TURNOVER OF THE #YT BC COM PLEX IS VERY IMPORTANT .ITROSOMONAS HAS A SOLUBLE PERIPLASMIC COPPER PROTEIN WHICH CATALYSES OXIDATION OF DYES BY OXYGEN OR NITRITE -ILLER AND 7OOD $I3PIRITO ET AL B )TS POSSIBLE ROLE IN REDUCTION OF DIOXYGEN AS A TERMINAL ELECTRON ACCEPTOR IS UNDER INVESTIGATION %FOJUSJàDBUJPO 4HE ./ AND ./ PRODUCED DURING OXIDATION OF AM MONIA BY .ITROSOMONAS AT LOW CONCENTRATIONS OF OXYGEN CAN ACCOUNT FOR OR MORE OF THE .( n. OXIDIZED ,IPSCHULTZ ET AL 0RODUCTION OF ./ OR ./ IN VIVO APPEARS TO OCCUR BY THE REDUCTION OF NITRITE PRODUCED BY (!/ RATHER THAN THE INCOMPLETE OXIDATION OF HYDROXYLAMINE BY (!/ 0OTH AND &O CHT (OOPER ET AL 2EMDE AND #ONRAD ! SOLUBLE ENZYME WITH NITRITE REDUCTASE AND DYE OXIDASE ACTIVITY HAS BEEN ISOLATED $I3PIRITO ET AL B )NTERESTINGLY THE GENE FOR THIS BLUE COPPER OXIDASENITRITE REDUCTASE IS IMMEDIATELY UPSTREAM OF GENES FOR A MONOHEME C CYTOCHROME AND A DIHEME C CYTOCHROME BOTH OF WHICH ARE ALSO EXPRESSED AND SOLUBLE AND THEN A NIR+ TYPE GENE FOR A COPPER CONTAINING NITRITE REDUCTASE 4HE ROLE OF THE PROTEINS ENCODED BY THIS @NIR+GENE CLUSTER IS
UNDER INVESTIGATION "EAUMONT ET AL CURRENT SPECULATION IS THAT BOTH ENZYMES CAN CONTRIBUTE TO DENITRIlCATION )N KEEPING WITH PHYSIOLOGICAL OBSER VATIONS GENES ENCODING A ./ REDUCTASE ARE IN THE GENOME WHEREAS GENES FOR NITRATE OR ./ REDUCTASE ARE NOT #HAIN ET AL 1PTTJCMF#ZQBTTPGUIF$ZUCD $PNQMFY )N VIVO INHIBITION OF THE OXIDATION OF AMMONIA OR HYDROXYLAMINE BY ANTIMYCIN ! MYXOTHIAZOL OR STIGMATELLIN IS NOT COMPLETE (ENCE UNDER CERTAIN CIRCUMSTANCES A SMALL FRACTION OF THE mUX OF ELECTRONS FROM (!/ CAN POSSIBLY BYPASS THE #YT BC COMPLEX !LTHOUGH THE 8 RAY STRUCTURE OF CYTOCHROME CSHOWS A DOCKING SITE FOR (!/ BUT NOT FOR CYTOCHROME C )VERSON ET AL IN VITRO ELECTRON mOW FROM HYDROXYLAMINE TO CYTOCHROME C IS EXTREMELY RAPID IN THE PRESENCE OF CATALYTIC AMOUNTS OF (!/ AND CYTOCHROME C 9AMANAKA AND 3HINRA 4HIS COULD IN THEORY PROVIDE A BYPASS OF THE BC COMPLEX (OWEVER THE RESPONSE OF CELLS TO INHIBITORS OF THE BC COMPLEX INDICATES THAT THIS ACTIVITY IS NOT NEARLY AS RAPID IN VIVO AS IN VITRO ! SECOND CANDIDATE FOR A BYPASS OF THE #YT BC COMPLEX WOULD BE ELECTRON TRANSFER THROUGH A CYTOCHROME C LIKE PROTEIN WHICH IS PRESENT IN MEMBRANES OF .ITROSOMONAS AT ON A MOLAR BASIS RELATIVE TO THE CORE PROTEINS OF . OXIDATION 7HITTAKER ET AL %XAMPLES OF CYTOCHROME C IN OTHER ORGANISMS ARE THOUGHT TO PARTICIPATE IN ELECTRON TRANSFER FROM UBIQUINOL TO CYTOCHROMES AS PART OF A PATH LEADING TO A TERMINAL OXIDASE
&IG !LTERNATE FATES OF THE HYPOTHETICAL @USABLE TWO ELECTRONS FROM THE (!/ REACTION @5SABLE REFERS TO THE OF ELECTRON EQUIVALENTS PER CATALYTIC CYCLE OF (!/ WHICH ARE NOT OBLIGATORILY CONSUMED BY THE !-/ REACTION $ASHED LINES INDICATE ELECTRON TRANSFER STEPS WHICH HAVE NOT BEEN CONCLUSIVELY ESTABLISHED BY EXPERIMENT
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB .PMBS3BUJPPG3FEPY$PNQPOFOUT 4HE RELATIVE AMOUNTS OF ELECTRON TRANSFER COMPONENTS ARE APPROXIMATELY (!/ SUBUNIT CYTOCHROME C CYTOCHROME C- UBIQUINONE #YT BC COMPLEX TOGETHER WITH ./ REDUCTASE CYTOCHROME C CYTOCHROME AA 7HITTAKER ET AL )T IS INTRIGUING THAT THERE ARE ROUGHLY EQUAL AMOUNTS OF CYTOCHROME C CYTOCHROME C- #YT BC COMPLEX AND CYTOCHROME AA PER SUBUNIT OF (!/ )N CONTRAST THE MOBILE ELECTRON CARRIERS MEMBRANE UBIQUINONE AND THE SOLUBLE PERIPLASMIC CYTOCHROME C ARE IN CONSIDERABLE EXCESS 4HE EQUAL AMOUNTS OF CYTOCHROME C AND CYTOCHROME C- PER SUBUNIT OF (!/ PROMPT SPECULATION THAT THESE COMPONENTS MIGHT FORM A STOICHIOMETRIC AGGREGATE RADIATING FROM THE (!/ TRIMER '0UIFSSFEPYQSPUFJOT /JUSPTPDZBOJO .ITROSOCYANIN IS A NOVEL SOLUBLE RED COPPER PROTEIN LOCATED IN THE PERIPLASM OF .ITROSOMONAS !RCIERO ET AL )T IS FOUND IN QUANTITIES EQUIVALENT TO (!/ AND SO IS THOUGHT TO POSSIBLY HAVE A ROLE IN A CENTRAL . OXIDATION PATHWAYS "ASED ON ITS PRIMARY AND TER TIARY STRUCTURE IT BELONGS TO THE CUPREDOXIN FAMILY OF REDOX PROTEINS ,IEBERMAN ET AL (OWEVER IT DIFFERS FROM OTHER CUPREDOXINS IN BINDING COPPER IN A @TYPE SITE HAVING AN OPEN COPPER COORDINATION SITE 4HUS THE POSSIBILITY OF EITHER AN ELECTRON TRANSPORT OR CATALYTIC ROLE IS BEING CONSIDERED $ZUPDISPNF1 #YTOCHROME 0 A SOLUBLE PERIPLASMIC MONO HEME C CYTOCHROME WITH HYDROXYLAMINE OXIDIZING ACTIVITY IS ALSO FOUND IN SMALL QUANTITY %RICKSON AND (OOPER .UMATA ET AL "ERGMANN AND (OOPER B )T IS CHARACTERIZED BY AN OPTICAL SPECTRUM WITH A BROAD 3ORET BAND AT NM IN THE FERRIC FORM AND NM IN THE FERROUS FORM _ AND ` ABSORPTION BANDS OF TYPICAL C HEMES ARE NOT SEEN WHEREAS THE HEME 0 OF (!/ IS A C HEME ALSO COVALENTLY BOUND TO A PEPTIDE TYROSINE THE C HEME OF CYTOCHROME 0 IS CROSS LINKED TO A PEPTIDE LYSINE !RCIERO AND (OOPER )TS PHYSIOLOGICAL ROLE IS NOT KNOWN 2ECENT WORK SUGGESTS THAT ITS ROLE IS IN THE DETOXIlCATION OF ./ OR RELATED COMPOUNDS )TS SEQUENCE IS HOMOLOGOUS TO THAT OF A CYTOCHROME 0
WITH SIMILAR OPTICAL PROPERTIES AND REACTIVITY WITH . OXIDES FOUND IN THE METHANE OXIDIZING BACTERIA "ERG MANN ET AL 3IGNIlCANTLY THE LATTER ALSO HAS GREAT SEQUENCE HOMOLOGY WITH CYTOCHROME C| FROM THE SAME ORGANISM A PROTEIN WHOSE ROLE IS THOUGHT TO BE SEQUESTRATION OF ./ &URTHER REPLACEMENT OF THE CROSSLINKED LYSINE WITH !RG !LA OR 4YR RESULTS IN HETEROLOGOUSLY EXPRESSED MUTANT PROTEIN WITH LIGAND BINDING ABILITY WITHOUT CATALYTIC ACTIVITY AND WITH OPTICAL ABSORPTION PROPERTIES DISSIMILAR TO THE WILD TYPE PROTEIN BUT RESEMBLING THOSE OF CYTOCHROME C| "ERGMANN AND (OOPER %JIFNF$ZUPDISPNFD1FSPYJEBTF .ITROSOMONAS CONTAINS A PERIPLASMIC DIHEME CYTO CHROME C PEROXIDASE WHICH MAY SCAVENGE HYDROGEN PEROXIDE !RCIERO AND (OOPER #YT C SERVES AS AN EFFECTIVE ELECTRON DONOR 4HE DI FERRIC FORM OF THE DI HEME CYTOCHROME C PEROXIDASE OF .ITRO SOMONAS HAS THE NOVEL ABILITY TO REACT WITH (/ TO FORM A STABLE FERRYL OXYGEN DERIVATIVE COMPOUND ) AND (/ 4HIS CONTRASTS WITH THE MORE COMMONLY OBSERVED TYPE OF DIHEME CYTOCHROME C PEROXIDASE FROM 0 AERUGINOSA WHICH REQUIRES REDUCTION OF THE HIGH POTENTIAL ELECTRON TRANSFER HEME BEFORE THE CATA LYTIC HEME BECOMES REACTIVE WITH (/ AND CAN THEN FORM COMPOUND ) #OMPARISON OF THE CRYSTAL DERIVED STRUCTURES OF THE TWO PROTEINS &IG INDICATES THAT A HISTIDINE IS THE SIXTH AXIAL LIGAND OF THE CATALYTIC HEME OF THE ENZYME FROM 0 AERUGINOSA WHEREAS IN THE ENZYME FROM .ITROSOMONAS THE LOOP CONTAINING THE HOMOLOGOUS HISTIDINE IS HELD AWAY FROM THE HEME THUS PROVIDING ACCESS OF PEROXIDE TO THE REACTIVE IRON 3HIMIZU ET AL 4HIS HISTIDINE CONTAINING LOOP OF THE ENZYME FROM 0 AERUGINOSA IS PRESUMED TO FOLD AWAY UPON REDUCTION OF THE ELECTRON TRANSFER HEME TO ALLOW CATALYSIS TO COMMENCE )T IS POSSIBLE THAT THIS PROPERTY OF THE ENZYME FROM .ITROSOMONAS ALLOWS SEQUESTRATION OF (/ AT RELATIVELY HIGH STEADY STATE REDOX POTENTIALS WITHIN THE CELL &ULL REDUCTION OF THE SECOND OXYGEN AND COMPLETION OF TURNOVER OF ##0 OF .ITROSOMONAS STILL REQUIRES THE ENTRY OF TWO ADDITIONAL ELECTRONS (1SPUPO(SBEJFOU (FOFSBUJPOPGUIF(SBEJFOU 4HE STOICHIOMETRY OF PROTON GRADIENT GENERATED PER ELECTRON PAIR FROM HYDROXYLAMINE IS HARD TO PREDICT
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&IG #OMPARISON OF THE STRUCTURES OF THE RESTING OXIDIZED DIHEME CYTOCHROME C PEROXIDASES ##0 OF .ITROSOMONAS EUROPAEA AND 0SEUDOMONAS AERUGINOSA 4HE TWO ENZYMES HAVE SIMILAR DOMAINS ENCLOSING A LOW POTENTIAL ,0 CATALYTIC C HEME AND HIGH POTENTIAL (0 ELECTRON TRANSFERING C HEME (IS OF THE 0SEUDOMONAS ENZYME IS THE SIXTH AXIAL LIGAND OF THE FERRIC ,0 HEME WHEREAS THE LOOP CONTAINING THE HOMOLOGOUS (IS OF THE .ITROSOMONAS ENZYME IS RELATIVELY DISPLACED RESULTING IN AN OPEN CATALYTIC SITE AND PENTA COORDINATE CATALYTIC C HEME
)T IS FAIR TO ASSUME THAT OXIDATION OF UBIQUINOL BY THE CYTOCHROME BC COMPLEX AND CYTOCHROME OXIDASE WOULD YIELD THE NET TRANSLOCATION OF SIX PROTONS FROM . TO 0 SIDE &IG 4HE REACTION OF (!/ RELEASES FOUR PROTONS ON THE 0 SIDE TWO PER UBIQUINONE REDUCED 4HE IONIZA TION OF NITRITE MIGHT BE CONSIDERED AS CONTRIBUTING A lFTH PROTON (OWEVER THE ACIDIlCATION OF THE MEDIA DURING GROWTH IS APPROXIMATELY STOICHIOMETRIC WITH NITRITE PRODUCTION INDICATING THAT THIS SHOULD NOT BE CONSIDERED AN ENERGETICALLY USEFUL PROTON IE ONE WHICH COULD BE TRANSLOCATED TO . SIDE DURING TURNOVER OF AN ENZYME SUCH AS !40 SYNTHASE 4HE NET TRANS LOCATION OF PROTONS IN THE REDUCTION OF UBIQUINONE BY ELECTRONS FROM HYDROXYLAMINE WOULD DEPEND ON THE MEMBRANE LOCATION OF THE SITE OF REDUCTION OF AND UPTAKE OF PROTONS BY UBIQUINONE #YTOCHROME C- IS ATTACHED TO THE 0 SIDE OF THE MEMBRANE BY AN . TERMINAL TRANSMEMBRANE _ HELIX )F THE GLOBULAR TETRAHEME REGION LIES ON THE MEMBRANE SURFACE AND REDUCES UBIQUINONE DURING CATALYSIS PROTONS WOULD BE ABSORBED INTO MEMBRANE QUINOL ON THE 0 SIDE )N THIS CASE THE NET PROTON TRANSLOCATION DURING THE REDUC TION OF UBIQUINONE BY ELECTRONS FROM HYDROXYLAMINE WOULD BE )NTERESTINGLY THIS ORIENTATION OF #YT C- COULD ALLOW THE REDUCTION OF UBIQUINONE n M6 BY THE M6 HEMES OF CYTOCHROME C !RCIERO ET AL B TO BE FACILITATED BY THE STEADY STATE TRANSMEMBRANE CHARGE DIFFERENTIAL )F ON THE OTHER
HAND CYTOCHROME C- PROJECTS INTO THE MEMBRANE FAR ENOUGH TO REDUCE UBIQUINONE ON THE . SIDE TWO . SIDE PROTONS COULD BE TAKEN UP )N THAT CASE THE REDUCTION OF ONE MOLECULE OF UBIQUINONE BY TWO ELEC TRONS FROM (!/ WOULD RESULT IN THE EQUIVALENT OF THE TRANSLOCATION OF TWO PROTONS FROM . TO 0 SIDE )T IS NOT KNOWN WHETHER NET TRANSLOCATION OF PROTONS OCCURS IN THE !-/ REACTION )T DEPENDS ON WHETHER THE RELEASE OF TWO PROTONS PRODUCED IN THE OXIDATION OF UBIQUINOL UBIQUINOL A UBIQUINONE En ( AND THE UPTAKE OF THE SUBSTRATE PROTONS OF THE MONOOXY GENASE REACTION .( / En ( TAKE PLACE ON THE 0 OR . SIDE 4HE LARGE NUMBER OF TRANSMEMBRANE _ HELICES PREDICTED IN !-/ SEE !-/ BELOW HAVE THE POTENTIAL TO PROVIDE THE SCAFFOLDING TO SUPPORT A SYSTEM FOR MOVING EITHER PROTONS OR ELECTRONS ACROSS THE MEMBRANE AND COULD THEORETICALLY SUPPORT SEVERAL SCENARIOS 2ELEASE OF TWO PROTONS ON THE 0 SIDE AND UPTAKE OF TWO PROTONS ON THE . SIDE WOULD RESULT IN THE EQUIVALENT OF THE TRANSLOCATION OF TWO PROTONS TO THE 0 SIDE WHEREAS THE REVERSE ORIENTATION OF THE REACTIONS WOULD RESULT IN A NET TRANSLOCATION OF TWO PROTONS TO THE . SIDE )NTERESTINGLY THE LATTER ORIENTA TION OF REACTIONS WOULD ALLOW THE !-/ REACTION TO BE DRIVEN BY THE STEADY STATE TRANSMEMBRANE CHARGE DIFFERENTIAL )F THIS WERE SO IT COULD ACCOUNT FOR THE GREAT SENSITIVITY OF THE !-/ REACTION TO UNCOUPLERS )F BOTH THE PROTON YIELDING AND PROTON UTILIZING REAC TIONS OF !-/ ARE ON THE SAME SIDE OF THE MEMBRANE
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB THE NET TRANSLOCATION OF PROTONS WOULD BE ZERO 5NTIL MORE IS KNOWN REGARDING THE HYDROXYLAMINE TO UBIQUINONE STEPS AND THE !-/ REACTION ONE CAN ONLY GUESS THE NUMBER OF PROTONS WHICH ARE TRANSLO CATED TO THE 0 SIDE PER TRANSFER OF FOUR ELECTRONS FROM .(/( TWO OF WHICH THEN REDUCE / AND TWO OF WHICH REDUCE !-/ TO REPLACE THE OXIDIZED .(/( 4HE EQUIVALENT OF SIX PROTONS ARE TRANSLOCATED BY #YT BC AND #YT AA IN THE REOXIDATION OF A MOLECULE OF UBIQUINOL 4HE HIGH AND LOW VALUES OF THE POSSIBLE CONTRIBUTION OF THE ELECTRON HYDROXYLAMINE TO UBIQUINONE STEP ARE AND PROTONS 4HE HIGH AND LOW VALUES OF THE CONTRIBUTION OF THE TWO ELECTRON !-/ REACTION ARE AND n (ENCE THE THEORETICAL OVERALL YIELD RANGES FROM BY (!/QUINONE BY #YT BC#YT AA AND BY !-/ TO BY (!/QUINONE #YT BC AND #YT AA AND n FROM !-/
TASE AND .A ( ANTIPORTER WHICH MIGHT HAVE ROLES IN SODIUM SYMPORT OR ANTIPORT ***)ZESPYZMBNJOF0YJEPSFEVDUBTF "0WFSWJFXPG3FBDUJPO 4HE REACTION CATALYZED BY (!/ CAN BE THOUGHT OF AS A DEHYDROGENATION OF HYDROXYLAMINE TO NITROSONIUM .(/( A ./ En (
FOLLOWED BY HYDROLYSIS TO NITROUS ACID ./ (/ A (./ (
(!/ WILL ALSO RAPIDLY OXIDIZE HYDRAZINE TO DINI TROGEN
6UJMJ[BUJPOPGUIF(SBEJFOU B"514ZOUIBTF !40 SYNTHASE IS DRIVEN BY A PROTON GRADIENT WHICH CAN BE EXPERIMENTALLY DEMONSTRATED DURING OXIDATION OF HYDROXYLAMINE +UMAR AND .ICHOLAS ! STEADY STATE VALUE OF 6P OF M6 INSIDE NEGATIVE HAS BEEN ESTIMATED FOR .ITROSOMONAS (OLLOCHER ET AL !LL GENES FOR A PROTON DEPENDENT !40 SYNTHASE ARE PRESENT #HAIN ET AL )T WOULD PRESUMABLY TRANSLOCATE APPROXIMATELY FOUR PROTONS TO THE . SIDE PER !40 FORMED .OTE THAT THIS MAY CORRESPOND TO ALL OR MORE THAN THE PROTON GRADIENT YIELD OF THE OXIDA TION OF ONE MOLECULE OF AMMONIA C3FEVDUJPOPG1ZSJEJOF/VDMFPUJEFT ! GENE CLUSTER ENCODES ALL SUBUNITS OF A PROTON DEPENDENT .!$ DEHYDROGENASE COMPLEX ) WHICH IS PRESUMED TO FUNCTION PREDOMINANTLY OR ONLY AS A .!$ REDUCTASE IN THIS ORGANISM )NTERESTINGLY EACH TURNOVER OF THIS ENZYME MAY REQUIRE !-/ AND (!/ TO TURNOVER TWICE ONCE TO REDUCE UBIQUINONE AND THEN .!$ AND ONCE TO REPLENISH THE ( GRADIENT EQUIVALENTS UTILIZED IN THE REDUCTION OF .!$ BY UBIQUINOL !NOTHER GENE CLUSTER ENCODES A PROTON GRADIENT DEPENDENT .!$.!$0 TRANSHYDROGENASE PRESUMABLY FOR THE GENERATION OF .!$0( (ENCE .!$0( IS VERY ENERGETICALLY EXPENSIVE POSSIBLY REQUIRING THREE TURNOVERS OF THE !-/(!/ SYSTEM 4HE GENOME ENCODES A .A DEPENDENT .!$ REDUC
.( .( A . E (
4HE OXIDATION OF HYDRAZINE BY (!/ HAS BEEN EXPERIMENTALLY USEFUL BUT HAS NO KNOWN FUNCTION IN .ITROSOMONAS 4HE REACTION AS CATALYZED BY AN (!/ WITH SIMILAR CATALYTIC AND MOLECULAR PROPER TIES HAS AN IMPORTANT ROLE IN THE BACTERIAL ANAEROBIC TRANSFORMATION OF AMMONIUM AND NITRITE TO DINITROGEN DISCUSSED LATER 3CHALK ET AL 4HE TURNOVER OF THE ENZYME IS COMMONLY MEASURED IN THE PRESENCE OF AN ARTIlCIAL ELECTRON ACCEPTOR SUCH AS PHENAZINE METHOSULFATE CATALYTIC IN THE PRESENCE OF / DICHLO ROPHENOLINDOPHENOL MAMMALIAN CYTOCHROME C OR CYTOCHROMES OF .ITROSOMONAS EG CYTOCHROME C IN THE PRESENCE OF CATALYTIC AMOUNTS OF CYTOCHROME C 9AMANAKA AND 3HINRA 3EVERAL ELEMENTS OF THE POSSIBLE CATALYTIC MECHA NISM MAY BE SUGGESTED #ATALYSIS MUST INVOLVE SELECTIVE BINDING OF SUBSTRATE . INTERMEDIATES MUST BE STABILIZED TO PREVENT THE RELEASE OF SUCH POSSIBLE COMPOUNDS AS (./ OR ./ !CTIVATION OF SUBSTRATE AND . ( INTERMEDIATES MAY INVOLVE CONVERSION TO ELECTRON RICH FORMS BY DEPROTONATION 7ATER MAY ALSO BE ACTIVATED BY DEPROTONATION %LECTRONS MAY BE REMOVED IN SINGLE STEPS OR PAIRS POSSIBLY AS A HYDRIDE OR A COMBINATION EG AS A HYDRIDE IN ONE STEP FOLLOWED BY TWO ONE ELECTRON STEPS %LECTRONS MUST BE WITHDRAWN AND ALSO EFFECTIVELY REMOVED FROM THE SITE OF CATALYSIS TO HIGH POTENTIAL REDOX CENTERS SO AS TO PREVENT @BACK mOW OF ELECTRONS TO POSSIBLE OXIDANTS SUCH AS DIOXYGEN OR HYDROGEN PEROXIDE OR TO
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PREVENT REVERSAL OF THE . OXIDATION REACTION 2EMOVAL OF PROTONS COULD OCCUR BY WAY OF AN INTRA PROTEIN RELAY OR A CHANNEL 4HE PRODUCT NITROUS ACID MAY EXIT THROUGH A CHANNEL OR THROUGH SEPARATE NITRITE CHANNEL AND PROTON PATH 3UBSTRATE BINDS WITH HIGH AFlNITY (!/ HAS A +M FOR HYDROXYLAMINE OR HYDRAZINE IN THE LOW +- RANGE (OOPER AND .ASON 4HE MEASURED RATE OF TRANSFER OF ELECTRONS FROM SUBSTRATE TO HEMES OF THE ENZYME IS AT LEAST 3n (OOPER ET AL B !RCIERO ET AL A 5NDER SOME REACTION CONDITIONS THE ISOLATED ENZYME PRODUCES SOME ./ POSSIBLY THE PRODUCT OF THE DIMERIZATION AND HYDROLYSIS OF TWO NITROXYLS (./ HENCE THE lRST STEP MAY BE REMOVAL OF TWO ELECTRONS OR A HYDRIDE (OOPER ./ CAN ALSO BE A PRODUCT INDICATING THAT THE SECOND STAGE CAN SOMETIMES OCCUR IN VITRO IN SINGLE ELECTRON STEPS (OOPER ET AL (OOPER AND 4ERRY ./ WILL NOT SERVE AS SUBSTRATE FOR THE PRODUCTION OF NITRITE (ENDRICH ET AL HOWEVER ./ WILL RE OXIDIZE FERROUS C HEMES 2ELEASE OF INTERMEDIATES DOES NOT OCCUR IN VIVO BUT CAN OCCUR IN VITRO DEPENDING ON THE NATURE OF THE ARTIlCIAL ELECTRON ACCEPTOR AND THE ASSAY CONDITIONS #0WFSWJFXPGUIF4USVDUVSFPG)"0 $IFNJDBM$IBSBDUFSJ[BUJPO 0URIlED (!/ IS A RICH RED IN COLOR DUE TO ITS HIGH CON TENT OF HEME (OOPER ET AL )N FACT CELLS HAVE A STRIKING BROWNISH RED COLOR DUE TO THE PRESENCE OF LARGE AMOUNTS OF (!/ AND OTHER C TYPE CYTOCHROMES 3EVERAL YEARS OF RIGOROUS PROTEIN CHEMISTRY REVEALED (!/ TO BE AN OLIGOMER IN WHICH EACH MONOMER CON TAINED SEVEN C HEMES EACH COVALENTLY ATTACHED BY TWO THIOETHER BONDS FROM VINYL GROUPS TO CYSTEINES AND AN UNUSUAL C HEME DERIVATIVE HEME 0 4HIS WAS DETERMINED BY PROTEOLYSIS AND ISOLATION OF ALL HEME CONTAINING PEPTIDES !RCIERO AND (OOPER !S REVEALED BY ANALYSIS OF AN UNUSUAL CROSSLINKED DIPEP TIDE RECOGNIZED BY HAVING TWO . TERMINAL AMINO ACID RESIDUES HEME 0 HAS AN ADDITIONAL COVALENT BOND FROM A RING CARBON OF A PEPTIDE TYROSINE TO A METHYNE CARBON OF A HEME !RCIERO ET AL 4REATMENT OF FERRIC (!/ WITH HYDROGEN PEROXIDE SPECIlCALLY AND IRREVERSIBLY ALTERS THE ENZYME SO THAT THE NM OPTICAL BAND IS NO LONGER SEEN IN THE FERROUS ENZYME )N ADDITION THE TURNOVER OF THE ENZYME AND THE REDUC IBILITY OF C HEMES BY SUBSTRATE IS LOST (ENCE HEME
0 IS CRITICAL TO CATALYSIS (OOPER AND 4ERRY !LL EVIDENCE INDICATES THAT IN FACT HEME 0 BINDS TO SUBSTRATE 4HE SEQUENCE DEDUCED FROM THE CLONED GENE RESULTED IN NUMBERING OF HEMES n FROM THE . TERMINUS 3AYAVEDRA 3OTO ET AL 4HE OPTICAL SPECTRUM OF FERROUS (!/ CONSISTS OF A LARGE 3ORET BAND A SHOULDER AT NM OF HEME 0 AND _ AND ` BANDS AT AND NM DUE TO C HEMES &IG 4HE FERRIC SPECTRUM CONTAINS ONLY THE BROAD 3ORET BAND AND _ AND ` BANDS OF THE C HEMES AND A WEAK BROAD BAND AT NM DUE TO HEME 0 #OLLINS ET AL /PTICAL SPECTRO ELECTROCHEMISTRY &IG INSET REVEALED THE REMARKABLE RANGE OF MIDPOINT POTENTIALS 4ABLE TO BE M6 TO n M6 4HE REDUCTION POTENTIAL OF HEME 0 DECREASED BY M6 PER P( UNIT IE PROTONATION OF A MOIETY ON THE ENZYME PROMOTES UPTAKE OF ELECTRONS BY THE CATALYTIC IRON 4HE HEMES WITH POTENTIALS AT AND n M6 ARE REDUCED EITHER AT EQUILIBRIUM IN THE PRESENCE OF HYDROXYLAMINE OR IN THE STEADY STATE IN THE PRESENCE OF HYDROXYLAMINE AND ELECTRON ACCEPTORS 5IF$SZTUBM4USVDUVSFPG)"0 4HE DETERMINATION OF THE CRYSTAL STRUCTURE OF (!/ HAS ADVANCED THE lELD REMARKABLY )GARASHI ! SIDE VIEW OF A MONOMER &IG ! REVEALS C HEMES CONCENTRATED IN THE LOWER PORTION OF THE MOLECULE 4HE MOLECULE IS WIDE AT THE BASE AND A SPINE OF LONG _ HELICAL DOMAINS RISE AT THE BACK &IG " MORE CLEARLY SHOWS THE ARRANGEMENT IN SPACE OF THE HEMES OF A MONOMER )N THIS VIEW HEME HEME 0 AS IDENTIlED BY ITS CROSS LINKING TO TYROSINE IS THE HIGHEST HEME "Y THEIR SPACING THE HEMES OF EACH SUBUNIT APPEAR TO GROUP INTO FOUR CLUSTERS A TRIMER OF HEMES AND DIMERS OF HEMES AND AND AND AND A MONOMERIC HEME )GARASHI ET AL 4HE TRIMER OF (!/ HAS A THREE CLOVE @GARLIC SHAPE !S SEEN FROM THE SIDE VIEW &IG THE HEMES ARE CONCENTRATED IN THE WIDE BOTTOM HALF OF THE MOLECULE AROUND THE TOWER OF LONG _ HELICES ! SHALLOW CAVITY PROJECTS INTO THE BASE OF THE TRIMER 4HE THREE CATALYTIC HEME 0S ARE AT THE TOP OF THE CLOUD OF C HEMES IN THE (!/ TRIMER 2EMARKABLY THE HEME CROSSLINK OF HEME 0 IS TO A TYROSINE IN THE ADJACENT SUBUNIT (ENCE (!/ IS A COVALENTLY LINKED ENTITY CONTAINING HEMES &ROM THE TOP VIEW &IG THE HEMES APPEAR IN A RING WITH A HEME PROJECT ING RADIALLY FROM EACH SUBUNIT
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&IG /PTICAL ABSORPTION SPECTRUM OF (!/ OF .ITROSOMONAS EUROPAEA !BSORBANCE AS A FUNCTION OF WAVELENGTH $ARKER LINE OXIDIZED ENZYME ,IGHTER LINE REDUCED ENZYME )NSET REDOX TITRATION OF THE SEVEN C HEMES OF (!/ DATA FOR HEME 0 NOT SHOWN !BSORBANCE AS A FUNCTION OF POTENTIAL H $ATA BASED ON #OLLINS ET AL 4ABLE 2EDUCTION POTENTIALS FOR THE HEMES OF (!/ #OLLINS ET AL %|M .(%
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&IG 3TRUCTURE OF A SUBUNIT OF (!/ ;BASED ON )GARASHI ET AL = 3IDE VIEW (EME NUMBERS ARE BASED ON THE PRIMARY STRUCTURE AND BEGIN AT THE . TERMINAL ! HEMES AND RIBBON VIEW OF PROTEIN " (EMES ALONE
1PTTJCMF1BUIPG&MFDUSPOTGSPN)FNF1 !CCESS TO SOLVENT BY THE HEMES IS LIMITED TO A SUB STRATE ACCESSIBLE CHANNEL LEADING TO AN OPEN AXIAL POSITION OF THE CATALYTIC HEME 0 HEME TO THE PORPHYRIN EDGE OF HEME MOST DISTAL FROM THE
CENTER OF THE HEME CLUSTER AND TO A REGION IN THE BASE CAVITY NEAR HEMES AND /N THIS BASIS HEME WAS PREDICTED TO BE THE POINT OF EXIT OF ELECTRONS FROM (!/ )GARASHI ET AL 4HE IRONS OF ALL HEMES ARE WITHIN ¯ FROM THE NEAREST HEME IRON (ENCE THERE IS NO APPARENT KINETIC BARRIER TO ELECTRON TRANSFER
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&IG 3TRUCTURE OF TRIMER OF (!/ WITH ONE #YT C DOCKED ;BASED ON )AGARASHI ET AL AND )VERSON ET AL = 3IDE VIEW
(EMES ARE ALSO CLOSE ENOUGH TO HEMES OF THE ADJA CENT SUBUNIT TO ALLOW RAPID ELECTRON TRANSFER (ENCE A LOW RESISTANCE ELECTRON PATH COULD BEGIN AT HEME 0 OF A GIVEN SUBUNIT AND AT HEME BIFURCATE TO A PATH THROUGH HEME OF THAT SUBUNIT AND A SECOND PATH THROUGH HEME AND AND HEMES AND OF THE ADJACENT SUBUNIT INTERESTINGLY THE SUBUNIT TO WHICH HEME 0 IS COVALENTLY BOUND &IG &MFDUSPOJDBOE3FEPY1SPQFSUJFTPGUIF )FNFT 4HE FERRIC ENZYME ANALYZED BY PERPENDICULAR MODE %02 IS SEEN TO CONTAIN A VARIETY OF LOW SPIN AXIAL BIS (IS HEME IRONS WITH G VALUES OF APPROXIMATELY AND PAIRS OF HEMES EXHIBITING ELECTRONIC EXCHANGE &IG /NE OF THE ELECTRONICALLY COUPLED PAIRS EXHIBITS SIGNALS AT G AND !N %02 SIGNAL FOR THE SECOND PAIR IS NOT SEEN EXCEPT BY PARALLEL MODE
%02 BY WHICH AN INTEGER SPIN SIGNAL AT G IS SEEN (ENDRICH ET AL 2EDUCTION OF THE ENZYME WITH SUBSTRATE ,IPSCOMB AND (OOPER OR REDOX TITRATION OF THE ENZYME 0RINCE AND (OOPER RESULTS IN AN ARRAY OF SHIFTING %02 SIGNALS REmECTING INTERACTION BETWEEN HEMES !N EXHAUSTIVE SERIES OF STUDIES HAVE BEEN CARRIED IN WHICH %02 SPECTRA WERE MEASURED ON POISED SAMPLES OF (!/ -EASUREMENT OF %02 SPECTRA AT SEVERAL FREQUENCIES AND ANALYSIS OF THE HEME GEOMETRIES FROM THE CRYSTAL STRUCTURE ALLOWED ASSIGNMENT OF %02 PROPERTIES AND REDOX POTENTIALS TO SEVERAL HEMES (ENDRICH ET AL 4HE CATALYTIC HEME 0 n M6 IS EXCHANGE COUPLED TO THE NEIGHBORING HEME 4HE VALUES OF MIDPOINT REDUCTION POTENTIAL OF n M6 AND n M6 WERE ASSIGNED TO HEME 0 AND HEME RESPEC TIVELY 4HESE LOW VALUES OF MIDPOINT POTENTIAL ARE IN KEEPING WITH THE INABILITY TO EXPERIMENTALLY OBSERVE REDUCTION OF HEME 0 BY SUBSTRATE IN THE STEADY
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&IG 3PATIAL ARRANGEMENT OF HEMES OF TRIMER OF (!/ ;BASED ON )GARASHI ET AL = 4OP VIEW (EMES 0 OF THE THREE SUBUNITS ARE NUMBERED | AND || AND ARE DARKER
STATE OR BY STOPPED mOW MEASUREMENTS IN THE MSEC RANGE (OOPER ET AL B )N THE FERRIC ENZYME THE COUPLING BETWEEN THESE TWO HEMES CAN BE DISRUPTED BY BINDING OF SUBSTRATE OR CYANIDE TO HEME 0 (ENDRICH ET AL #YANIDE IS A LIGAND TO HEME 0 AND AN INHIBITOR OF THE ENZYME ACTIVITY 4ABLE ,OGAN ET AL 4HE TWO HEMES WITH MIDPOINT POTENTIALS NEAR ARE ALSO EXCHANGE COUPLED AND HAVE BEEN IDENTIlED AS HEMES AND .OTABLY THESE ARE TWO OF THE HEMES THAT ARE REDUCED DURING REACTION OF (!/ WITH HY DROXYLAMINE OR HYDRAZINE 4HE ELECTRONIC COUPLING BETWEEN HEMES IN THESE TWO PAIRINGS IMPLIES THAT ELECTRONS EQUILIBRATE RAPIDLY BETWEEN THE TWO HEME IRONS 4HIS PROPERTY MIGHT MAKE THEM ESPECIALLY SUIT ABLE AS ELECTRON REDOX MEDIATORS OR SINKS (EME HAS A MIDPOINT POTENTIAL OF n M6 4HE OTHER THREE HEMES AND HAVE NOT YET BEEN MATCHED WITH THE UNASSIGNED VALUES OF MIDPOINT POTENTIAL n AND n )NTERESTINGLY BECAUSE THE ELECTRON PATHS IN THE ENZYME DIVIDE AND REJOIN &IG THE HIGHEST PO TENTIAL HEMES OF THE (!/ TRIMER SIX HEMES OF ^ M6 AND THREE OF M6 CAN IN THEORY PROVIDE A RESERVOIR FOR AT LEAST ELECTRONS AND THUS ALLOW COM PLETE TURNOVER AND RELEASE OF NITRITE ONCE A SUBSTRATE MOLECULE IS BOUND
&IG $IAGRAM OF POSSIBLE ELECTRON PATHS FROM THE THREE ACTIVE SITES OF THE (!/ TRIMER THROUGH HEMES OF (!/ #YT C AND #YT C- (EMES ARE SHOWN AS CIRCLES
&IG %02 SPECTRUM OF (!/ OF .ITROSOMONAS #ONDITIONS ARE AS DESCRIBED IN (ENDRICH ET AL $ARKER LINE PERPEN DICULAR MODE %02 ,IGHTER LINE PARALLEL MODE %02
4QFDVMBUJPO3FHBSEJOHUIF.FDIBOJTNPG $BUBMZTJT B#JOEJOHBOE%FQSPUPOBUJPOPG4VCTUSBUF &IGURE SHOWS THE ORIENTATION OF AMINO ACID RESIDUES OF THE ACTIVE SITE AS DETERMINED BY CRYSTALLOGRAPHY )GARASHI ET AL MODELED WITH A MOLECULE OF HYDROXYLAMINE BOUND BY ITS NITROGEN TO THE IRON !TOMS OF SEVERAL RESIDUES PROJECT INTO THE ACTIVE
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SITE WITHIN HYDROGEN BONDING DISTANCES n¯ OF HYDROGENS OF HYDROXYLAMINE AND ARE THUS PO TENTIALLY CAPABLE OF FACILITATING SELECTIVE BINDING OF SUBSTRATE STABILIZATION OF INTERMEDIATES AND ACTIVATION OF THE SUBSTRATE BY DEPROTONATION 4HEY INCLUDE THE CROSSLINKED TYROSINE AS WELL AS HISTIDINE ASPARTATE AND TYROSINE WHICH ARE ABOVE THE SUBSTRATE ( BONDS COULD EXTEND FROM THE HYDROXYL ( OF SUBSTRATE TO THE EPSILON . OF HISTIDINE FROM ONE SUBSTRATE AMINO ( TO THE RING OXYGEN OF TYROSINE FROM THE OTHER SUBSTRATE AMINO ( TO THE CARBOXYLATE / OF ASPARTATE (YDROXYLAMINE AND HYDRAZINE REDUCE THE C HEMES OF THE ENZYME . METHYLHYDROXYLAMINE WILL ALSO REDUCE C HEMES OF (!/ BUT / METHYL HYDROXYLAMINE IS NOT REACTIVE 2ITCHIE AND .ICHOLAS EMPHASIZING THE POS SIBLE IMPORTANCE OF BEING ABLE TO FORM AN OXYANION DURING OXIDATION OF HYDROXYLAMINE
&IG (EME 0 OF (!/ SHOWING NEARBY PEPTIDE RESIDUES AND PUTATIVE MODE OF BINDING OF SUBSTRATE $ATA BASED ON )GARASHI ET AL 3HOWN ARE THE CROSSLINKED TYROSINE AND THREE RESIDUES THAT PROJECT INTO THE ACTIVE SITE ASPARTATE TYROSINE AND HISTIDINE (YDROXYLAMINE IS MODELED AS HAVING AN . &E BOND TO THE HEME 4HE AXIAL HISTIDINE APPEARS BELOW THE HEME
C&MFDUSPO3FNPWBM 4HE ELECTRONIC COUPLING OF THE CATALYTIC HEME PAIR AND MAKE IT A REASONABLE HYPOTHESIS THAT THE REACTION OCCURS IN TWO ELECTRON STEPS )N THE lRST STEP DEPROTONATION FOLLOWED EITHER BY REMOVAL OF A HYDRIDE OR A SECOND DEPROTONATION AND REMOVAL OF TWO ELECTRONS WOULD PRODUCE IRON (./ )N THE SECOND STEP HYDRIDE TRANSFER OR DEPROTONATION AND REMOVAL OF TWO ELECTRONS WOULD PRODUCE IRON NITROSONIUM ./
4HE LOW VALUES OF REDOX POTENTIAL FOR THE CATALYTIC HEME PAIR MAKE IT LIKELY THAT ELECTRONS QUICKLY PASS TO THE M6 PAIR HEMES AND IN KEEPING WITH THE INABILITY TO EXPERIMENTALLY DEMONSTRATE REDUCTION OF HEME 0 DURING CATALYSIS
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB D5IF3FBDUJPOPG)"0JT6OJRVFGPSB$BUB MZUJD)FNF )N THE REACTION CATALYZED BY (!/ SUBSTRATE lRST BINDS TO THE IRON OF A HEME )N THE COURSE OF THE REACTION ELECTRONS ARE REMOVED FROM SUBSTRATE AND ARE MOVED OUT OF THE SYSTEM THROUGH THE OTHER C HEMES OF THE PROTEIN 4HIS IS THE ONLY REACTION OF THIS TYPE IN NA TURE !LL OTHER REDOX REACTIONS OF SUBSTRATES BOUND TO THE IRON OF A HEME INVOLVE TRANSFER OF ELECTRONS INTO THE CATALYTIC SYSTEM AND THEN TO THE IRON BOUND SUBSTRATE 4ABLE 4HIS DESCRIBES THE REACTIONS OF THE SIROHEME CONTAINING NITRITE OR SULlTE REDUCTASE THE CYTOCHROME CD NITRITE REDUCTASE THE PENTA HEME NITRITE REDUCTASE .I2 AND THE NITRIC OXIDE REDUC TASE AS WELL AS REDUCTION OF OXYGEN BY CYTOCHROME OXIDASE OR CYTOCHROME C PEROXIDASE )N THE CASE OF CYTOCHROME OXIDASE ELECTRONS MAY BE THOUGHT OF AS ENTERING THE CATALYTIC SYSTEM AND REDUCING ONE OF THE OXYGEN ATOMS TO WATER !T THE SAME TIME THE IRON AND PORPHYRIN RING DONATE ELECTRONS AS THE FERRYL OXYGEN INTERMEDIATE IS FORMED 4HE LATTER IS REDUCED TO WATER AS TWO ADDITIONAL ELECTRONS ENTER THE SYSTEM )N THE CASE OF CYTOCHROME 0 MONOOXYGENASE ELECTRONS MAY ALSO BE THOUGHT OF AS ENTERING THE CATALYTIC SYS TEM AND REDUCING ONE OXYGEN TO WATER ELECTRONS ARE ALSO WITHDRAWN FROM THE IRON AND PORPHYRIN RING AS THE FERRYL OXYGEN INTERMEDIATE IS FORMED 4HE LATTER DECAYS AS SUBSTRATE IS HYDROXYLATED )N THE REACTION OF CATALASE WITH A MOLECULE OF HYDROGEN PEROXIDE THE PORPHYRIN AND IRON DONATE ELECTRONS AS THE FER RYL OXYGEN INTERMEDIATE AND WATER ARE FORMED 4HIS IS FOLLOWED BY REACTION OF THE OXYGEN WITH A SECOND MOLECULE OF HYDROGEN PEROXIDE 3INCE THE TRAIT OF ELECTRON WITHDRAWAL FROM SUBSTRATE WHICH IS BOUND TO THE IRON OF HEME 0 IS SO NOVEL WE STRONGLY SUSPECT THAT IT IS RELATED TO THE MOST NOVEL ASPECT OF THE STRUCTURE OF (!/ THE TYROSINE CROSS LINKED TO THE CATALYTIC HEME 4HIS IS REINFORCED BY A COMPARISON OF (!/ WITH THE PENTA C HEME NITRITE REDUCTASE )N BOTH ENZYMES CATALYSIS TAKES PLACE AT THE IRON OF A C HEME AND ELECTRONS ARE EITHER WITHDRAWN TO (!/ OR DONATED BY .I2 AUXILIARY C HEMES OF THE ENZYME 2EMARKABLY ALTHOUGH SIGNIlCANT HOMOLOGY OF AMINO ACID SEQUENCE IS NOT OBSERVED THE TERTIARY STRUCTURE OF SUBUNITS OF (!/ AND .I2 ARE VERY SIMILAR AND THE HEMES OF .I2 HAVE EXACTLY THE SAME ORIENTATION IN SPACE RELATIVE TO ONE ANOTHER AS HEMES n OF (!/ %INSLE ET AL 4HE CATALYTIC HEME IRON OF .I2 DIFFERS FROM HEME 0 IN HAVING A LYSINE RATHER THAN HISTIDINE AS THE
AXIAL LIGAND -OST NOTABLY HOWEVER THE CATALYTIC HEME OF .I2 LACKS THE TYROSINE CROSS LINK FOUND IN (!/ 7ITH THIS IN MIND DETERMINATION OF THE ROLE OF THE TYROSINE CROSS LINK IN (!/ IS A HIGH PRIORITY FOR FUTURE WORK "Y SHOWING THAT THE PLANE OF THE TYROSINE RING IS PERPENDICULAR TO THE PORPHYRIN PLANE &IG RATHER THAN IN THE SAME PLANE THE CRYSTAL STRUCTURE OF (!/ ELIMINATED MECHANISMS DEPENDENT ON A CONTINUOUS CONJUGATED / ELECTRON SYSTEM BETWEEN PORPHYRIN AND TYROSINE RINGS &OR EXAMPLE ONE HYPOTHETICAL MECHANISM HAD INVOLVED THE OXIDATION OF A 3CHIFFS BASE WHICH WULD HAVE BEEN PREVIOUSLY FORMED BY REACTION OF HYDROXYLAMINE WITH A CARBONYL OXYGEN OF TYROSINE CONJUGATED WITH THE HEME RING !RCIERO AND (OOPER )N FACT THE ORIENTATION OF THE TYROSINE TO PORPHYRIN COVALENT BOND INDICATES THAT THE RELEVANT PORPHYRIN CARBON IS A TETRAHEDRAL CENTER BONDED TO THE TWO ADJACENT PYRROLE CARBONS THE TYROSYL CARBON AND PRESUMABLY HYDROGEN IE THE PORPHYRIN RING COULD NOT BE UNSATURATED AT THAT POINT 4HIS STRUCTURE A DIHYDROPORPHYRIN COULD THEORETICALLY PARTICIPATE IN HYDRIDE REMOVAL FROM IRON BOUND HYDROXYLAMINE !RCIERO AND (OOPER )N THAT MODEL THE ROLE OF IRON WAS THE STABILIZATION AND ACTIVATION OF SUBSTRATE RATHER THAN ELECTRON TRANSFER /THER POSSIBLE MODELS WHICH ARE UNDER CONSIDERATION CALL FOR A CLEARER DETERMINATION OF THE NATURE OF THE TYROSYL PORPHYRIN STRUCTURE !RCIERO AND (OOPER E3FBDUJWJUZPGUIF"DUJWF4JUFPG)"0 /RGANOHYDRAZINES ARE SUICIDE SUBSTRATES OF (!/ ,OGAN AND (OOPER )T IS HYPOTHESIZED THAT ORGANOHYDRAZINE IS OXIDIZED TO DINITROGEN AND A RADICAL CATION WHICH SUBSEQUENTLY REACTS WITH HEME 0 !S NOTED ABOVE HYDROGEN PEROXIDE REACTS WITH AND DESTROYS HEME 0 OF (!/ (YDROGEN PEROXIDE IS HYPOTHESIZED TO BIND AT THE IRON UNDERGO DEPROTONATION AND REACT WITH FERRIC HEME 0 TO FORM A FERRYL OXYGEN INTERMEDIATE WHICH MAY DECAY OR DISMUTATE TO REACTIVE FORMS OF OXYGEN THAT OPEN THE PORPHYRIN RING 4HIS INACTIVATION IS APPARENTLY PREVENTED IN VIVO %ITHER / OR (/ ARE CATALYTICALLY REDUCED TO (/ OR (/ RESPECTIVELY AT HEME 0 OF FULLY FERROUS (!/ OR IN THE PRESENCE OF AN EXCESS OF REDUCTANT IE THE ENZYME CAN ACT AS AN OXIDASE OR PEROXIDASE (OOPER AND "ALNY (OOPER ET AL 4ABLE 4HE REACTIONS ARE RAPID ONLY DURING THE OXIDATION OF THE LOW POTENTIAL HEMES OF THE ENZYME 4HESE REAC
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HAVE REDOX POTENTIALS OF %M| VS .(% n AND n M6 !RCIERO ET AL B $URING TURNOVER WITH (!/ THE TWO HEMES WITH MIDPOINT POTENTIALS OF M6 ARE REDUCED !RCIERO ET AL B 4HE MAGNETIC PROPERTIES OF THE C HEMES HAVE BEEN PARTIALLY CHARACTERIZED AND EXHIBIT{ ELEC TRONIC INTERACTIONS SIMILAR TO THOSE OBSERVED IN (!/ !NDERSSON ET AL -OST OF #YT C IS SOLUBLE IN EXTRACTS (OWEVER A SIGNIlCANT FRACTION OF #YT C IS MEMBRANE ASSOCIATED AND CAN BE WASHED OFF WITH HIGH CONCENTRATIONS OF .A#L 4HE DISSOCIATED #YT C RE BINDS IN APPROXIMATELY STOICHIOMETRIC AMOUNTS -C4AVISH ET AL 4HE BINDING CAPABILITIES OF #YT C IN THE INITIAL SUPERNATANT THE SALT WASH AND HIGHEST LEVEL OF PURIlCATION ARE THE SAME 4HE AMINO ACID SEQUENCE "ERGMANN ET AL (OM MES ET AL AND THE 8 RAY STRUCTURE HAVE BEEN COMPLETED )VERSON ET AL A ! SIDE VIEW OF THE 8 RAY STRUCTURE IS SHOWN IN &IG (EMES ARE NUMBERED )n)6 STARTING FROM THE . TERMINUS 2EDUCTION POTENTIALS AND SPECTROSCOPIC PROPERTIES AND HEME HEME INTERACTIONS HAVE BEEN ASSIGNED TO THE HEMES OF THE PROTEIN 5PADHYAY ET AL 4HE DISTRIBUTION OF SURFACE CHARGE AND THE SURFACE GEOMETRY OF (!/ AND CYC REVEAL A REASONABLE DOCKING SITE WHICH BRINGS THE EXPOSED EDGE OF HEME OF (!/ WITHIN ¯ OF THE SOLVENT EXPOSED EDGE OF HEME ) OF #YT C &IG )VERSON ET AL 4HE
TIONS APPARENTLY DO NOT OCCUR IN VIVO AND IN FACT MUST BE AVOIDED !S EXPECTED FOR A FERROUS HEME WHICH REACTS WITH DIOXYGEN FERROUS HEME 0 BINDS #/ CAUSING THE 3ORET BAND TO SHIFT TO NM !S DETER MINED BY OPTICAL SPECTROSCOPY FERRIC (!/ BINDS CYANIDE BUT DOES NOT BIND THE ANIONS AZIDE CYANATE OR THIOCYANATE mUORIDE CHLORIDE OR "ROMIDE ,OGAN ET AL #YANIDE INHIBITS ENZYME ACTIVITY ./ FORMS A DERIVATIVE WITH FERRIC HEME 0 IN THE RESTING ENZYME AND CAUSES THE REOXIDATION OF FERROUS HEMES OF THE PARTIALLY REDUCED ENZYME (ENDRICH ET AL 3EVERAL LINES OF EVIDENCE INDICATE THAT FERROUS (!/ REDUCES NITRITE 4HE STOICHIOMETRIC CONVERSION OF HYDROXYLAMINE AND NITRITE TO NITROUS OXIDE IN THE PRESENCE OF (!/ POSSIBLY INVOLVES THE INTERMEDI ATE PRODUCTION OF (./ FROM NITRITE (OOPER &URTHER THE REDUCED ENZYME IS REOXIDIZED IN THE PRESENCE OF NITRITE 9AMANAKA ET AL 3CHALK ET AL 4ABLE *75SBOTGFSPG&MFDUSPOTGSPN )"0UP6CJ RVJOPOF "$ZUPDISPNFD %LECTRONS FROM (!/ PASS TO #YT C 9AMANAKA AND 3HINRA 4HE C HEMES OF THIS CYTOCHROME
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IRON IRON DISTANCE BETWEEN THE TWO HEMES IS ¯ 7ITHIN #YT C A POSSIBLE ELECTRON TRANSPORT CHAIN THROUGH HEMES ) ))) )6 AND )) WOULD HAVE IRON IRON DISTANCES OF n ¯ AND ALLOW RAPID EQUILIBRIUM OF ELECTRONS AMONG THE HEMES OF #YT C AND BOUND (!/ )N THE STEADY STATE OR AT EQUILIBRIUM ELECTRONS COULD ACCUMULATE IN THE TWO M6 HEMES OF #YT C AS WELL AS THE ^ M6 AND M6 HEMES OF (!/ 2ECALLING THE APPARENT CAPACITY FOR ELECTRON TRANSPORT BETWEEN SUBUNITS A SINGLE ACTIVE SITE OF (!/ IN A FULLY OCCUPIED ;(!/#YT C=COMPLEX THEORETICALLY HAS AN ELECTRON RESERVOIR OF HIGH POTENTIAL C HEMES ON (!/ AND ON #YT C &IG 4HE UTILITY OF THIS ARRANGEMENT MAY BE THE ASSURANCE OF COMPLETE TURN OVER ONCE A SUBSTRATE MOLECULE HAS BOUND #$ZUPDISPNFD. "1VUBUJWF$ZUPDISPNF D6CJRVJOPOF3FEVDUBTF )N A GENE CLUSTER CONTAINING (!/ 3AYAVEDRA 3OTO ET AL THE GENE FOR #YT C SHARES AN OPERON AND IS CO TRANSCRIBED WITH THE GENE ENCODING THE MEMBRANE TETRAHEME #YT C- "ERGMANN AND (OOPER 4HE . TERMINAL SEQUENCE OF THE PURIlED DETERGENT SOLUBILIZED PROTEIN IS THE ONLY TRANSMEMBRANE DOMAIN 6ANNELLI ET AL "ASED ON ITS GENE SEQUENCE
THIS PROTEIN IS A MEMBER OF THE @.IR4.AP# FAM ILY OF TETRAHEME MEMBRANE ANCHORED CYTOCHROMES WHICH ARE IMPLICATED IN ELECTRON TRANSFER FROM MEMBRANE QUINOLS TO PERIPLASMIC TERMINAL ELECTRON ACCEPTORS 4HIS FAMILY IS EXEMPLIlED BY .IR4 .AP# AND .RF( WHICH DIRECT ELECTRONS TO THE #U CONTAINING NITRITE REDUCTASE *UNGST ET AL THE NITRATE RE DUCTASE 2OLDAN ET AL OR THE PENTA HEME NITRITE REDUCTASE 3IMON ET AL RESPECTIVELY %LECTRON TRANSFER FROM QUINOL TO THE APPROPRIATE PERIPLASMIC CYTOCHROME HAS BEEN EXPERIMENTALLY DEMONSTRATED WITH .RF( )N ALL THE MEMBERS OF THIS FAMILY THE SUBSEQUENT PERIPLASMIC ELECTRON TRANSFER STEP IS MEDIATED BY A SOLUBLE PERIPLASMIC C CYTOCHROME OR A lFTH C HEME IN AN ADDITIONAL VERY LARGE # TERMINAL DOMAIN OF THE QUINOL DEHYDROGENASE )T IS PROPOSED THAT #YT C- OF .ITROSOMONAS TRANSFERS ELECTRONS FROM THE PERIPLASMIC OXIDATION OF HYDROXYLAMINE BY (!/C TO UBIQUINONE IN THE MEMBRANE !LTHOUGH MEMBERS OF THE .IR4.AP# FAMILY HAVE SIMILAR LARGE CENTRAL AMINO ACID SEQUENCE DOMAINS CONTAINING THE FOUR C HEME BINDING MOTIFS THEY DIFFER VERY SIGNIlCANTLY IN THE SEQUENCE OF # TERMINAL DOMAINS 3INCE THE FAMILY OF PROTEINS ARE ALL THOUGHT TO REACT WITH MEMBRANE QUINONES THE VARIABLE
&IG 3TRUCTURE OF #YT C OF .ITROSOMONAS ;BASED ON )VERSON ET AL = 3IDE VIEW 4HE PROTEIN IS ARRANGED AS IF PREPARED TO DOCK AT THE LEFT SIDE OF THE (!/ TRIMER OF &IG (EME NUMBERS ARE BASED ON THE PRIMARY STRUCTURE AND BEGIN AT THE . TERMINAL
# TERMINAL DOMAINS MAY PLAY A ROLE IN THE SPECIlCITY OF PERIPLASMIC REDOX PARTNER 4HE # TERMINAL DOMAIN OF THE .ITROSOMONAS #YT C- IS REMARKABLY ACIDIC OF THE AMINO ACIDS ARE ASPARTATE OR GLUTAMATE )F THE ENZYMATIC ROLE OF #YT C- IS CONlRMED THE TRANSFER OF ELECTRONS FROM AN (!/ BOUND .(/( TO UBIQUINONE WILL CONTAIN HEMES PER (!/ SUBUNIT OR IN THE HYPOTHETICAL FULLY OCCUPIED ;(!/#YT C#YT C-= COMPLEX AS IN &IG /BVIOUSLY ONE ASKS WHY SO MANY HEMES ARE REQUIRED BY THIS PROCESS 4HE ANSWER MIGHT INCLUDE RAPID AND IRREVERSIBLE REMOVAL OF ELECTRONS TO A SAFE DISTANCE 74QFDVMBUJPOPOUIF&WPMVUJPOPGUIF )ZESPYZMBNJOF0YJEJ[JOH4ZTUFN $URING ANALYSIS OF THE CRYSTAL STRUCTURE OF #YT C PAIRS OF HEMES WITHIN (!/ AND #YT C WERE SEEN TO HAVE THE SAME SPATIAL ORIENTATION RELATIVE TO ONE ANOTHER IE THE PAIRS FROM THE TWO PROTEINS COULD BE SUPERIMPOSED WITH HIGH PRECISION AND WERE THUS RECOGNIZED AS DIHEME DIMENSIONAL MOTIFS )VERSON ET AL B 4HE MOTIFS WERE SEEN TO OCCUR IN SEVERAL OTHER MULTIHEME CYTOCHROMES WHICH HAD
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AN ELECTRON TRANSFER ROLE IN THE OXIDATION OR REDUCTION OF INORGANIC NITROGEN OR SULFUR COMPOUNDS -OST DRAMATICALLY THE ENTIRE TETRAHEME MOTIF OF #YT C HEMES )) )6 ))) ) WAS PRECISELY SUPERIMPOSABLE ON HEMES OF (!/ 4HIS CAN BE VISUALIZED BY LIFTING THE COLLECTIVE HEMES OF #YT C FROM THE PROTEIN IN ITS DOCKING SITE WITH (!/ TURNING THEM END TO END AND SETTING THEM ON THE HEMES OF (!/ &IG 4HE SIMILARITY EXTENDS TO MUCH OF THE SECONDARY STRUCTURE SURROUNDING THE HEMES IN SPITE OF AN ALMOST COMPLETE LACK OF HOMOLOGY IN PRIMARY STRUCTURE )VERSON ET AL LB 'IVEN THE FUNCTIONAL INTERRELATIONSHIP BETWEEN THESE PROTEINS AND THEIR PRESENCE IN THE SAME GENOME THE HYPOTHESIS OF COMMON ANCESTRY IS SIMPLER AND PERHAPS MORE LIKELY THAN CONVERGENT EVOLUTION )NTERESTINGLY HEME )) OF #YT C WHICH IS HOMOLOGOUS TO THE CATALYTIC HEME 0 HEME OF (!/ AS REVEALED IN THE ALIGNMENT OF THE TETRAHEME MOTIF HAS THE UNUSUAL PROPERTY OF BEING PENTACOORDINATE AND YET BEING PROTECTED FROM ACCESS TO SOLVENT BY A PROTEIN FOLD 4HIS PROPERTY IS CORRELATED WITH ITS LACK OF REACTIVITY WITH LIGANDS AND THE SEEMINGLY PARADOXICAL POSSESSION OF A 3ORET OPTICAL BAND CHARACTERISTIC OF A HIGH SPIN %02 CEN TER #YT C COULD IN THEORY HAVE EVOLVED FROM A PRIMITIVE (!/ BY GENE DUPLICATION FOLLOWED BY THE LOSS OF HEME BINDING #89#( MOTIFS AND SOME OF THE PROTEIN DOMAINS FOR HEMES AND 4HIS WOULD HAVE BEEN ACCOMPANIED BY ACQUISITION OF A PROTEIN FOLD COVERING HEME )) AND LOSS OF SURFACE
PROPERTIES RELATED TO THE TRIMER STRUCTURE AND THE TYROSINE CROSSLINK !LTERNATIVELY THE MORE PRIMITIVE MOLECULE MAY HAVE BEEN AN ENZYME RESEMBLING #YT C WHICH POSSESSED A CATALYTIC SITE OPEN TO SOLVENT 4HIS ANCESTRAL ENZYME COULD HAVE EVOLVED TO (!/ AND THE ELECTRON TRANSPORT MEDIATOR #YT C FOLLOW ING GENE DUPLICATION 4HE COMPLETION OF THE CRYSTAL STRUCTURE OF THE PENTAHEME CYTOCHROME C NITRITE REDUCTASE FROM SEVERAL SPECIES ADDED ANOTHER COMPLEX MULTIHEME C CYTOCHROME TO THE FAMILY )T SHARES ITS HEME MOTIF WITH HEMES n OF (!/ AND HEMES ))n)6 OF #YT C %INSLE ET AL 4HESE RELATION SHIPS ARE DIAGRAMED IN &IG 2EMARKABLY THE THREE CATALYTIC OR PENTACOORDINATE HEMES HEME 0 OF (!/ HEME OF .I2 AND HEME )) OF #YT C ARE HOMOLOGOUS BY THIS CRITERION OF ORIENTATION IN SPACE 4HE RELATIONSHIP BETWEEN (!/ AND .I2 IS ESPECIALLY INTERESTING SINCE THE REACTIONS INVOLVE THE SIX ELECTRON REDUCTION OF NITRITE BY .I2 OR THE PRODUCTION OF NITRITE BY THE EXTRACTION OF FOUR ELECTRONS FROM SUBSTRATE BY (!/ REPECTIVELY 4HE SUBUNIT SIZE AMINO ACID COMPOSITION (OOPER ET AL AND MUCH OF THE SECONDARY STRUCTURE IS SIMILAR IN THE TWO ENZYMES THOUGH THEY HAVE LITTLE HOMOLOGOUS SEQUENCE &URTHER .I2 IS A DIMER AND LACKS THE INTER MOLECULAR TYROSINE CROSSLINK TO THE CATALYTIC HEME )N FACT STERIC CONSID ERATIONS MIGHT MAKE FORMATION OF TWO SUCH CROSSLINKS IN A DIMER OF (!/ SUBUNITS IMPOSSIBLE )F SO THE TRIMERIC (!/ MIGHT BE THE SMALLEST OLIGOMER ALLOW
&IG !LIGNMENT OF HEMES OF A SUBUNIT OF (!/ AS IN &IG " AND THE HEMES OF #YT C AS ROTATED IN THE PLANE OF THE PAGE FROM THEIR ARRANGEMENT IN &IG $ATA OF )VERSON ET AL A B
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&IG (YPOTHETICAL EVOLUTIONARY RELATIONSHIPS BETWEEN #YT C (!/ AND .I2 BASED ON $ HEME MOTIFS "ASED ON DATA OF )GARASHI ET AL )VERSON ET AL A B AND %INSLE ET AL 4HE SIMILARITY OF HEME SPATIAL ARRANGEMENTS FOR (!/ AND #YT C IS ILLUSTRATED IN &IG .UMBERS IN PARENTHESES ARE HOMOLOGOUS HEMES OF (!/ AS CATEGORIZED BY ORIENTATION IN SPACE RELATIVE TO OTHER C HEMES IN THE SAME MOLECULE
ING EQUIVALENT CROSSLINKS TO ALL THREE CATALYTIC HEMES (EME OF .I2 IS THE lRST EXAMPLE OF A C HEME WITH A n#89#+ MOTIF WITH THE LYSINE AS AXIAL LIGAND TO THE IRON )N THE DIMER OF .I2 SUBUNITS ASSOCIATE AT THE BASE NEAR HEME EQUIVALENT TO HEME OF (!/ (EME OF .I2 WHICH IS PROPOSED TO BE THE POINT OF ENTRY OF ELECTRONS INTO THE ENZYME %INSLE ET AL CORRESPONDS TO HEME OF (!/ )NTERESTINGLY HEME IN (!/ IS THE APPARENT ELECTRON DONOR TO HEMES AND WHOSE HOMOLOGUES ARE NOT FOUND IN .I2 OF THE EXITT PATH FROM (!/ !S WITH (!/ AND #YT C (!/ AND .I2 COULD RELATIVELY EASILY HAVE ARISEN FROM A COMMON ANCESTOR 4HIS WOULD REQUIRE EITHER ACQUISITION OR LOSS OF THE BINDING SITES FOR HEMES AND OF (!/ AND THE HEME CROSSLINK AND TUNING OF THE CATALYTIC SITE FOR DEPROTONATION AND OXIDATION OR PROTONATION AND REDUCTION RESPECTIVELY OF . OXIDES 4HE EVOLUTIONARY RELATIONSHIPS BETWEEN GENES FOR THESE PROTEINS ARE SUBJECT FOR FUTURE SPECULATION ,OOKING AT EXAMPLES OF MEMBERS OF THIS FAMILY IT IS INTERESTING TO NOTE THAT BECAUSE THE ORDER OF SPATIALLY
HOMOLOGOUS HEMES ARE CO LINEAR WITH THE AMINO ACID SEQUENCE 4ABLE MOST EVOLUTIONARY TRANSFORMA TIONS SUGGESTED CAN OCCUR WITH SIMPLE MUTATION OF A HEME LIGATION SITE ANDOR DELETIONADDITION OF TERMINAL DOMAINS AND DO NOT REQUIRE EXCISION OR INSERTION OF DOMAINS INTERNAL TO THE GENE )MPORTANTLY THE LACK OF SEQUENCE HOMOLOGY INDICATES THAT THESE ARE OLD ENZYMES WHICH DIVERGED FROM COMMON ANCESTRY LONG AGO #AUTION MUST ACCOMPANY THE HYPOTHESIS THAT THESE PARTICULAR HEME MOTIFS ARE EVOLUTIONARILY RELATED )T HAS BEEN POINTED OUT THAT GIVEN THE TWO THIOETHER HEME TO PEPTIDE LINKAGES THE HISTIDINE AXIAL IRON LIGATION FROM THE n#89#( PRIMARY SEQUENCE MOTIF AND AXIAL LIGATION OF EACH HEME WITH A SECOND (IS STERIC HINDRANCE ALLOWS FEW ORIENTATIONS OF TWO HEMES IF PACKED WITHIN A HYDROPHOBIC ENVIRONMENT SUCH THAT THE HEMES ARE LESS THAN ¯ APART "RIGE ET AL &URTHER THE OBSERVED ORIENTATIONS WITHIN THE DIHEME MOTIFS OF THE CHARACTERIZED CYTOCHROMES ARE THE ONES PREDICTED FROM THESE STERIC CONSIDERATIONS
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#YT C ) )) ))) )6 .UMBERS IN ROWS REFER TO ORDER OF HEME COVALENT BINDING MOTIFS IN PRIMARY STRUCTURE OF PROTEIN (EMES IN THE SAME COLUMN ARE HOMOLOGOUS BASED ON THE CRITERION OF SPATIAL ORIENTATION RELATIVE TO OTHER HEMES IN THE SAME PROTEIN $ATA PENTAHEME NITRITE REDUCTASE $ARWIN ET AL (!/ 3AYAVEDRA 3OTO ET AL #YT C "ERGMANN ET AL A (OMMES ET AL
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"4USVDUVSFPG".04VCVOJUT 4HE REACTION BASED INACTIVATOR ACETYLENE LABELS A K$A MEMBRANE PROTEIN SEEN IN DENATURING GELS (YMAN AND 7OOD !N APPARENT COMPLEX CON SISTING OF THE ACETYLENE DERIVATIZED PROTEIN !-/ ! AND A K$A PROTEIN !-/ " WAS ISOLATED "ASED ON THE . TERMINI AN OPERON CONTAINING THE GENES AMO! AND AMO" FOR THE PUTATIVE SUBUNITS !-/ ! AND n" WAS CLONED AND SEQUENCED -C4AVISH ET AL A "ERGMANN AND (OOPER B (OMOLO GOUS GENES FOR SUBUNITS OF THE PARTICULATE METHANE MONOOXYGENASE P--/ WERE SUBSEQUENTLY FOUND IN -ETHYLOCOCCUS CAPSULATUS "ATH .GUYEN ET AL TOGETHER WITH A THIRD UPSTREAM GENE PMMO# ! HOMOLOGOUS GENE AMO# WAS THEN FOUND IN THE AMMONIA OXIDIZING NITRIlERS B UPSTREAM FROM AMO" +LOTZ ET AL 4HERE ARE NO OTHER OPEN READING FRAMES IN THE AMO GENE CLUSTER OF .ITRO SOMONAS #HAIN ET AL "ASED ON AMINO ACID SEQUENCE THE SUBUNITS OF !-/ AND P--/ ARE CLEARLY IN THE SAME EVOLUTIONARY FAMILY OF ENZYMES AND DISTINCT FROM THE SOLUBLE METHANE MONOOXYGEN ASE OF THE METHYLOTROPHIC BACTERIA (OLMES ET AL (YDROPHOBICITY PLOTS OF DEDUCED AMINO ACID SEQUENCES OF THE THREE SUBUNITS PREDICT A LARGE NUMBER OF TRANSMEMBRANE SEGMENTS IN BOTH !-/ ! AND
# AND IN !-/ " &IG 2EGIONS WITH SIGNIlCANT IDENTITY FOR !-/ AND P--/ ARE FOUND IN THE LARGE . AND # TERMINAL PERIPLASMIC DOMAINS OF SUBUNIT " AS WELL AS THE MOST # TERMINAL PERIPLASMIC LOOP OF SUBUNIT ! OF !-/ 4HE LATTER LOOP IS THOUGHT TO BE CYTOPLASMIC IN A THEORETICAL SEQUENCE BASED ANALYSIS OF THE HOMOLOGOUS SUBUNIT IN P--/ OF - CAPSULATUS WHERE SEVEN TRANSMEMBRANE SEGMENTS WERE PREDICTED 4UKHVATULLIN ET AL
4HE ELECTRON DONOR TO !-/ HAS NOT BEEN IDENTIlED $UROQUINOL SERVES AS ELECTRON DONOR IN CRUDE !-/ OR PURIlED P--/ SYSTEMS 3HEARS AND 7OOD :AHN AND $I3PIRITO 4HUS THE DONOR IS HYPOTH ESIZED TO BE UBIQUINOL -ANY OBSERVATIONS SUGGEST A ROLE FOR #U ION IN !-/ 7ITH CELLS THE COPPER BINDING AGENTS ALLYLTHIOUREA +#. OR DIETHYLDITHIOCARBAMATE INHIBIT AMMONIA OXIDATION (OOPER AND 4ERRY #OPPER IONS STIMULATE AMMONIA OXIDATION IN VITRO %NSIGN ET AL 3PECIlC PHOTO INACTIVATION OF AMMONIA OXIDIZING ACTIVITY BUT NOT HYDROXYLAMINE OXIDIZING ACTIVITY IN CELLS OF .ITROSOMONAS IS CAUSED BY NM LIGHT (OOPER AND 4ERRY %NZYME ACTIVITY IS LOW IN EXTRACTS (ENCE THE PRESENT LIMITED UNDERSTANDING OF THE MECHANISM OF !-/ HAS COME FROM STUDIES WITH INTACT CELLS )N CELLS !-/ IS REACTIVE WITH A REMARKABLE VARIETY OF NON POLAR SUBSTRATES WITH A SUSCEPTIBLE # ( BOND OR / ELECTRONS 4HE LIST INCLUDES .( #/ METHANE METHANOL PROPYLENE CYCLOHEXANE BENZENE PHENOL ETHYLENE HALOGENATED ALIPHATIC COMPOUNDS HALOBEN ZENES NAPHTHALENE AND CHLORO TRICHLOROMETHYL PYRIDINE (YMAN AND 7OOD 6ANNELLI AND (OOPER +EENER AND !RP REVIEWED IN (OOPER ET AL "ASED ON THE P( OPTIMUM FOR OXIDATION AMMONIA RATHER THAN AMMONIUM MAY BE THE PREFERRED SUBSTRATE 3UZUKI ET AL 0OLAR COMPOUNDS IN GENERAL ARE NOT GOOD SUBSTRATES SUG GESTING THAT THE ACTIVE SITE IS NON POLAR 4HE BROAD RANGE OF SUITABLE SUBSTRATES SUGGESTS THAT TURNOVER IS INITIATED BY THE ACTIVATION OF OXYGEN RATHER THAN THE ACTIVATION OF SUBSTRATE )N THEORY / ACTIVATION COULD INVOLVE BINDING AND ELECTRON REDUCTION OF / BY A METAL CENTER 4HE HYDROXYLATING AGENT COULD BE THE RESULTING PEROXYL DERIVATIVE OR A METAL / DERIVATIVE PRODUCED BY HETEROLYTIC CLEAVAGE
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OF PEROXYL WITH RELEASE OF WATER - E ( / A - / (/
! HYDROGEN MAY BE EXTRACTED FROM SUBSTRATE FORM ING A PLANAR TRICOORDINATE INTERMEDIATE BY EITHER THE PEROXIDE RELEASING WATER OR THE METAL / SPECIES TO FORM A METAL HYDROXIDE AND A SUBSTRATE RADICAL 4HE @REBOUND REACTION OF THE METAL HYDROXYL AND SUBSTRATE RADICAL WOULD GENERATE METAL AND PRODUCT .( - / A ;.(= - /(
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!-/ IN CELLS OF .ITROSOMONAS CATALYZES TWO REAC TIONS IN ADDITION TO MONOOXYGENATION WHICH CAN BE RATIONALIZED IN TERMS OF THE THESE MECHANISMS 4HE DEHYDROGENATION OF ETHYLBENZENE TO FORM STYRENE +EENER AND !RP COULD INVOLVE FORMATION OF THE - / INTERMEDIATE FOLLOWED BY TWO CYCLES OF DEHYDROGENATION OF SUBSTRATE AND RELEASE OF WATER 4HE ANAEROBIC REDUCTIVE DECHLORINATION OF CHLORO TRICHLOROMETHYL PYRIDINE TO CHLORO DICHLORO METHYL PYRIDINE 6ANNELLI AND (OOPER MIGHT HAVE RESULTED FROM ORIENTATION OF THE PYRIDINE IN A WAY THAT THE RING . WAS REDUCED INSTEAD OF DIOXYGEN 4QFDVMBUJPOPOUIF(FPNFUSZPGUIF"DUJWF 4JUF #OMPARISON OF THE REACTIVITY OF VARIOUS SUBSTRATES
ALSO SUGGESTS THAT THE OXYGENATING SITE IS DEEP IN A SOMEWHAT mATTENED TUBULAR POCKET &OR EXAMPLE ALKANES OF TO CARBONS IN LENGTH ARE PREFERENTIALLY HYDROXYLATED AT THE OR POSTION (YMAN ET AL &URTHER THE SUBSTITUTED AROMATIC COMPOUND ANISOLE IS OXIDIZED PREFERENTIALLY AT THE SUBSTITUENT GROUP AND WHEN THE RING IS HYDROXYLATED DURING THE REACTION THE PROCESS OCCURS PREFERENTIALLY AT THE P RATHER THAN O OR M POSITIONS +EENER AND !RP IE THE SUBSTITUTEnD AROMATIC COMPOUNDS ENTERS THE ACTIVE SITE LEADING WITH EITHER ONE END OR THE OTHER ! SERIES OF OBSERVATIONS INDICATE THAT THE AROMATIC RING OF THE SUBSTRATE IS IMMOBILIZED IN THE ACTIVE SITE SO AS TO LIMIT ROTATION OF THE BENZYL MOIETY AND TO LIMIT ROTATION AROUND THE # BENZYL BOND &URTHER THE DATA SUGGEST THAT THE ACTIVATED OXYGEN OF THE ACTIVE SITE IS POSITIONED ASYMMETRICALLY RELATIVE TO THE TWO # #ARBONS 6ANNELLI ET AL !-/ EXHIBITS STRONGLY REGIOSELECTIVE HYDROXYLATION OF # IN PREFERENCE TO # OF ETHYLBENZENE IE THE SUBSTRATE IS ORIENTED SO THAT THE ACTIVE OXYGEN IS CLOSER TO # &IG 3UBSTITUTION OF DEUTERIUM FOR HYDROGEN ON # CHANGED THE REGIOSELECTIVITY # RATHER THAN # WAS PREFERENTIALLY HYDROXYLATED 4HIS DEUTERIUM EFFECT INDICATED THAT A # ( BOND IS BROKEN DURING CATALYSIS )N TERMS OF THE CATALYTIC MODEL THIS WOULD BE THE ABSTRACTION OF AN ( OR $ BY THE REACTIVE OXYGEN 4HE RESULT SUGGESTS THAT ALTHOUGH THE ORIENTATION OF THE SUBSTRATE BY THE ACTIVE SITE PLACED # DEUTERIUM CLOSEST TO THE REACTIVE OXYGEN AS INDICATED BY THE REGIOSELECTIVITY OF THE NON DEUTERATED SUBSTRATE THE # HYDROGEN WAS CLOSE ENOUGH THAT THE LATTER WAS
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AND .ITROSOCOCCUS OXIDIZE METHANE TO #/ AND THE ENTIRE PROCESS IS LIKELY TO BE CATALYZED BY !-/ *ONES AND -ORITA 6OYSEY AND 7OOD )T MAY BE NUTRITIONALLY SIGNIlCANT THAT THE ORGANISM ALSO INCORPORATES A SIGNIlCANT FRACTION OF THE METHANE # INTO BIOMASS *ONES AND -ORITA 7ARD !-/ OF .ITROSOMONAS IS ALSO VERY MUCH MORE REAC TIVE WITH AROMATIC COMPOUNDS THAN P--/ ,ONTOH ET AL 7**"OBFSPCJD3FTQJSBUJPOCZ"VUPUSPQIJD /JUSJàFST 6ERY SLOW GROWTH OF .ITROSOMONAS EUROPAEA UNDER ANAEROBIC CONDITIONS HAS BEEN REPORTED WITH PYRUVATE AS REDUCTANT AND NITRITE AS TERMINAL ELECTRON ACCEP TOR !BIELOVICH AND 6ONSHAK 4HE DATA IN THE GENOME REVEAL THE PRESENCE OF GENES ENCODING MANY OF THE ENZYMES NECESSARY FOR THIS PROCESS AS MEDIATED BY PYRUVATE DEHYDROGENASE THE CITRIC ACID CYCLE AND NITRITE REDUCTASE #HAIN ET AL !NAEROBICALLY .ITROSOMONAS EUTROPHA AND .ITROSOMONA EUROPAEA ARE REPORTED TO OXIDIZE HYDROGEN WHILE REDUCING NITRITE "OCK ET AL ! GENE FOR HYDROGENASE IS NOT SEEN IN THE CHROMOSOME OF THE STRAIN OF . EUROPAEA WHICH HAS BEEN SEQUENCED .ITROSOMONAS EUTROPHA IS ALSO ABLE TO GROW AN AEROBICALLY WHILE CONSUMING EQUAL AMOUNTS OF .( AND ./ AND PRODUCING A MIXTURE OF ./ ./n AND . 3CHMIDT ET AL 4HE INITIATING REACTION IS THOUGHT TO INVOLVE ./ AS OXYGEN DONOR TO !-/ RESULTING IN THE PRODUCTION OF .(/( AND ./
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VAN DE 'RAF ET AL 3TROUS ET AL 5SE OF .( WITH (./ RESULTED IN THE PRODUC TION OF ONLY . 4HIS ELEGANT OUTCOME INDICATED THAT A SINGLE ACTIVE SITE MUST HAVE SIMULTANEOUSLY BOUND AMMONIA AND NITRITE OR THEIR RESPECTIVE INTERMEDIATE PRODUCTS -EASURABLE AMOUNTS OF HY DRAZINE AND HYDROXYLAMINE ACCUMULATED DURING THE PROCESS 4HE HYPOTHESIS WHICH IS FAVORED BY THESE INVESTIGATORS INVOLVES THREE REACTIONS REDUCTION OF NITRITE TO HYDROXYAMINE REACTION OF AMMONIA WITH HYDROXYLAMINE TO FORM HYDRAZINE AND lNALLY THE DEHYDROGENATION OF HYDRAZINE (./ En ( A .(/( (/
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4HE REACTIONS ARE PROPOSED TO BE ARRANGED IN SEPARATE MEMBRANE COMPARTMENTS SO AS TO DRIVE FORMATION OF A CHEMIOSMOTIC GRADIENT 4HE ORGANISM HAS AN UNUSUAL INTERNAL MEMBRANE SYSTEM WHICH IS EX TREMELY DIFlCULT TO ANALYZE ,INDSAY ET AL )T IS DESCRIBED AS CONSISTING OF A PERIPHERAL CYTO PLASMIC MEMBRANE INSIDE THE CELL WALL 7ITHIN THE ENCLOSED @CYTOPLASM IS A REGION CALLED THE @RIBOPLASM WHICH IS ENCLOSED BY A SECOND INTERNAL MEMBRANE THE INTRACYTOPLASMIC MEMBRANE 4HE RIBOPLASM CONTAINS RIBOSOMES AND A NUCLEOID 4HE EXTENSIVE SPACE BETWEEN THE INTRACYTOPLASMIC MEMBRANE AND CYTOPLASMIC MEMBRANE IS TERMED @PARYPHOPLASM 4HE LATTER IS SEEN TO FORM A BALLOON LIKE INTRUSION INTO THE RIBOPLASM AND PERHAPS PINCH OFF PRODUCING YET A THIRD LAYER OF CONCENTRIC VACUOLES 4HE CELLS ARE RED IN COLOR DUE TO THE PRESENCE OF AN (!/ WHICH IS ABLE TO OXIDIZE HYDROXYLAMINE OR HYDRAZINE 3CHALK ET AL -OST OF THE MO LECULAR PROPERTIES ARE VERY SIMILAR TO THOSE OF (!/ OF .ITROSOMONAS AND .ITROSOCOCCUS 4HE REDUCED AND OPTICAL SPECTRA OF THE (!/ HAVE MAJOR 3ORET
AND _ AND ` BANDS ATTRIBUTABLE TO THE APPROXIMATELY C HEMES IN A TRIMER OF K$A 4HE FERROUS SPECTRUM HAS A WEAK 3ORET BAND AT NM WHICH IS NOT SEEN IF THE FERRIC ENZYME HAD BEEN TREATED WITH HYDROGEN PEROXIDE RESULTING IN LOSS OF ENZYME ACTIV ITY 4HIS APPEARS TO CORRESPOND TO HEME 0 OF THE .ITROSOMONAS ENZYME 4HE %02 SPECTRUM CONTAINS SOME SIGNALS FROM IRON OF LOW SPIN C HEMES AS WELL AS ATYPICAL SIGNALS POSSIBLY REPRESENTING ELECTRONI CALLY COUPLED HEME IRONS 4HE ENZYME IS THOUGHT TO AT LEAST ACCOUNT FOR THE OXIDATION OF HYDRAZINE TO DINITROGEN IN THE ORGANISM AND POSSIBLY CATALYZE THE REDUCTION OF NITRITE TO ./ 4HE HEME COMPOSITION AND CATALYTIC AND %02 PROPERTIES OF THE (!/S OF !.!--/8 AND OF .I TROSOMONAS ARE SIMILAR ENOUGH TO SUGGEST THAT THE HEMES OF THE TWO PROTEINS MIGHT ULTIMATELY BE FOUND TO ALIGN IN SIMILAR SPATIAL CONlGURATIONS *9)FUFSPUSPQIJD/JUSJàDBUJPO 4HE OXIDATION OF AMMONIA TO NITRITE ALSO OCCURS IN THE HETEROTROPHIC BACTERIA 4HE RATES OF OXIDATION OF AMMONIA PER CELL ARE LOWER THAN WITH THE AUTOTROPHIC NITRIlERS "IOCHEMICAL CHARACTERIZATION OF THE REAC TIONS INVOLVED HAS NOT BEEN EXTENSIVE !N AMMONIA MONOOXYGENASE FROM MEMBRANES OF 0ARACOCCUS DENITRIlCANS HAS BEEN SOLUBILIZED AND PURIlED TO A FORM CONTAINING SUBUNITS OF AND K$A -OIR ET AL 4HE ASSAY EMPLOYED DUROQUINOL AS REDUCTANT 4HE ENZYME RESEMBLED !-/ FROM .ITRO SOMONAS IN SENSITIVITY TO LIGHT AND COPPER BINDING AGENTS 4HE OXIDATION OF AMMONIA TO HYDROXYLAMINE NITRITE AND NITRATE BY 0SEUDOMONAS PUTIDA WAS SEEN TO BE INACTIVATED BY ACETYLENE $AUM ET AL !N OPEN READING FRAME IDENTIlED BY ITS HYBRIDIZATION WITH A PROBE FOR AMO! CONTAINED SEQUENCE SIMILARITY TO THE GENE IN AUTOTROPHIC NITRIlERS 4HUS IT APPEARS THAT !-/ OF THE !-/P--/ FAMILY IS UTILIZED BY MANY ORGANISMS )N CONTRAST THE HYDROXYLAMINE OXIDIZING ENZYMES THAT HAVE BEEN ISOLATED THUS FAR FROM HETEROTROPHIC BACTERIA DO NOT RESEMBLE (!/ OF THE AUTOTROPHIC NITRIlERS !N ENZYME FROM !R R THROBACTER GLOBIFORMIS +UROKAWA ET AL HAS HYDROXYLAMINE DYE REDUCTASE ACTIVITY OR HYDROXYL AMINE CYTOCHROME C REDUCTASE ACTIVITY BUT DOES NOT CONTAIN HEME ! SIMILAR ACTIVITY FROM 0ARACOCCUS DENITRIlCANS APPEARS TO BE CATALYZED BY AN IRON SULFUR PROTEIN -OIR ET AL
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"DLOPXMFEHNFOUT 4HE AUTHORS THANK -ARK 7HITTAKER 4OM 0OULUS 4ODD 6ANNELLI AND 0AUL +LUGE FOR DISCUSSIONS AND HELP IN PREPARATION OF THE MANUSCRIPT 4HIS WORK WAS SUPPORTED BY FUNDS FROM THE 53 .ATIONAL 3CIENCE &OUNDATION .3&-#" AND 53 $EPART MENT OF %NERGY $% &' %2 TO !"( AND FUNDS FROM .)( TO -0( 3FGFSFODFT !BIELOVICH ! AND 6ONSHAK ! !NAEROBIC METABOLISM OF .ITROSOMONAS EUROPAEA !RCH -ICROBIOL n !NDERSON )# AND ,EVINE *3 L 2ELATIVE RATES OF NITRIC OXIDE AND NITROUS OXIDE PRODUCTION BY NITRIlERS DENITRIlERS AND NITRATE RESPIRERS !PPL AND %NVIRON -ICROBIOL n !NDERSSON ++ AND (OOPER !" / AND (/ ARE EACH THE SOURCE OF ONE / OF (./ PRODUCED FROM .( BY .ITROSOMONAS . .-2 EVIDENCE &%"3 ,ETT n !NDERSSON ++ ,IPSCOMB *$ 6ELENTINE - -UNCK % AND (OOPER !" 4ETRAHEME CYTOCHROME C FROM .ITROSOMONAS EUROPAEA HEME HEME INTERACTIONS AND LIGAND BINDING * "IOL #HEM n !RCIERO $- AND (OOPER !" A (YDROXYLAMINE OXIDO REDUCTASE FROM .ITROSOMONAS EUROPAEA IS A MULTIMER OF AN OCTA HEME SUBUNIT * "IOL #HEM n !RCIERO $- AND (OOPER !" ! DI HEME CYTOCHROME C PEROXIDASE FROM .ITROSOMONAS EUROPAEA CATALYTICALLY ACTIVE IN BOTH THE OXIDIZED AND HALF REDUCED STATES * "IOL #HEM n !RCIERO $- AND (OOPER !" %VIDENCE FOR A PEPTIDYL LY SINE TO C HEME CROSSLINK IN CYTOCHROME 0 OF .ITROSOMONAS EUROPAEA &%"3 ,ETT n !RCIERO $- AND (OOPER !" #ONSIDERATION OF A PHLORIN STRUCTURE FOR HAEM 0 OF HYDROXYLAMINE OXIDOREDUCTASE AND ITS IMPLICATIONS REGARDING REACTION MECHANISM "IOCHEM 3OC 4RANS n !RCIERO $- AND (OOPER !" (EME 0 OF HYDROXYLAMINE OXIDOREDUCTASE -ODELS FOR CATALYSIS BASED ON DIHYDROPORPHYRIN AND ISOPORPHYRIN RING SYSTEMS * )NORG "IOCHEM !RCIERO $ "ALNY # AND (OOPER !" A 3PECTROSCOPIC AND RAPID KINETIC STUDIES OF REDUCTION OF CYTOCHROME C BY HYDROXYLAMINE OXIDOREDUCTASE FROM .ITROSOMONAS EUROPAEA "IOCHEMISTRY n !RCIERO $- #OLLINS - (ALADJIAN * "IANCO 0 AND (OOPER !" B 2ESOLUTION OF THE FOUR HEMES OF CYTOCHROME C FROM .ITROSOMONAS EUROPAEA BY REDOX POTENTIOMETRY AND OPTICAL SPECTROSCOPY "IOCHEMISTRY n !RCIERO $- (OOPER !" #AI - AND 4IMKOVICH 2 %VIDENCE FOR THE STRUCTURE OF THE ACTIVE SITE HEME 0 IN HYDROXYLAMINE OXIDOREDUCTASE OF .ITROSOMONAS "IOCHEMISTRY n !RCIERO $- 0IERCE "3 (ENDRICH -0 AND (OOPER !" .ITROSOCYANIN A RED CUPREDOXIN LIKE PROTEIN FROM M .ITROSOMONAS EUROPAEA "IOCHEMISTRY n "EAUMONT (%" (OMMES .' 3AYAVEDRA 3OTO ,! !RP $* !RCIERO $* (OOPER !" 7ESTERHOFF (6 AND 6AN 3PANNING
2*- .ITRITE REDUCTASE OF .ITROSOMONAS EUROPAEA IS NOT ESSENTIAL FOR PRODUCTION OF GASEOUS NITROGEN OXIDES AND CONFERS TOLERANCE TO NITRITE * "ACTERIOL n "ERGMANN $* AND (OOPER !" A 4HE PRIMARY STRUCTURE OF CYTOCHROME 0 OF .ITROSOMONAS EUROPAEA 0RESENCE OF A C HEME BINDING MOTIF &%"3 ,ETT n "ERGMANN $* AND (OOPER !" B 3EQUENCE OF THE GENE AMO" WHICH ENCODES THE K$A POLYPEPTIDE OF AMMONIA MONOXYGENASE OF .ITROSOMONAS EUROPAEA "IOCHEM "IOPHYS 2ES #OMMUN n "ERGMANN $* AND (OOPER !" #YTOCHROME 0 OF .ITRO SOMONAS EUROPAEA &ORMATION OF THE HEME LYSINE CROSSLINK IN A HETEROLOGOUS HOST AND MUTAGENIC CONVERSION TO A NON CROSS LINKED CYTOCHROME C| %UR * "IOCHEM n "ERGMANN $* !RCIERO $ AND (OOPER !" /RGANIZATION OF THE (!/ GENE CLUSTER OF .ITROSOMONAS EUROPAEA 'ENES FOR TWO TETRAHEME CYTOCHROMES * "ACTERIOL n "ERGMANN $* :AHN *! (OOPER !" AND $I3PIRITO !! #YTOCHROME 0 GENES FROM THE METHANOTROPH -ETHYLOCOCCUS CAPSULATUS BATH * "ACTERIOL n "OCK % 3CHMIDT ) 3TàVEN 2 AND :ART $ .ITROGEN LOSS CAUSED BY DENITRIFYING .ITROSOMONAS CELLS USING AMMONIUM OR HYDROGEN AS ELECTRON DONORS AND NITRITE AS ELECTRON ACCEPTOR !RCH -ICROBIOL n "RANTNER #! 2EMSEN ## /WEN (! "UCHHOLZ ,! AND #OLLINS -,0 )NTRACELLULAR LOCALIZATION OF THE PARTICULATE METHANE MONOOXYGENASE AND METHANOL DEHYDROGENASE IN -ETHYLOMI CROBIUM ALBUM "' !RCH -ICROBIOL n "RIGE ! ,EYS $ -EYER 4% #USANOVICH -! AND 6AN "EEUMEN ** 4HE ¯ RESOLUTION STRUCTURE OF THE DIHEME .AP" SUBUNIT OF SOLUBLE NITRATE REDUCTASE REVEALS A NOVEL CYTOCHROME C FOLD WITH A STACKED HEME ARRANGEMENT "IOCHEMISTRY n #HAIN 0 ,AMERDIN * ,ARINER & 2EGALA 7 ,AO 6 ,AND - (AUSER , (OOPER !" +LOTZ - .ORTON * 3AYAVEDRA 3OTO , !RCIERO $ (OMMES . 7HITTAKER - AND !RP $ #OMPLETE GENOME SEQUENCE OF THE AMMONIA OXIDIZING BACTERIUM AND OBLIGATE CHEMOLITHOAUTOTROPH .ITROSOMONAS EUROPAEA * "ACTERIOL n #OLLINS - !RCIERO $- AND (OOPER !" /PTICAL SPEC TROPHOTOMETRIC RESOLUTION OF THE HEMES OF HYDROXYLAMINE OXIDOREDUCTASE (EME QUANTITATION AND P( DEPENDENCE OF %M * "IOL #HEM n #ONRAD 2 3OIL MICROORGANISMS AS CONTROLLERS OF AT MOSPHERIC TRACE GASES ( #/ #( /#3 ./ AND ./ -ICROBIOL 2EV n $ARWIN ! (USSAIN ( 'RIFlTHS , 'ROVE * 3AMBONGI 9 "USBY 3 AND #OLE * 2EGULATION AND SEQUENCE OF THE STRUC TURAL GENE FOR CYTOCHROME C FROM %SCHERICHIA COLI .OT A HEXAHAEM BUT A K$A TETRAHAEM NITRITE REDUCTASE -OLE# -ICROBIOL n $AUM - :IMMER 7 0APEN ( +LOOS + .AWRATH + AND "OTHE ( 0HYSIOLOGICAL AND MOLECULAR BIOLOGICAL CHARACTERIZATION OF AMMONIA OXIDATION OF THE HETEROTROPHIC NITRIlER 0SEUDOMO NAS PUTIDA #URRENT -ICROBIOL n $I3PIRITO !! 4AAFFE ,2 AND (OOPER !" A ,OCALIZATION AND CONCENTRATION OF HYDROXYLAMINE OXIDOREDUCTASE AND CYTO CHROMES C C C- C- AND A IN .ITROSOMONAS EUROPAEA "IOCHIM "IOPHYS !CTA n $I3PIRITO !! 4AAFFE ,2 ,IPSCOMB *$ AND (OOPER !" B ! @BLUE COPPER OXIDASE FROM .ITROSOMONAS EUROPAEA "IOCHIM
$IBQUFS #BDUFSJBM0YJEBUJPOPG"NNPOJB "IOPHYS !CTA n $I3PIRITO !! ,IPSCOMB *$ AND (OOPER !" #YTO CHROME AA FROM .ITROSOMONAS EUROPAEA * "IOL #HEM n %INSLE / -ESSERSCHMIDT ! 3TACH 0 "OURENKOV '" "ARTUNIK ($ (UBER 2 AND +RONECK 0-( 3TRUCTURE OF CYTOCHROME C NITRITE REDUCTASE .ATURE n %INSLE / 3TACH 0 -ESSERSCHMIDT ! 3IMON * +ROGER ! (UBER 2 AND +RONECK 0-( #YTOCHROME C NITRITE REDUCTASE FROM 7OLINELLA SUCCINOGENES 3TRUCTURE AT ¯ RESOLUTION INHIBITOR BINDING AND HEME PACKING MOTIFS * "IOL #HEM n %NSIGN 3! (YMAN -2 AND !RP $* )N VITRO ACTIVATION OF AMMONIA MONOXYGENASE FRO- NITROSOMONAS BY COPPER * "ACTERIOL n %RICKSON 2( AND (OOPER !" 0RELIMINARY CHARACTERIZA TION OF A VARIANT #/ BINDING HEME PROTEIN FROM .ITROSOMONAS "IOCHIM "IOPHYS !CTA n 'OREAU 4* +APLAN 7! 7OFSY 3# -C%LROY -" 6ALOIS &7 AND 7ATSON 37 0RODUCTION OF ./ AND ./ BY NITRIFYING BACTERIA AT REDUCED CONCENTRATIONS OF OXYGEN !PPL %NVIRON -ICROBIOL n (ENDRICH - ,OGAN -30 !NDERSSON ++ !RCIERO $- ,IPSCOMB *$ AND (OOPER !" 4HE ACTIVE SITE OF HYDROXYLAMINE OXIDOREDUCTASE %VIDENCE FROM INTEGER SPIN %02 * !M #HEM 3OC n (ENDRICH -0 0ETASIS $ !RCIERO $- AND (OOPER !" #ORRELATIONS OF STRUCTURE AND ELECTRONIC PROPERTIES FROM %02 SPECTROSCOPY OF HYDROXYLAMINE OXIDOREDUCTASE * !M #HEM 3OC n (ENDRICH -0 5PADHYAY !+ 2IGA * !RCIERO $- AND (OOPER !" 3PECTROSCOPIC CHARACTERIZATION OF THE ./ ADDUCT OF HY DROXYLAMINE OXIDOREDUCTASE "IOCHEMISTRY n (OLLOCHER 4# +UMAR 3 AND .ICHOLAS $*$ 2ESPIRATION DEPENDENT PROTON TRANSLOCATION IN .ITROSOMONAS EUROPAEA AND ITS APPARENT ABSENCE IN .ITROBACTER AGILIS DURING INORGANIC OXIDATIONS * "ACTERIOL n (OLMES !* #OSTELLO ! ,IDSTROM -% AND -URRELL *# %VIDENCE THAT PARTICULATE METHANE MONOXYGENASE AND AMMONIA MONOXYGENASE MAY BE EVOLUTIONARILY RELATED &%-3 -ICROBIOL ,ETT n (OMMES .' 3AYAVEDRA 3OTO ,! AND !RP $* 3EQUENCE OF HCY A GENE ENCODING CYTOCHROME C IN .ITROSOMONAS EUROPAEA 'ENE n (OOPER !" ! NITRITE REDUCING ENZYME FROM .ITROSOMONAS EUROPAEA PRELIMINARY CHARACTERIZATION WITH HYDROXYLAMINE AS ELECTRON DONOR "IOCHIM "IOPHYS !CTA n (OOPER !" "IOCHEMICAL BASIS OF OBLIGATE AUTOTROPHY IN .ITROSOMONAS EUROPAEA * "ACTERIOL n (OOPER !" AND .ASON ! #HARACTERIZATION OF HYDROX YLAMINE CYTOCHROME C REDUCTASE FROM THE CHEMOAUTOTROPHS .ITROSOMONAS EUROPAEA AND .ITROSOCYSTIS OCEANUS * "IOL #HEM n (OOPER !" AND 4ERRY +2 3PECIlC INHIBITORS OF AMMONIA OXIDATION IN .ITROSOMONAS * "ACTERIOL n (OOPER !" AND 4ERRY +2 0HOTOINACTIVATION OF AMMONIA OXIDATION IN .ITROSOMONAS * "ACTERIOL n (OOPER !" AND 4ERRY +2 (YDROXYLAMINE OXIDOREDUCTASE OFF .ITROSOMONAS )NACTIVATION BY HYDROGEN PEROXIDE "IOCHEM ISTRY n (OOPER !" AND 4ERRY +2 (YDROXYLAMINE OXIDOREDUCTASE
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!UTHOR FOR CORRESPONDENCE EMAIL DRICHARDSON UEAACUK $AVIDE :ANNONI ED 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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4VNNBSZ 2ESPIRATORY REACTIONS INVOLVING INORGANIC NITROGEN SPECIES PROVIDE A RICH VARIETY OF SYSTEMS WITH WHICH TO STUDY BACTERIAL BIOENERGETICS AND BIOLOGICAL CHEMISTRY 4HESE RESPIRATORY REACTIONS ENCOMPASS THE REDUCTION OF NITRATE NITRITE NITRIC OXIDE AND NITROUS OXIDE IN ALL CASES CATALYSIS INVOLVES REDOX CHEMISTRY THAT TAKES PLACE AT METAL CENTERS 4HESE CATALYTIC METAL CENTERS INCLUDE MONO NUCLEAR TYPE )) COPPER C HEME OR D HEME IN NITRITE REDUCTASES THE -O BIS MOLYBDOPTERIN GUANINE DINUCLEOTIDE COFACTOR IN NITRATE REDUCTASES A NON HEME &E HEME &E DINCUCLEAR CENTER IN NITRIC OXIDE REDUCTASES AND A TETRA NUCLEAR COPPER SULlDE CENTER IN NITROUS OXIDE REDUC TASE 2ECENT STRUCTURES AND NEW SPECTROSCOPIC DATA FOR THESE ENZYMES HAVE RAPIDLY ADVANCED OUR KNOWLEDGE OF THE MOLECULAR AND DYNAMIC PROPERTIES OF THE CATALYTIC CENTERS AS WELL AS HOW ELECTRONS ARE TRANSFERRED TO THEM MODELS OF CATALYSIS CAN THUS BE PROPOSED "IOENERGETIC STUDIES HAVE ALSO PROVIDED AN UNDERSTANDING OF THE WAY IN WHICH THESE ENZYMES ARE COUPLED TO ENERGY CONSERVING ELECTRON TRANSPORT PATHWAYS
**OUSPEVDUJPO %O| M6 OF 4HE REDUCTION OF NITRATE TO NITRITE % %O| M6 OR AMMONIUM NITRITE TO NITRIC OXIDE % %O| M6 OF NITRIC OXIDE TO NITROUS OXIDE % % %O| M6 AND OF THE LATTER TO NITROGEN GAS % %O| M6 ARE EACH REACTIONS WITH %O| VALUES THAT ARE SUFlCIENTLY POSITIVE FOR THE REACTIONS TO BE LINKED TO BACTERIAL ENERGY CONSERVING RESPIRATORY CHAINS 4HIS THEN ALLOWS OXIDATIVE PHOSPHORYLATION TO OCCUR UNDER ANAEROBIC CONDITIONS ALTHOUGH IT SHOULD BE NOTED THAT MANY OF THE ORGANISMS THAT USE THESE REACTIONS DO SO AS A SECOND CHOICE WHEN OXYGEN IS NOT AVAILABLE AS THE TERMINAL ELECTRON ACCEPTOR 4HE REACTIONS CAN OCCUR EITHER INDIVIDUALLY WITHIN A BACTERIAL SPECIES OR IN VARIOUS COMBINATIONS 4HUS IN A DENITRIFYING BACTERIUM NITRATE CAN BE SEQUENTIALLY REDUCED THROUGH TO NITROGEN GAS IN WHICH CASE THE PRODUCT OF NITRITE REDUCTION WILL BE NITRIC OXIDE 4YPICAL EXAMPLES OF ORGANISMS EQUIPPED IN THIS WAY INCLUDE 0ARACOCCUS DENITRIlCANS 0ARACOCCUS PANTOTROPHUS 0SEUDOMO NAS STUTZERI AND 0SEUDOMONAS AERUGINOSA /N THE OTHER HAND SOME ORGANISMS FOR EXAMPLE %SCHERICHIA COLI HAVE ENZYMES THAT SEQUENTIALLY REDUCE NITRATE TO AMMONIUM VIA NITRITE HENCE THIS ORGANISM IS NOT A DENITRIlER BUT IS SOMETIMES CALLED AN AMMONIlER /THER ORGANISMS MAY HAVE OTHER COMBINATIONS OF THESE ENZYME ACTIVITIES FOR EXAMPLE THE PATHOGEN .EISSERIA GONORRHOEAE HAS A NITRITE REDUCTASE AND NITRIC OXIDE REDUCTASE ONLY (OUSEHOLDER ET AL !BBREVIATIONS -O BIS -'$ n MOLYBDENUM BIS MOLYBDOP TERIN GUANINE DINUCLEOTIDE .AP n PERIPLASMIC NITRATE REDUCTASE .AR n MEMBRANE BOUND NITRATE REDUCTASE .IR n NITRITE REDUCTASE .OS n NITROUS OXIDE REDUCTASE .OR n NITRIC OXIDE REDUCTASE Q E n NUMBER OF POSITIVE CHARGES MOVED ACROSS THE MEMBRANE PER TWO ELECTRONS TRANSFERRED
,ISSENDEN ET AL 4HE PHOTOSYNTHETIC BACTERIA 2HODOBACTER SPHAEROIDES AND 2HODOBACTER CAPSULA TUS ALWAYS APPEAR TO HAVE THE NITRIC OXIDE REDUCTASE BUT THE PRESENCE OF ONE OR MORE OF THE REDUCTASES FOR NITRATE NITRITE AND NITROUS OXIDE VARIES BETWEEN STRAINS "ELL ET AL (ISTORICALLY THE ORGANISMS RECOG NIZED AS INVOLVED IN THESE REACTIONS HAVE BEEN FROM A LIMITED NUMBER OF EUBACTERIA 0ERSPECTIVE ON THE OC CURRENCE OF THESE ENZYMES IS RAPIDLY CHANGING OWING TO THE ACQUISITIONS OF MANY GENOME SEQUENCES 4HE LATTER ARE ESTABLISHING THAT THESE ENZYMES ESPECIALLY THE NITRIC OXIDE REDUCTASE ARE MORE WIDESPREAD THAN HITHERTO SUSPECTED AND SOME AT LEAST ARE NOW KNOWN TO OCCUR IN !RCHAEA (ENDRIKS ET AL 2ICHARDSON ET AL 4HE ROLES OF THESE ENZYMES WITHIN THE NITROGEN CYCLE ARE PRESENTED IN &IG 4HE RELATIVELY POSITIVE REDOX POTENTIALS OF ALL THE REACTIONS INVOLVED IN REDUCTION OF NITRATE AND SPECIES FORMED THEREFROM MEANS THAT THE ENZYMES FOR EACH OF THE REACTIONS CAN BE LINKED TO BACTERIAL ELECTRON TRANSPORT SYSTEMS AT EITHER THE QUINONE OR C TYPE CYTOCHROMECUPREDOXIN REGION 2EDUCTION OF NITRATE TO NITRITE IS ALWAYS LINKED TO THE QUINONE LEVEL % %O| IN REGION OF n TO M6 WHEREAS REDUCTION OF NITROUS OXIDE IS ONLY KNOWN TO BE ASSOCIATED WITH THE CYTOCHROME CCUPREDOXIN LEVEL % %O| IN REGION OF M6 OF AN ELECTRON TRANSFER CHAIN 4HIS MAKES SENSE IN TERMS OF THE RELATIVE REDOX POTENTIALS OF THE REACTIONS 4HE LINKAGE OF THE NITRITE AND NITRIC OXIDE REDUCTION REACTIONS SHOWS MORE VARIATION 7HEN NITRITE IS REDUCED TO AMMONIA THE CONNECTION IS AT THE LEVEL OF QUINONE (OWEVER CYTOCHROMES C OR CU PREDOXINS USUALLY PROVIDE ELECTRONS TO THE REDUCTASES THAT HAVE NITRIC OXIDE AS REACTION PRODUCT ALTHOUGH AN EXCEPTION MAY BE IN SOME 0SEUDOMONAS SPECIES WHERE THE CONNECTION MAY BE AT THE LEVEL OF QUINOL
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.ITRIC OXIDE REDUCTASES HAVE A MORE COMPLEX PATTERN WITH SOME HAVING QUINOLS AS ELECTRON DONORS AND OTHERS C TYPE CYTOCHROMES !LTHOUGH AT lRST SIGHT THIS SEEMS UNEXPECTED IT SHOULD BE RECALLED THAT THE SAME VARIATION IS FOUND AMONGST CYTOCHROME OXIDASES A POINT OF COMPARISON THAT IS REINFORCED BY THE STRUCTURAL SIMILARITY BETWEEN OXIDASES AND NITRIC OXIDE REDUCTASES THAT WILL BE DISCUSSED BELOW !LTHOUGH THESE ENZYMES ALL FUNCTION IN CONJUNC TION WITH THE MEMBRANE BOUND RESPIRATORY ELECTRON TRANSPORT SYSTEMS NOT ALL THE ENZYMES ARE THEMSELVES MEMBRANE BOUND 7HILE ONE TYPE OF NITRATE REDUCTASE &IG AND ALL TYPES OF NITRIC OXIDE REDUCTASE &IG HAVE SUBUNITS THAT ARE INTEGRAL MEMBRANE PROTEINS IN 'RAM NEGATIVE BACTERIA A SECOND TYPE OF NITRATE REDUCTASE ALL THREE TYPES OF NITRITE REDUCTASES AND THE NITROUS OXIDE REDUCTASE ARE WATER SOLUBLE PERIPLASMIC PROTEINS &IGS AND 'RAM POSITIVE ORGANISMS DO NOT HAVE A PERIPLASM AND THUS THEY TEND TO HAVE VARIANT FORMS OF NITRITE AND NITROUS OXIDE REDUCTASES THAT ARE ANCHORED TO THE CYTOPLASMIC MEMBRANE WHILE
THE PERIPLASMIC TYPE OF NITRATE REDUCTASE IS ABSENT FROM 'RAM POSITIVE ORGANISMS 4HE ULTRASTRUCTURE OF THE !RCHAEA MEANS THAT THEY DO NOT HAVE A FORMAL PERIPLASM HENCE ALL THEIR ENZYMES HAVE SOME FORM OF MEMBRANE ANCHOR ON THE OUTER SURFACE OF THE CYTOPLASMIC MEMBRANE !N IMPORTANT BIOENERGETIC POINT CONCERNING THE RESPIRATION OF NITRATE NITRITE NITRIC OXIDE AND NITROUS OXIDE IS TO ESTABLISH THE STOICHIOMETRY OF CHARGE TRANSLOCATION THAT ACCOMPANIES EACH REACTION %LEC TRONS PASSING FROM UBIQUINOL OR MENAQUINOL TO NITRATE WILL BE COUPLED TO CHARGE TRANSLOCATION ONLY IF THE NITRATE REDUCTASE ITSELF IS CATALYZING NET PROTON MOVEMENT ACROSS THE MEMBRANE !S WE SHALL SEE ONE KIND OF NITRATE REDUCTASE THAT IN THE PERIPLASM CANNOT DO THIS AND THUS THE Q E WHEREAS THE SECOND TYPE OF NITRATE REDUCTASE WHICH IS MEMBRANE BOUND ACHIEVES Q E 4HE PASSAGE OF ELECTRONS VIA THE CYTOCHROME BC COMPLEX FROM QUINOL TO THE C TYPE CYTOCHROME LEVEL IN THE RESPIRATORY CHAIN WILL RESULT IN A Q E BUT NO ADDITIONAL PROTON TRANSLOCATION
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&IG 4HE ORGANIZATION OF THE .RF( AND .RF" DEPENDENT CYTOCHROME C NITRITE REDUCTASE SYSTEMS
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&IG 4HE DIFFERENT SUBGROUPS OF NITRIC OXIDE REDUCTASES AND OXIDASES
OCCURS AS THE ELECTRONS PASS ON TO THE NITRITE NITRIC AND NITROUS OXIDE REDUCTASES 7HEN DIRECT OXIDA TION OF QUINOL BY A NITRIC OXIDE REDUCTASE OCCURS THEN Q E (OWEVER IN GENERAL THE SITUATION IS THAT THE Q E IS NEVER GREATER THAN FOR ANY OF THE ELECTRON ACCEPTORS WE ARE CONSIDERING HERE WHEN QUINOL IS THE ELECTRON DONOR 4HIS CONTRASTS WITH THE USE OF OXYGEN BY ELECTRON TRANSFER CHAINS BECAUSE Q E VALUES OF BETWEEN AND ARE FOUND FOR MOST PATHWAYS OF ELECTRON TRANSFER LEADING TO A CYTOCHROME OXIDASE 4HESE HIGHER STOICHIOMETRIES CORRELATE WITH THE GENERAL OBSERVATION THAT CELLS MAKE MOST !40 PER PAIR OF ELECTRONS WHEN THEY REDUCE OXYGEN AND THUS THE LATTER IS THE PREFERRED ELECTRON ACCEPTOR 7E NOTE THAT IN MANY CIRCUMSTANCES THE ELECTRONS WILL REACH UBIQUINONE VIA A PROTON TRANSLOCATING ENZYME 4HIS CAN BE AN .!$( DEHYDROGENASE FOR WHICH USUALLY Q E OR ANOTHER TYPE OF ENZYME FOR EXAMPLE A FORMATE DEHYDROGENASE FOR WHICH Q E 4HUS EVEN WHEN ELECTRONS ARE ULTIMATELY DELIVERED FROM QUINOL TO THE ACCEPTOR BY THE PERIPLASMIC NITRATE REDUCTASE OR A QUINOL DEPENDENT NITRIC OXIDE REDUC TASE THEY ARE STILL OFTEN ACCOMPANIED BY SOME PROTON TRANSLOCATION &INALLY IT IS IMPORTANT TO NOTE THAT THERE IS NOT A SIMPLE CORRELATION BETWEEN THE REDOX POTENTIAL OF THE ACCEPTOR COUPLE AND THE Q E RATIO IF THERE WERE THEN THE RATIO SHOULD BE HIGHER FOR NITROUS OXIDE %O| CA M6 REDUCTION THAN THAT FOR OXY GEN %O| CA M6 &OR SOME REASON IT SEEMS THAT A PROTON TRANSLOCATING AND THUS MEMBRANE BOUND
NITROUS OXIDE REDUCTASE HAS NOT EVOLVED 4HE AIM OF THIS CHAPTER IS TO SUMMARIZE CURRENT KNOWLEDGE AND UNCERTAINTIES CONCERNING I THE DELIV ERY OF ELECTRONS TO REDUCTASES THAT CATALYZE REDUCTION OF NITRATE NITRITE NITRIC OXIDE AND NITROUS OXIDE AND II THE STRUCTUREFUNCTION RELATIONSHIPS FOR THE ENZYMES &OR THE MOST PART WE DEAL ONLY WITH RECENT DEVELOP MENTS AND REFER THE READER TO SEVERAL PREVIOUS REVIEWS EG "ERKS ET AL A &ERGUSON A 2ICHARD SON AND 7ATMOUGH -OURA AND -OURA FOR BACKGROUND INFORMATION 0ARTICULAR EMPHASIS IS PLACED ON THE CYTOCHROME CD NITRITE REDUCTASE AS THIS SYSTEM PROVIDES AN EXCELLENT EXAMPLE OF HOW 8 RAY CRYSTALLOGRAPHY IS BEING COMBINED WITH A RANGE OF SPECTROSCOPIC AND BIOPHYSICAL TECHNIQUES TO INFORM ON THE ENZYME STRUCTURE AND FUNCTION )T ALSO DEM ONSTRATES WHY IT IS IMPORTANT TO STUDY AN INDIVIDUAL ENZYME SYSTEM IN MORE THAN ONE SPECIES OF BACTERIA AS RATHER UNEXPECTED DIFFERENCES CAN EMERGE **3FTQJSBUPSZ/JUSBUF3FEVDUBTFT 4HERE ARE TWO TYPES OF RESPIRATORY NITRATE REDUCTASE IN BACTERIA &IG EACH OF WHICH SHOULD BE DISTIN GUISHED FROM AN ASSIMILATORY AND CYTOPLASMIC NITRATE REDUCTASE THAT IS FOUND IN MANY ORGANISMS !LL THREE CLASSES OF BACTERIAL NITRATE REDUCTASE CONTAIN A -O BIS GUANINE DINUCLEOTIDE COFACTOR -O BIS -'$ AT THE ACTIVE SITE AND CATALYZE THE REACTION
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO ./n ( En A ./n (/
$ISCUSSION OF THE ASSIMILATORY ENZYME IS BEYOND THE SCOPE OF THIS CHAPTER BUT DETAILS HAVE RECENTLY BEEN DESCRIBED ,IN AND 3TEWART 2ICHARDSON ET AL "5IF1FSJQMBTNJD/JUSBUF3FEVDUBTF /BQ
4HE GENES FOR THIS ENZYME CAN BEEN FOUND IN THE GENOMES OF A NUMBER OF _ ` b ¡ AND a PROTEOBACTERIA 2ICHARDSON 2ICHARDSON ET AL 4HE ENZYME CAN SERVE A RANGE OF PHYSIOLOGICAL FUNCTIONS THAT INCLUDE EXCESS ENERGY DISSIPATION UNDER AEROBIC CONDITIONS IN 0 DENITRIlCANS REDOX POISING OF THE PHOTOSYNTHETIC ELECTRON TRANSPORT SYSTEM IN 2HODO BACTER CAPSULATUS ANAEROBIC HIGH AFlNITY NITRATE RESPIRATION IN % COLI AND CATALYZING THE lRST STEP IN ANAEROBIC DENITRIlCATION IN 0SEUDOMONAS SP ' 2ICHARDSON 0OTTER ET AL 2ICHARDSON ET AL 4HE REGULATION OF NAP GENE EXPRESSION REmECTS THESE DIFFERENT PHYSIOLOGICAL FUNCTIONS 4HUS IN 0 PANTOTROPHUS NAP EXPRESSION IS MAXIMUM DURING AEROBIC GROWTH ON HIGHLY REDUCED CARBON SUBSTRATES 3EARS ET AL %LLINGTON ET AL WHILE IN % COLI NAP EXPRESSION IS HIGHEST UNDER ANAEROBIC NITRATE LIMITED GROWTH CONDITIONS 7ANG ET AL 4USVDUVSFBOE4QFDUSPTDPQZPGUIF1FSJ QMBTNJD/JUSBUF3FEVDUBTF )N MOST CASES THE PERIPLASMIC NITRATE REDUCTASE PURIlES AS A TWO SUBUNIT ENZYME COMPLEX CONTAINING A K$A DI HEME .AP" AND A K$A CATALYTIC SUBUNIT .AP! THAT BINDS AN ;&E 3= CLUSTER AND THE BIS MOLYBDOP TERIN GUANINE DINUCLEOTIDE -O BIS-'$ COFACTOR "ERKS ET AL C (OWEVER THE ENZYME FROM $ DESULFURICANS PURIlES AS A SINGLE SUBUNIT ENZYME AND THE 8 RAY CRYSTAL STRUCTURE OF THIS HAS RECENTLY BEEN SOLVED $IAS ET AL 4HE PROTEIN FOLDS INTO FOUR DOMAINS /NE OF THESE IS FORMED ENTIRELY FROM AN . TERMINAL SEGMENT OF THE POLYPEPTIDE AND BINDS THE ;&E 3= CLUSTER 4HE REMAINING THREE DOMAINS HAVE _` STRUCTURE AND FOLD AROUND THE -O BIS-'$ COFACTOR PROVIDING NUMEROUS ( BONDS TO THE -'$ MOIETIES 4HE -'$ COFACTOR LIES AT THE BOTTOM OF A SUBSTRATE FUNNEL BETWEEN DOMAINS )) AND ))) WHICH HAVE A SIMILAR FOLD THAT EXHIBITS SOME RESEMBLANCE TO THE TYPICAL .!$ BINDING FOLD OF DEHYDROGENASES 2ESIDUES LINING THIS CLEFT AND AT ITS BASE WILL PLAY ROLES IN DElNING SUBSTRATE SPECIlCITY AND ORIENTATING THE
SUBSTRATE ONTO THE -O /F PARTICULAR SIGNIlCANCE IS A CONSERVED ARGININE AT THE BASE OF THE POCKET THAT MAY INTERACT WITH NITRATO OXYGENS OF THE SUBSTRATE 4HIS ARGININE IS CONSERVED IN THE HOMOLOGOUS &DH ( SUBUNIT OF THE FORMATE HYDROGEN LYASE SYSTEM OF % COLI FOR WHICH THE CRYSTAL STRUCTURE OF A NITRITE INHIB ITED FORM SHOWS THE ARGININE TO BIND A NITRITE OXYGEN "OYINGTON ET AL 3PECTROSCOPIC STUDIES OF 0 PANTOTROPHUS .AP!" COMPLEX AND $ESULFOVIBRIO DESULFURICANS .AP! HAVE REVEALED %02 SIGNALS IN THE DITHIONITE REDUCED ENZYME THAT ARE CHARACTERISTIC OF A S ;&E3= CENTER "RETON ET AL "URSAKOV ET AL "UTLER ET AL 4HE ;&E3= CENTER IS OF CATALYTIC RELEVANCE AS IT IS OXIDIZED TO AN %02 SILENT ;&E 3= STATE FOLLOWING ADDITION OF NITRATE TO INITIATE ENZYME TURNOVER 4HE ;&E 3= CENTER IS ¯ AWAY FROM THE -O ION OF THE -O BIS -'$ CENTER /NE OF THE -'$ MOIETIES LIES BETWEEN THE &E3 CENTER AND THE -O RAISING THE POSSIBILITY THAT IT MEDIATES ELECTRON TRANSFER 4HE MIDPOINT REDOX POTENTIAL OF THIS &E3 CENTER HAS BEEN DETERMINED FROM %02 REDOX TITRATIONS TO BE n M6 "RETON ET AL 4HIS IS MUCH LOWER THAN EXPECTED FOR A REDOX CENTER INVOLVED IN ELECTRON TRANSFER FROM UBIQUINOL %M M6 TO NITRATE %M M6 AND PROVIDES AN EXAMPLE OF AN ENDERGONIC ELECTRON TRANSFER STEP IN AN ELECTRON TRANSFER PROCESS THAT IS EXERGONIC OVERALL !LTHOUGH THIS MAY SEEM ODD IT IS MORE IMPORTANT FOR KINETIC REASONS TO POSITION A REDOX CENTER ON A PATHWAY SO AS TO OFFER THE ELECTRON A ROUTE THAT INVOLVES TUNNELING UP TO ¯ BETWEEN CENTERS THAN IT IS TO SET THE REDOX POTENTIAL TO MATCH THOSE OF OTHER COMPONENTS ON THE PATHWAY &ERGUSON B 0AGE ET AL 7E WILL RETURN TO THIS POINT IN THE CONTEXT OF THE MEMBRANE BOUND NITRATE REDUCTASE 4HE CRYSTAL STRUCTURE OF .AP! FROM $ DESULFURI CANS $IAS ET AL HAS CONlRMED THE EARLIER PRE DICTIONS BASED ON AMINO ACID SEQUENCE COMPARISONS "ERKS ET AL C THAT THE MOLYBDENUM AMINO ACID LIGAND IN PERIPLASMIC NITRATE REDUCTASES IS A CYSTEINE RESIDUE 4HE -O ION IS ADDITIONALLY COORDINATED BY FOUR SULFUR LIGANDS PROVIDED BY THE TWO -'$ MOI ETIES AND A WATERHYDROXO LIGAND 4HE STRUCTURE HAS BEEN INTERPRETED AS BEING THAT OF -O6) REDOX STATE "ASED UPON THIS OXIDIZED STRUCTURE IT WAS PROPOSED THAT THE ENZYME CYCLES BETWEEN DES OXO -O)6 AND MONO OXO -O6) WHICH IS THEN PROTONATED TO FORM THE WATERHYDROXO LIGAND $IAS ET AL 4HIS SCHEME IS DIFFERENT FROM THAT DERIVED FROM 8 RAY ABSORPTION STUDIES ON THE 0 PANTOTROPHUS .AP!"
ENZYME -O + EDGE %8!&3 OF FERRICYANIDE OXIDIZED ENZYME SUGGESTED A DI OXO -O6) SPECIES WITH lVE SULFUR LIGANDS WHICH CHANGED TO MONO OXO -O )6 SPECIES WITH THREE SULFUR LIGANDS ON REDUCTION WITH DITHIONITE &IG 4HE ADDITION OF NITRATE TO THE REDUCED ENZYME RESULTED IN RE OXIDATION TO A DI OXO -O6) SPECIES SIMILAR TO THE FERRICYANIDE OXIDIZED ENZYME "UTLER ET AL 4HUS THE 0 PANTOTROPHUS .AP!" ENZYME MAY CYCLE BETWEEN MONO OXO AND DI OXO STATES DURING THE CATALYTIC CYCLE RATHER THAN THE MONO OXODES OXO STATES PREDICTED FOR $ DESULFURI CANS .AP! /F COURSE THIS DIFFERENCE MAY LIE IN THE DIFFERENT EXPERIMENTAL METHODS USED TO PROVIDE THE DATA ON WHICH THE CATALYTIC CYCLE IS PROPOSED (OW EVER IT SHOULD ALSO BE NOTED THAT BASED ON PRIMARY SEQUENCE SIMILARITY .AP! FROM $ DESULFURICANS IS MORE CLOSELY RELATED TO &DH FROM % COLI THAN IS .AP! FROM 0 PANTOTROPHUS 2ICHARDSON ET AL &URTHERMORE UNLIKE .AP! FROM 0 PANTOTROPHUS .AP! FROM $ DESULFURICANS HAS BEEN DEMONSTRATED TO HAVE LOW LEVELS OF FORMATE DEHYDROGENASE ACTIVITY "URSAKOV ET AL 4HUS THE POSSIBILITY EXISTS THAT THE SUBTLE DIFFERENCES BETWEEN THE TWO ENZYMES MAY ACCOUNT FOR DIFFERENT OXYGEN CO ORDINATION AT -O DURING THE CATALYTIC CYCLE 4HE %8!&3 DATA ON 0 PANTOTROPHUS .AP!" ALSO SUGGEST THAT UPON REDUCTION BY DITHIONITE TWO SULFUR ATOMS PRESUMABLY FROM ONE PTERIN ARE LOST FROM THE
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO CO ORDINATION SPHERE OF -O)6 BUT RE LIGATE FOLLOW ING REOXIDATION BY NITRATE 4HE CATALYTIC RELEVANCE OF CHANGES IN THE CO ORDINATION OF THE PTERINS TO THE -O ION IN -O BIS-'$ ENZYMES IS DEBATED &OR EXAMPLE THE LABILITY OF ONE OF THE PTERIN DITHIOLENE LIGANDS HAS BEEN OBSERVED FOR THE CRYSTAL STRUCTURES OF THE $-3/ REDUCTASE FROM 2 SPHAEROIDES AND 2BA CAPSULATUS 3CHINDELIN ET AL 3CHNEIDER ET AL (OWEVER AT LEAST IN THE CASE OF NON PHYSIOLOGICAL DITHIONITE DRIVEN TURNOVER OF .AP IT WOULD APPEAR THAT REDOX STATE DEPENDENT PTERIN DIS SOCIATION CAN BE PART OF A CATALYTIC CYCLE "UTLER ET AL 4HE POSSIBILITY THAT THE -O COORDINATION SPHERE OF .AP CAN ADOPT A RANGE OF STATES IS REmECTED IN THE %02 SPECTRA OF THIS ENZYME WHICH SHOW THAT THE -O6 BIS -'$ OF 0 PANTOTROPHUS .AP! CAN EXHIBIT A NUMBER OF DIFFERENT %02 SIGNALS "UTLER ET AL REVIEWED IN 0OTTER ET AL $ISTINCT SIGNALS THOUGHT TO ARISE FROM -O6 COORDINATED BY lVE SULFURS AND BY THREE SULFURS HAVE BEEN DESCRIBED AS WELL AS SIGNALS ARISING FROM ENZYME WITH NITRATE AZIDE CYANIDE OR THIOCYANATE IN THE CATALYTIC POCKET "UTLER ET AL 0 PANTOTROPHUS .AP! HAS ALSO BEEN STUDIED BY 8 BAND PROTON %.$/2 WHICH TOGETHER WITH %02 ANALYSIS SUGGESTED THAT NITRATE DOES NOT BIND DIRECTLY TO -O6 DURING TURNOVER BUT MOST LIKELY BINDS TO THE -O)6 STATE "UTLER ET AL ! MODEL FOR THE CATALYTIC CYCLE OF 0
&IG 4HE STRUCTURAL ORGANIZATION OF THE .AP! SUBUNIT OF THE PERIPLASMIC NITRATE REDUCTASE ! THE -O BIS -'$ FACTOR " AND AN OXO TRANSFERASE MECHANISM FOR NITRATE REDUCTION # .OTE THE QUESTION OF WHETHER .OP -O 6) IS A DI OXO OR MONO OXO SPECIES IS DEBATABLE HENCE THE QUESTION MARK ON THE SECOND OXO GROUP 4HE BIS -'$ AND CYSTEINE 3 LIGANDS ARE NOT SHOWN IN #
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO PANTOTROPHUS .AP! -O BIS -'$ BASED ON %02 %8!&3 AND %.$/2 DATA IS GIVEN IN &IG &MFDUSPO5SBOTQPSUUP/BQ" %LECTRON TRANSPORT TO .AP! &IG ILLUSTRATES A MORE GENERAL QUESTION IN BACTERIAL RESPIRATION OF HOW ELECTRONS ARE TRANSFERRED FROM THE QUINOL POOL TO SOME PERIPLASMIC OXIDO REDUCTASES WITHOUT THE PARTICIPATION OF AN ENERGY CONSERVING CYTOCHROME BCL COMPLEX 4HE NAP OPERON CONTAINS A GENE NAP# WHICH IS PREDICTED TO ENCODE A MEMBRANE ANCHORED TETRA HEME C TYPE CYTOCHROME HOMOLOGUES OF WHICH ARE FOUND IN A NUMBER OF GENE CLUSTERS ASSOCIATED WITH PERIPLASMIC OXIDOREDUCTASES "ERKS ET AL C 0RIMARY STRUCTURE ANALYSIS OF THIS PROTEIN SUGGESTS THAT IT IS A MEMBER OF A LARGE FAMILY OF BACTERIAL TETRA HEME AND PENTA HEME CYTOCHROMES THAT IS PRO POSED TO PARTICIPATE IN ELECTRON TRANSFER BETWEEN THE QUINOLQUINONE POOL AND PERIPLASMIC REDOX ENZYMES 2OLDAN ET AL 4HESE ENZYMES INCLUDE THE TRIMETHYLAMINE . OXIDE REDUCTASE DIMETHYLSULFOXIDE REDUCTASE SOLUBLE FUMARATE REDUCTASE SOME NITRITE REDUCTASES AND HYDROXYLAMINE OXIDOREDUCTASE 4HE QUINOL OXIDATION ACTIVITY OF SOME OF THESE FAMILY MEMBERS HAS BEEN DEMONSTRATED 2OLDAN ET AL 3HAW ET AL 3IMON ET AL 3IMON ET AL 4OPOLOGICAL ANALYSIS SUGGESTS THAT IN THEIR NATIVE STATE THESE CYTOCHROMES INCLUDE A SINGLE . TERMINAL TRANSMEMBRANE _ HELIX THAT SERVES TO ANCHOR A GLOBULAR MULTI HEME DOMAINS ONTO THE PERIPLASMIC SURFACE OF THE CYTOPLASMIC MEMBRANE 4O FACILITATE BIOCHEMICAL AND SPECTROSCOPIC ANALYSES .AP# HAS BEEN EXPRESSED AS A WATER SOLUBLE PERIPLASMIC PROTEIN .AP#SOL 2OLDAN ET AL 3TUDIES USING MAGNETIC CIRCULAR DICHROISM -#$ AND ELECTRON PARAMAGNETIC RESONANCE %02 SUGGESTED THAT ALL FOUR HEMES ARE LOW SPIN AND FEATURE BIS HISTIDINE AXIAL LIGATION 2OLDAN ET AL 2EDOX POTENTIOMETRIC ANALYSIS INDICATED THAT THE HEMES HAD LOW MIDPOINT REDOX POTENTIALS %M OF nM6 nM6 nM6 nM6 0RIMARY STRUCTURE AND MUTATIONAL ANALYSIS OF .AP# AND MULTIPLE SEQUENCE ALIGNMENTS OF OTHER MEMBERS OF THE .AP# FAMILY LED TO THE IDENTIlCATION OF FOUR HISTIDINE RESIDUES AS CANDIDATES FOR THE DISTAL HEME LIGANDS OF THE FOUR COVALENTLY BOUND BOUND HEMES -UTATION OF EACH OF THESE RESULTS IN A FOLDED PROTEIN THAT BINDS A HIGH SPIN (IS (//( LIGATED HEME #ARTRON ET AL 4HIS PRIMARY STRUCTURE ANALYSIS AND THE PROTEOLYSIS PATTERN OF .AP# HAS LED TO THE PROPOSAL THAT THE GLOBULAR REGION COMPRISES TWO STRUCTURALLY SIMILAR DI HEME BINDING DOMAINS
2OLDAN ET AL #ARTRON ET AL !ROUND AMINO ACIDS OF THE . TERMINAL AND # TERMINAL SEGMENTS EACH CONTAINING TWO #88#( MOTIFS CAN BE ALIGNED 4HE TWO #88#( MOTIFS IN EACH SEG MENT ARE SPACED AROUND AMINO ACIDS APART AND A CONSERVED (IS RESIDUE LIES BETWEEN THE TWO MOTIFS IN EACH SEGMENT 4HESE ARE CANDIDATES FOR THE DISTAL LIGAND OF TWO OF THE BIS (IS CO ORDINATED HEME IRONS PREDICTED FROM THE %02-#$ ! SECOND CONSERVED (IS RESIDUE LIES TO AMINO ACIDS AFTER THE SECOND #88#( MOTIF IN EACH SEGMENT AND IS A CANDIDATE FOR THE LIGAND OF THE SECOND BIS (IS LIGATED HEME IN EACH DOMAIN 4HE PARALLEL DI HEME PAIRS ENVISAGED WOULD BE SIMILAR TO THAT IDENTIlED IN THE RECENT 8 RAY CRYSTAL STRUCTURE .AP" SEE BELOW /THER MEMBERS OF THE .AP# FAMILY WHICH HAVE BEEN PURIlED INCLUDE .RF( FROM 7 SUCCINOGENES #YM! FROM 3HEWANELLA PUTREFACIENS AND THE PENTA HEME $OR# FROM 2HODO BACTER CAPSULATUS 3PECTROSCOPIC ANALYSIS OF #YM! HAS ALSO DEMONSTRATED THAT THE FOUR HEMES ARE BIS HISTIDINE COORDINATED AND THE FOUR CANDIDATES FOR THE .AP# DISTAL HEME LIGANDS ARE CONSERVED IN #YM! &IELD ET AL (OWEVER THIS IS NOT THE CASE FOR .RF( AND $OR# 3EQUENCE ANALYSIS SUGGESTS THAT THESE CYTOCHROMES LIE IN DIFFERENT SUB GROUPS OF THE FAMILY TO .AP# AND #YM! AND SO THE POSSIBILITY ARISES THAT NOT ALL MEMBERS OF THE .AP# FAMILY HAVE EQUIVALENT HEME LIGATION PATTERNS !N IMPORTANT UNANSWERED QUESTION CONCERNING .AP# AND RELATED PROTEINS IS HOW IT INTERACTS WITH QUINONES AND QUINOLS WHICH ARE WIDELY BELIEVED TO BE CONlNED TO THE HYDROPHOBIC ENVIRONMENT OF THE BILAYER 4HE SINGLE TRANSMEMBRANE HELIX PREDICTED FOR THIS CLASS OF PROTEIN IS UNLIKELY TO PROVIDE A QUI NONE BINDING SITE ANALOGOUS TO THOSE THAT HAVE BEEN ESTABLISHED IN THE TRANSMEMBRANE HELICAL REGIONS OF PHOTOSYNTHETIC REACTION CENTERS AND THE CYTOCHROME BC COMPLEX )T IS MORE LIKELY THAT .AP# CONTAINS SOME AMPHIPHILIC HELICES THAT CAN DIP INTO THE MEMBRANE BILAYER ANALOGOUSLY FOR EXAMPLE TO THE PERIPHERAL MEMBRANE PROTEIN $ LACTATE DEHYDROGENASE IN % COLI $YM ET AL )T IS NOTABLE THAT @LINKER REGION BETWEEN THE TWO PUTATIVE DOMAINS OF .AP# CONTAINS A CONSERVED %7 SEQUENCE 3UCH A MOTIF HAS BEEN PROPOSED TO CONTRIBUTE A GENERAL QUINONE BINDING SITE IN SOME PROTEINS &ISHER AND 2ICH 4HE DI HEME .AP" CYTOCHROME SUBUNIT OF THE .AP!" COMPLEX IS THE ELECTRON ACCEPTING REDOX PARTNER FOR .AP# 3TUDIES USING MAGNETIC CIRCULAR DICHROISM -#$ AND ELECTRON PARAMAGNETIC RESO NANCE %02 SUGGESTED THAT BOTH HEMES ARE LOW SPIN AND FEATURE BIS HISTIDINE AXIAL LIGATION "UTLER ET AL
0RIMARY STRUCTURE ANALYSIS OF .AP" LED TO THE IDENTIlCATION OF TWO CONSERVED HISTIDINE RESIDUES AS CANDIDATES FOR THE DISTAL HEME LIGANDS 2EDOX POTENTIOMETRIC ANALYSIS OF 0 PANTOTROPHUS .AP!" INDICATED THAT THE HEMES HAD MIDPOINT REDOX POTEN TIALS OF M6 AND n M6 "ERKS ET AL 4HE REDOX CYCLING OF THE HEMES HAS BEEN OBSERVED BY BOTH %02 AND VISIBLE SPECTROSCOPIES DURING TURNOVER OF THE .AP!" COMPLEX WITH NITRATE DEMONSTRATING THE COMPETENCE OF THESE COFACTORS IN MEDIATING ELECTRON TRANSFER TO THE CATALYTIC SITE OF THE ENZYME "ERKS ET AL "UTLER ET AL (EMOPHILUS INmUENZAE .AP" HAS BEEN EXPRESSED INDEPENDENTLY FROM .AP! IN % COLI "RIGE ET AL 4HE MIDPOINT REDOX POTENTIALS OF THE TWO HEMES WERE DETERMINED TO BE n M6 AND n M6 4HESE ARE LOWER THAN THOSE DETERMINED FOR THE 0 PANTOTROPHUS .AP" AND COULD REmECT THE FACT THAT IN THE .AP SYSTEM IS DEPENDENT ON MENAQUINOL %| n M6 IN ( INmUENZAE RATHER THAN UBIQUINONE %| M6 AS IN 0 PANTOTRO PHUS 4HE CRYSTAL STRUCTURE OF A PROTEOLYZED FORM OF THE RECOMBINANT .AP" FROM ( INmUENZAE HAS BEEN SOLVED AND REVEALED A NEW PROTEIN FOLD FOR THE .AP" CLASS OF CYTOCHROMES "RIGE ET AL 4HE TWO HEME GROUPS HAVE NEARLY PARALLEL HEME PLANES AND ARE STACKED AT VAN DER 7AALS DISTANCES WITH AN IRON TO IRON DISTANCE OF ONLY ¯ 4HESE STRUCTURAL FEATURES HAVE NOW BEEN FOUND IN A NUMBER OF MULTI HEME ENZYMES "ARKER AND &ERGUSON 2ICHARDSON INCLUDING THE CYTOCHROME C NITRITE REDUCTASE AND HYDROXYLAMINE CYTOCHROME C OXIDOREDUCTASE THAT WILL BE DISCUSSED IN SECTION )))! /N THE BASIS OF THE ( INmUENZAE .AP" AND $ DESULFURICANS .AP! STRUCTURES A MODEL OF THE .AP!" COMPLEX HAS BEEN PROPOSED IN WHICH THE FOUR REDOX CENTERS ARE POSI TIONED IN A VIRTUALLY LINEAR CONlGURATION WHICH SPANS A DISTANCE OF NEARLY ¯ "RIGE ET AL (OWEVER A RECENT CRYSTAL STRUCTURE OF A .AP!" COMPLEX HAS NOT CONlRMED THIS MODEL !RNOUX ET AL 4HE COFACTORS ARE ARRANGED AS A SLIGHTLY BENT MOLECULAR WIRE WITH HEME )) OF .AP" BEING IN THE VICINITY OF THE IRON SULFUR CLUSTER IN .AP! (EME ) OF .AP" IS EXPOSED TO SOLVENT AND CAN BE PROPOSED TO BE THE ELECTRON ACCEPTOR OF .AP# WHICH IS THE PHYSIOLOGI CAL ELECTRON DONOR 5IF1PTTJCMF3PMFTPG"DDFTTPSZOBQ (FOF1SPEVDUT )N ADDITION TO THE NAP!"# # GENES SIX OTHER NAP GENES NAP$%+&'( ( ARE FOUND IN DIFFERENT COM
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO BINATIONS IN DIFFERENT BACTERIA &IG .AP+ AND .AP% ARE MONOTOPIC INTEGRAL MEMBRANE PROTEINS OF NO CURRENTLY KNOWN FUNCTION AND .AP$ MAY BE A PRIVATE CHAPERONE FOR .AP! THAT IS INVOLVED IN FOLD ING AND COFACTOR INSERTION PRIOR TO EXPORT VIA THE 4AT SYSTEM (OWEVER .AP & ' AND ( ARE PREDICTED TO BIND IRON SULFUR CLUSTERS RAISING THE POSSIBILITY OF A ROLE IN ELECTRON TRANSFER TO .AP! .AP& AND .AP' ARE PREDICTED IT TO BE K$A PROTEINS THAT BIND FOUR ;&E 3= CLUSTERS AND TO BE LOCATED IN THE CYTOPLASM AND PERIPLASM RESPECTIVELY .AP( K$A IS PRE DICTED TO BE AN INTEGRAL MEMBRANE PROTEIN WITH FOUR TRANSMEMBRANE HELICES A CYTOPLASMIC # TERMINAL DOMAIN THAT CONTAINS TWO ;&E 3= BINDING MOTIFS AND TWO CYTOPLASMIC #YS 8AA #YS 0RO MOTIFS THAT MAY BE REDOX ACTIVE OR BIND A METAL CENTER "ERKS ET AL A )NTRODUCTION OF IN FRAME DELETIONS TO YIELD NAP# NAP& & NAP'( AND NAP&'( MUTANTS IN ( UBIQUINOL ONLY AND MENAQUINOL ONLY BACKGROUNDS IN % COLI HAS DEMONSTRATED THAT .AP'( BUT NOT .AP& ARE ESSENTIAL FOR ELECTRON TRANSFER FROM UBIQUINOL TO .AP! AND THAT .AP# IS ESSENTIAL TO ELECTRON TRANSFER FROM BOTH UBIQUINOL AND MENAQUINOL "RONDIJK ET AL 4HIS RAISES THE POSSIBILITY THAT .AP'( FORM A QUINOL DEHYDROGENASE COMPLEX !LTHOUGH .AP& WAS NOT REQUIRED FOR ELECTRON TRANSFER TO .AP!" THERE WAS EVIDENCE FROM GROWTH RATE AND GROWTH YIELD ANALYSIS THAT .AP'(& MIGHT FORM A PROTONMOTIVE COMPLEX IN WHICH COUPLING WAS REGULATED BY .AP& 4HE POS SIBILITY THAT .AP'( CAN FORM A QUINOL OXIDIZING COMPLEX COULD RATIONALIZE THE ABSENCE OF .AP# BUT PRESENCE OF .AP'( IN THE NAP GENE CLUSTER OF #AMPYLOBACTER JEJUNI 0ARKHILL ET AL )T ALSO RAISES THE POSSIBILITY OF PARALLEL NON PROTONMOTIVE .AP# ONLY AND PROTON MOTIVE .AP'( ROUTES OF ELECTRON TRANSFER FROM 1( TO .AP!" IN BACTERIA THAT HAVE NAP# # AND NAP'( GENES &IG 4HE GROWTH ( CONDITIONS UNDER WHICH THE DIFFERENT ROUTES OPERATE SHOULD NOW BE STUDIED IN MORE DETAIL TO ASSESS THEIR PHYSIOLOGICAL SIGNIlCANCE #5IF.FNCSBOF#PVOE/JUSBUF3FEVDUBTF /BS
4HE MEMBRANE BOUND NITRATE REDUCTASE IS A COMPLEX THREE SUBUNIT QUINOL DEHYDROGENASE )T CONTAINS A CA K$A BIS -'$ CATALYTIC SUBUNIT .AR' _ CA K$A ELECTRON TRANSFER SUBUNIT .AR( ` WHICH BINDS FOUR IRON SULFUR CLUSTERS AND A CA K$A DI B HAEM INTEGRAL MEMBRANE QUINOL DEHYDROGENASE SUBUNIT .AR) a 4HE NAR'() GENES ENCODING THESE )
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO SUBUNITS ARE PRESENT IN ALL OF THE NARR GENE CLUSTERS SO FAR IDENTIlED &IG AS IS NAR** WHICH ENCODES A PRIVATE CHAPERONE 2ICHARDSON ET AL (OWEVER UPSTREAM AND DOWNSTREAM OF NAR'(*)) THE GENE CON TENT CAN VARY &OR EXAMPLE IN % COLI NAR'(*)) IS AN OPERON AND IS PRECEDED BY THE NAR+ GENE ENCODING + A NITROGEN OXYANION TRANSPORTER &URTHER UPSTREAM OF NAR+ THERE ARE GENES ENCODING THE TWO COMPONENT + NITRATE RESPONSIVE SENSOR REGULATOR SYSTEM .AR8, )N SOME GENE CLUSTERS EG 0SEUDOMONAS AERUGINOSA THERE ARE TWO NAR+ GENES UPSTREAM OF .AR' BUT IN + 0 PANTOTROPHUS AND PROBABLY ALSO 0 DENITRIlCANS THE NARR TRANSCRIPTIONAL UNIT IS NAR+'(*) WITH THE ) NAR+ + BEING A GENE FUSION OF TWO SMALLER NAR+ UNITS 7OOD ET AL )N -YCOBACTERIUM TUBERCULOSIS THE NAR+ GENE DOES NOT CLUSTER WITH THE + NAR'(*) GENES BUT THERE ARE FOUR ) NAR+ LIKE GENES + ELSEWHERE ON THE CHROMOSOME )N " SUBTILIS AND 0 PANTOTROPHUS GENES ENCODING TRANSCRIPTION FACTORS OF THE &NR FAMILY ARE ALSO PRESENT IN THE NARR GENE CLUSTER 7OOD ET AL )N % COLI &NR BINDS AN OXYGEN SENSITIVE IRON SULFUR CLUSTER THAT SERVES TO MEDIATE THE ANAEROBIC INDUCTION OF THE NAR'(*) OPERON ACTING IN CONJUNCTION WITH THE NITRATE RE SPONSIVE .AR8, SENSOR REGULATOR AND INTEGRATION HOST FACTOR TO MAXIMIZE NITRATE REDUCTASE EXPRESSION 0OTTER ET AL )N 0 PANTOTROPHUS THE SITUATION IS DIFFERENT NAR+'(*)) IS AGAIN UNDER THE CONTROL OF AN OXYGEN RESPONSIVE IRON SULFUR CLUSTER CONTAINING &NR PROTEIN &NR0 (OWEVER NO .AR8, SYSTEM HAS BEEN IDENTIlED )NSTEAD A &NR HOMOLOGUE .AR2 THAT LIES UPSTREAM OF NAR+'(*)) SERVES A NITRATE OR NITRITE RESPONSIVE TRANSCRIPTION FACTOR THAT CO REGULATES THE NAR+ OPERON 7OOD ET AL !NOTHER RE CENTLY DISCOVERED VARIATION IS THE PRESENCE OF NAR# CODING FOR A C TYPE CYTOCHROME AS THE lRST GENE IN THE OPERON CODING FOR .AR IN 4HERMUS THERMOPHILUS :AFRA ET AL .AR# HAS AN ESSENTIAL ROLE IN THE SYNTHESIS OF AN ACTIVE ENZYME AND ITS INTEGRATION INTO THE MEMBRANE :AFRA ET AL 7HY THIS EXTRA COMPONENT IS NEEDED IN THIS THERMOPHILE IS UNCLEAR .OTABLE DIFFERENCES BETWEEN .AP AND .AR ARE THAT ONLY THE LATTER USES CHLORATE OR BROMATE AS ALTERNATIVE SUBSTRATES AND IS STRONGLY INHIBITED BY AZIDE 5IF4USVDUVSFBOE4QFDUSPTDPQZPG/BS()* 4HIS ENZYME HAS BEEN THE SUBJECT OF A GREAT DEAL OF BIOCHEMICAL AND SPECTROSCOPIC ANALYSIS REVIEWED IN "LASCO ET AL WHICH CAN NOW BE MAPPED ON TO RECENT STRUCTURAL RESOLUTION *ORMAKKA ET AL
)N CONTRAST TO .AP IN WHICH A #YS 3 SERVES AS -O LIGAND THE TWO STRUCTURES OF THE % COLI .AR' REVEALS THAT AN !SP PROVIDES EITHER ONE *ORMAKKA ET AL OR TWO "ERTERO ET AL OXYGEN LIGANDS TO -O ! 3ER DERIVED OXYGEN LIGAND IS FOUND IN THE 2HODOBACTER CAPSULATUS $-3/ REDUCTASE SEE #HAPTER BY -C%WAN ET AL 6OL OF THIS BOOK WHICH HAS -O6 %02 SIGNALS THAT ARE MORE SIMILAR TO .AR THAN TO .AP "ENNETT ET AL 3EARS ET AL "UTLER ET AL #ONSIDERING THE .AR STRUCTURES IN CONJUNCTION WITH EARLY %8!&3 AND %02 STUDIES IT SEEMS MOST LIKELY THAT AT P( WHEN .AR IS MOST ACTIVE THE -O6) IS COORDINATED BY FOUR THIOLS PROVIDED BY THE TWO -'$ MOIETIES ONE !SP AND ONE OXO GROUP 4HE OXO GROUP WOULD CONVERT TO HYDROXIDE ON REDUCTION TO -O6 AND BE LOST AS WATER ON FURTHER REDUCTION TO -O)6 4HE SIGNIlCANCE OF THE MONODENTATE ) / LIGAND FROM ASPARTATE VERSUS THE BIDENTATE / LIGANDS FOR !SP -O COORDINATION REMAINS UNCERTAIN BUT THEY COULD REmECT DIFFERENT FORMS OF THE ENZYME ENTERED IN DIFFERENT REDOX STATES OR AT DIFFERENT P( AND IONIC STRENGTH BOTH OF WHICH CAN CAUSE SIGNIlCANT CHANGES IN THE -O6 %02 SPECTRA 4HIS WILL REQUIRE FURTHER STUDY 4HERE IS EVIDENCE THAT IN CONTRAST TO .AP THE -O6 STATE WILL BIND NITRATE AND THAT THIS SUBSTRATE BOUND STATE MAY BE RELEVANT TO THE CATALYTIC CYCLE AND A BIDENTATE COORDINATION MAY STERICALLY HINDER SUCH A BINDING PROCESS 'EORGE ET AL !NDERSON ET AL 4HE .AR' AND .AR( SUBUNITS OF .AR ARE PERIPHERAL TO THE CYTOPLASMIC SIDE OF THE MEMBRANE AND READILY RELEASED AS WATER SOLUBLE PROTEINS 4HESE TWO SUBUNITS SHOW NO ACTIVITY WITH QUINOLS AS ELECTRON DONORS ONLY WHEN THE TRANSMEMBRANE .AR) SUBUNIT IS PRESENT IS THE ABILITY TO OXIDIZE QUINOLS CONFERRED "ALLARD AND &ERGUSON 3EQUENCE ANALYSIS OF .AR) SHOWED THAT THERE ARE FOUR CONSERVED HISTIDINES TWO ON EACH OF TWO HELICES OUT OF THE lVE PREDICTED FOR THIS SUB UNIT "ERKS ET AL B 4HESE HISTIDINES BIND TWO HEMES STACKED ACROSS THE MEMBRANE AS OTHER WORK HAD SHOWN FOR THE ENZYME FROM 0 DENITRIlCANS THAT THE TWO HEMES HAD DISTINCT REDOX POTENTIALS M6 AND M6 "ALLARD AND &ERGUSON IT COULD BE SAFELY PROPOSED THAT THE FUNCTION OF THE .AR) SUB UNIT WAS TO OXIDIZE QUINOL AT THE OUTER 0 SIDE OF THE MEMBRANE AND TRANSFER ELECTRONS VIA THE TWO HEMES TO THE CATALYTIC SUBUNIT AT THE CYTOPLASMIC . SIDE OF THE MEMBRANE "ERKS ET AL B 4HIS ORGANIZA TION OF THE .AR ENZYME MEANS THAT IT WOULD ACT AS THE ELECTRON CARRYING ARM OF A REDOX LOOP SYSTEM FOR GENERATING PROTONMOTIVE FORCE AS ORIGINALLY PROPOSED
AS A GENERAL MECHANISM FOR RESPIRATORY CHAIN ACTION BY -ITCHELL 3UBSTANTIAL SUPPORT FOR THIS MODEL CAME FROM A RANGE OF SITE DIRECTED MUTAGENESIS STUDIES COUPLED WITH ANALYSIS OF REDOX POTENTIALS AND THE EFFECTS OF QUINOL BINDING TO .AR) SEE "LASCO ET AL 2OTHERY ET AL FOR REVIEWS 2ECENTLY STRUCTURAL BIOLOGY HAS PROVIDED THE lRST MOLECULAR DESCRIPTION OF A FULL PROTONMOTIVE REDOX LOOP COMPRISING THE % COLI NITRATE INDUCED FORMATE DEHYDROGENASE &DH . AND THE MEMBRANE BOUND NITRATE REDUCTASE *ORMAKKA ET AL "ERTERO ETAL 4HE STRUCTURE OF &DH . WAS THE lRST TO BE RESOLVED &DH . IS CLOSELY RELATED TO .AR ESPECIALLY IN RESPECT OF THE EQUIVALENT &E3 SUBUNIT B AND BIS -O -'$ CATALYTIC SUBUNIT A AND INITIALLY PROVIDED A GOOD MODEL FOR .AR *ORMAKKA ET AL 2ICHARDSON AND 3AWERS 4HIS TYPE OF FORMATE DEHYDROGENASE HAD ALSO BEEN PREDICTED TO HAVE TWO HEMES WITHIN AN INTEGRAL MEMBRANE SUBUNIT BUT WHICH DIFFER FROM THE COUNTERPART IN NITRATE REDUCTASE IN HAV ING ONLY FOUR HELICES THREE OF WHICH PROVIDE THE FOUR HISTIDINE LIGANDS "ERKS ET AL B ! CRITICAL DIF F FERENCE BETWEEN THE NITRATE REDUCTASE AND THE FORMATE DEHYDROGENASE IS TOPOLOGY BECAUSE IN THE FORMATE DEHYDROGENASE THE ALPHA AND BETA SUBUNITS ARE IN THE PERIPLASM 4HUS THIS DEHYDROGENASE CATALYSES THE INWARD MOVEMENT OF ELECTRONS FROM THE 0 SIDE TO THE . SIDE WHERE MENAQUINONE IS REDUCED 4HEREFORE IT RESEMBLES THE NITRATE REDUCTASE IN CONSTITUTING THE ELECTRON CARRYING ARM OF A REDOX LOOP )N &DH . THE ELECTRONS EXTRACTED FROM FORMATE AT THE PERIPLASMIC BIS -'$ ACTIVE SITE PASS DOWN A ¯ @LADDER OF REDOX CENTERS ULTIMATELY TO REDUCE MENAQUINONE AT THE . FACE OF THE CYTOPLASMIC MEMBRANE &IG 4HIS REDOX LADDER COMPRISES THE BIS -'$ lVE IRON SULFUR CLUSTERS AND TWO HEMES %ACH ARE WITHIN ¯ OF ITS NEAREST NEIGHBOR ENSURING RAPID ELECTRON TRANSFER ! LARGE ^ M6 POTENTIAL @DROP n M6 TO n M6 AT THE @. FACE ALLOWS EFlCIENT ELECTRON TRANSFER AGAINST THE MEMBRANE POTENTIAL TO OCCUR -UCH OF THE REDOX @LADDER IS MEMBRANE EXTRINSIC AND IT IS THE TWO MEMBRANE INTRINSIC HEMES THAT ARE CRITICAL TO CHARGE SEPARATION ACROSS THE MEMBRANE /F NOTE IS THE INTERACTION OF THE @. FACE HEME TO THE BOUND MENASEMIQUINONE ANALOGUE (1./ WHICH ACCEPTS A HYDROGEN BOND FROM ONE OF THE HISTIDINE LIGANDS 4HIS IS THE lRST TIME THAT A HEME LIGAND HAS BEEN SHOWN TO BE DIRECTLY INVOLVED IN QUINONE BINDING AND IT MAY BE THAT THIS WILL ALSO BE THE CASE IN THE NITRATE REDUCTASE a SUBUNIT 4HE BASIC &DH . FRAMEWORK OF AN INTEGRAL MEM BRANE DI HEME SUBUNIT A PERIPHERAL MEMBRANE FER
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO REDOXIN WITH FOUR ASSOCIATED IRON SULFUR CLUSTERS AND A PERIPHERAL MEMBRANE SUBUNIT WITH AN IRON SULFUR CLUSTER AND AN ACTIVE SITE -O BIS -'$ IS ALSO FOUND IN THE .AR'() )N THE &DH. BETA SUBUNIT THE FOUR IRON SULFUR CENTERS ALL ;&E 3= ARE ARRANGED IN TWO PAIRS IN EACH OF TWO DOMAINS 4HIS ARRANGEMENT WAS ALREADY PREDICTED FOR .AR FROM DETAILED SPECTROSCOPIC STUDIES ON % COLI .AR( THE ONLY DIFFERENCE BEING THAT ONE OF THE .AR &E3 CENTERS IS A ;&E 3= CLUSTER "LASCO ET AL )N THE CASE OF .AR( THE LOW REDOX POTENTIALS OF TWO OF THE CENTERS HAD RAISED THE POSSIBILITY THAT THEY ARE NOT DIRECTLY INVOLVED IN ELECTRON TRANSFER BETWEEN QUINOL AND NITRATE (OWEVER CONSIDERATION OF THE STRUCTURE OF THE .AR AND &DH . ` SUBUNITS LEAVES NO DOUBT THAT ALL FOUR CENTERS WILL BE DIRECTLY INVOLVED IN MEDIATING ELECTRON TRANSFER BETWEEN FORMATE AND NITRATE 4HUS IT SEEMS THAT THE EXTENDED ELECTRON TRANSFER CHAINS OF .AR'() AND &DH . ADD EXAMPLES TO A GROWING NUMBER OF CASES OF ELECTRON TRANSFER COMPLEXES IN BACTERIA THAT CONTAIN A MIXTURE OF ENDERGONIC AND EXERGONIC ELECTRON TRANSFER STEPS AND WHICH INCLUDE .AP! SEE SECTION ))"II THE .I &E HYDROGENASE AND FUMARATE REDUCTASE SEE /HNISHI ET AL FOR REVIEW 4HE ARRANGEMENT OF THE REDOX CENTERS IN THE &DH. .AR ELECTRON TRANSFER SYSTEM OF %COLI IS SHOWN IN &IG /NE QUESTION THAT HAS BEEN RESOLVED IS WHETHER LIKE THE ALPHA SUBUNIT OF &DH. .AR' BINDS AN IRON SULFUR CENTER IN ADDITION TO THE BIS -'$ 4HE POSSIBILITY THAT A (IS XX #YSXX#YSXN#YS MOTIF TOWARDS THE . TERMINAL OF .AR' BINDS AN IRON SULFUR CENTER HAD BEEN RAISED "ERKS ET AL A BUT SUCH A CENTER HAD NOT BEEN DETECTED SPECTROSCOPICALLY ALTHOUGH MUTATIONAL ANALYSIS HAS SUGGESTED THAT THESE #YS RESIDUES ARE IMPORTANT FOR ELECTRON TRANSFER SEE "LASCO ET AL (OWEVER THE .AR' STRUCTURE CONlRMS THAT THIS MOTIF DOES BIND A ;&E 3= CLUSTER FOR WHICH ELUCIDATION OF THE REDOX PROPERTIES MUST NOW AWAIT SPECTROSCOPIC INDENTIlCATION 4HE .AR AND &DH . STRUCTURES PROVIDE A FRAMEWORK UP ON WHICH STUDIES ON ELECTRON TRANSFER THROUGH THE ENZYME COMPLEXES CAN BE BUILT /NE OF THE TECHNIQUES BEING APPLIED IS PROTEIN lLM ELECTROCHEMISTRY %ARLY STUDIES BY #RASKE SHOWED THAT .AR'( CAN BE PURIlED SEPARATELY FROM .AR) RETAINING ENZYMATIC ACTIV ITY #RASKE AND &ERGUSON 2ECENTLY SOLUBLE .AR'( HAS BEEN SHOWN TO FORM lLMS ON GRAPHITE AND GOLD ELECTRODES WITHIN WHICH DIRECT AND FACILE EXCHANGE OF ELECTRONS BETWEEN THE ELECTRODE AND THE ENZYME OCCURS 0ROTEIN lLM VOLTAMMETRY HAS BEEN USED TO DElNE THE CATALYTIC BEHAVIOR OF .AR'( IN THE POTENTIAL DOMAIN AND A COMPLEX PATTERN OF REVERS IBLE NITRATE CONCENTRATION DEPENDENT MODULATION OF
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO
&IG 4HE ORGANIZATION OF THE &DH. .AR ELECTRON TRANSFER SYSTEM OF %SCHERICHIA COLI
ACTIVITY HAS BEEN RESOLVED !NDERSON ET AL 4HE STUDY SHOWS THAT .AR'( CAN CATALYZE NITRATE REDUCTION VIA TWO PATHWAYS EACH HAVING DISTINCT KCAT+ +- #ATALYSIS IS DIRECTED TO OCCUR VIA ONE OF THE PATHWAYS BY AN ELECTROCHEMICAL EVENT WITHIN .AR'( WHICH MAY LIE AT THE LEVEL OF THE -O BIS-'$ OR ONE OF THE IRON SULFUR CENTERS /JUSBUF5SBOTQPSUUP/BS /NE IMPORTANT QUESTION THAT THE ORIENTATION OF .AR POSES IS HOW IS THE SUBSTRATE DELIVERED TO THE ACTIVE SITE /NE OR MORE TRANSPORT PROTEINS ARE IMPLICATED BY THE lNDING THAT WHILE AND IN CONTRAST TO .AP THE PURIlED .AR ENZYME CAN USE CHLORATE AS AN ALTERNATIVE SUBSTRATE IN THE CELL .AR IS INACCESSIBLE TO CHLORATE UNLESS THE PERMEABILITY BARRIER OF THE CYTOPLASMIC MEMBRANE IS BREACHED THROUGH ADDITION OF A DETERGENT 4HE NITRATE ANION IS ENCOUNTERED IN THE EXTERNAL ENVI RONMENT OF THE BACTERIUM AND SO MUST BE TRANSPORTED INTO THE CELL AGAINST THE @NEGATIVE INSIDE MEMBRANE POTENTIAL TO SERVE AS THE SUBSTRATE FOR .AR'( 4HIS F COULD IN PRINCIPLE BE A PMF CONSUMING PROCESS THAT
WOULD THEN AFFECT THE NET ENERGETICS OF THE 1( .2 ELECTRON TRANSFER SYSTEM &IG )N THE ANAEROBIC DENITRIlCATION PATHWAY THERE IS A NEED TO TRANSPORT THE NITRATE ANION SUBSTRATE FOR .AR INTO THE CELL AND THE CYTOPLASMICALLY GENERATED NITRITE OUT OF THE CELL TO THE PERIPLASM WHERE IT CAN SERVE AS SUBSTRATE FOR NITRITE REDUCTASE &AILURE TO DO THIS WILL RESULT IN THE CELL BEING UNABLE TO DENITRIFY 4HUS THE NITRATENITRITE TRANSPORT PROCESS PROVIDES AN EXCELLENT LEVEL AT WHICH TO CONTROL DENITRIlCATION 4HIS WAS DEMONSTRATED IN A SERIES OF STUDIES IN THE S IN WHICH IT WAS DEMONSTRATED THAT NITRATE REDUCTION WAS INHIBITED BY OXYGEN IN INTACT CELLS BUT NOT IN MEMBRANE VESICLES OR TRITON 8 PERMEABILIZED CELLS !LEFOUNDER AND &ERGUSON 4HIS OXYGEN INHIBITION COULD ALSO BE MIMICKED BY NITROUS OXIDE OR FERRICYANIDE INDICATING THAT IT WAS NOT AN EFFECT OF MOLECULAR OXYGEN ITSELF BUT A REDOX EFFECT THAT COULD POSSIBLY BE MEDIATED BY THE 1 POOL WHICH WILL BE IN CONTACT WITH AN INTE GRAL MEMBRANE TRANSPORTER !LEFOUNDER ET AL 4HE BASIS OF OXYGENREDOX REGULATION OF NITRATE TRANSPORT HAS NOT YET BEEN RESOLVED BUT PROGRESS IS NOW BEING MADE THROUGH THE IDENTIlCATION OF THE
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
GENE NAR+ THAT ENCODES THE . OXYANION TRANSPORT SYSTEM AND IS REGULATED BY &NR0 AND .AR2 7OOD ET AL )N 0ARACOCCUS PANTOTROPHUS NAR+ + ENCODES A TRANSMEMBRANE HELIX PROTEIN THAT IS A FUSION OF TWO TRANSPORTERS .AR+ AND .AR+ 2ECENT WORK IN WHICH THE CONTRIBUTION THAT EACH DOMAIN MAKES TO NITRATE UPTAKE IN THE CELL HAS BEEN ASSESSED SEPA RATELY HAS SUGGESTED THAT .AR+ IS A NITRATE PROTON SYMPORTER AND .AR+ IS A NITRATE NITRITE ANTIPORTER 7OOD ET AL 4HIS CONlRMS EARLIER WORK ON 0 DENITRIlCANS "OOGERD ET AL THAT SUGGESTED THAT A NITRATE ( SYMPORTER INITIATES NITRATE RESPIRA TION AND THEN WHEN SUFlCIENT INTRACELLULAR NITRITE HAS BUILT UP A NITRATE NITRITE ANTIPORTER OPERATES 4HE LATER SYSTEM IS BIOENERGETICALLY MORE EFlCIENT AS IT DOES NOT CONSUME 0-& AND IT MAY BE THAT THE FUSED NATURE OF THE TWO TRANSPORTERS ALLOWS CO OPERATIVE REGULATION SO THAT @SWITCH ON OF THE NITRATE NITRITE ANTIPORTER SWITCHES OFF THE NITRATE PROTON SYMPORTER .AR+ AND .AR+ CLUSTER SEPARATELY IN PHYLOGENETIC TREES AND IT IS NOTABLE THAT A NUMBER OF BACTERIA EG 0S AERUGINOSA HAVE TWO SEPARATE GENES ENCODING .AR+ AND .AR+ HOMOLOGUES 7OOD ET AL WHILE % COLI BY CONTRAST HAS GENES ENCODING TWO .AR+ TYPE PROTEINS .AR+ AND .AR5 !LTHOUGH IT HAS BEEN ARGUED THAT % COLI NAR+ ASSOCIATED WITH + THE NAR'(*) GENE CLUSTER ENCODES A ) NITRITE ANTIPORTER 2OWE ET AL RECENT WORK HAS SUGGESTED THAT BOTH .AR+ AND .AR5 DO IN FACT CONTRIBUTE TO NITRATE UPTAKE #LEGG ET AL BUT THAT .AR5 THE GENE FOR WHICH IS ASSOCIATED WITH A SECOND NAR OPERON NAR: IN : % COLI MAY CONTRIBUTE MORE THAN .AR+ TO NITRITE EXCRETION #LEARLY THERE IS STILL MUCH TO BE LEARNT ABOUT THE INTERACTION AND PHYSIOLOGICAL ROLES OF DIFFERENT .AR+ TYPE PROTEINS IN DIFFERENT BACTERIA
CHARACTERISTIC OF C TYPE CYTOCHROME IS THAT THE HEME IS ATTACHED TO THE TWO CYSTEINES OF A #88#( MOTIF !S DISCUSSED BELOW ONE OF THE HEMES IN THIS TYPE OF NITRITE REDUCTASE IS EXCEPTIONAL IN BEING ATTACHED TO A #88#+ MOTIF AND IS THE CATALYTIC HEME /NE EXCEP TION TO THIS IS FOUND IN THE .RF! OF # JEJUNI IN WHICH ALL lVE HEMES ARE ATTACHED TO #88#( MOTIFS 4HE FUNCTIONAL SIGNIlCANCE OF THIS SOMEWHAT SURPRISING LATTER lNDING REMAINS TO BE DETERMINED 4HE SECOND REACTION CATALYSED BY NITRITE REDUCTASES IS THE REDUCTION OF NITRITE TO NITRIC OXIDE ./ E ( A ./ (/
4HIS IS CATALYSED BY TWO VERY DIFFERENT ENZYMES ONE CALLED CYTOCHROME CD THE OTHER COPPER NITRITE REDUCTASE 4HEY ARE LESS COMMONLY KNOWN BY THEIR GENE NAMES IE .IR3 AND .IR+ RESPECTIVELY !S THE NAME SUGGESTS CYTOCHROME CD CONTAINS A C TYPE CYTOCHROME CENTER AND A SECOND SPECIALIZED HEME WHICH IS NON COVALENTLY BOUND AND PROVIDES THE ACTIVE SITE /NE OF HEME CENTERS OF EACH TYPE IS ASSOCIATED WITH EACH POLYPEPTIDE CHAIN IN THE DIMERIC MOLECULE 4HE #U TYPE OF NITRITE REDUCTASE IS A TRIMERIC MOL ECULE WITH SIX COPPER IONS 4HREE OF THESE ARE 4YPE COPPERS AND SERVE TO COLLECT ELECTRONS FROM DONOR PROTEINS AND PASS THEM ON TO THE 4YPE COPPERS AT THE THREE ACTIVE SITES 4HE RECRUITMENT BY BIOLOGY OF BOTH IRON AND COPPER TO CATALYZE THE SAME REACTION IS COMMON IN BIOLOGY !S WE SHALL DESCRIBE BELOW THESE TWO TYPES OF NITRITE REDUCTASE HAVE VERY DIFFERENT REACTION MECHANISMS &INALLY WE NOTE THAT ONLY ONE OF THESE THREE TYPES OF NITRITE REDUCTASE HAS EVER BEEN FOUND CONCLUSIVELY IN ANY ONE ORGANISM "$ZUPDISPNFD/JUSJUF3FEVDUBTF /SG"
***3FTQJSBUPSZ/JUSJUF3FEVDUBTFT !S EXPLAINED IN THE )NTRODUCTION THERE ARE TWO TYPES OF REACTION CATALYSED BY NITRITE REDUCTASE /NE IS THE REDUCTION OF NITRITE TO AMMONIUM ./ E ( A .( (/
4HIS IS CATALYSED BY A DIMERIC ENZYME IN WHICH EACH MONOMER HAS lVE HEME GROUPS %ACH OF THE HEMES IS ATTACHED TO THE PROTEIN BY TWO THIOETHER BONDS AND SO THIS TYPE OF NITRITE REDUCTASE CAN BE TERMED A PENTAHEME C TYPE CYTOCHROME ALTHOUGH IT IS INCREASINGLY BEING CALLED .RF! AFTER ITS GENE 4HE
4HE REDUCTION OF NITRATE TO AMMONIUM IN THE PERI PLASM OF A NUMBER OF BACTERIA INCLUDING %SCHERICHIA COLI INVOLVES TWO ENZYMES PERIPLASMIC NITRATE REDUC TASE .AP! AND PERIPLASMIC NITRITE REDUCTASE .RF! 4HE PROCESS IS FOUND IN MANY ENTERIC BACTERIA AND IS IMPORTANT FOR ANAEROBIC NITRATE AND NITRITE RESPIRATION AT LOW NITRATE CONCENTRATIONS 0OTTER ET AL 4HE .RF! PROTEIN CATALYSES THE SIX ELECTRON REDUCTION OF NITRITE TO AMMONIUM ./n En ( A .( (/
.RF! CAN ALSO CATALYZE THE ELECTRON REDUCTION OF ./ AND ELECTRON REDUCTION OF HYDROXYLAMINE BOTH
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO OF WHICH MAY BE BOUND INTERMEDIATES IN THE CATALYTIC CYCLE FOR NITRITE REDUCTION "ERKS ET AL A )N TERESTINGLY IT HAS RECENTLY BEEN SHOWN THAT THE ./ REDUCTASE ACTIVITY OF .RF! MAY ALSO PLAY A PHYSIOLOGI CAL ROLE IN DETOXIlCATION OF EXOGENOUS ./ 0OOCK ET AL 0RIMARY STRUCTURE AND BIOCHEMICAL ANALYSIS REVEALED THAT THE % COLI .RF! PROTEIN IS A K$A PENTAHEME CYTOCHROME IN WHICH FOUR HEMES ARE COVALENTLY BOUND TO THE CONVENTIONAL MOTIF #88#( 4HE lFTH HEME IS ATTACHED TO THE NOVEL #88#+ MO TIF $ARWIN ET AL (USSAIN ET AL 4HIS ATTACHMENT REQUIRES SPECIALIZED HEME CYTOCHROME C MATURATION PROTEINS %AVES ET AL 5IF4USVDUVSFBOE4QFDUSPTDPQZPG/SG" 4HE CRYSTAL STRUCTURES OF CYTOCHROME C NITRITE RE DUCTASE FROM % COLI 3ULFUROSPIRILLUM DELEYIANUM AND 7OLINELLA SUCCINOGENES HAVE BEEN DETERMINED %INSLE ET AL "AMFORD ET AL )N ALL THREE SYSTEMS .RF! CRYSTALLIZED AS A HOMO DIMER &IG WITH THE HEMES WITHIN EACH MONOMER CLOSELY PACKED TO FORM ARRANGEMENTS OF NEAR PARALLEL AND NEAR PERPENDICULAR HEME PAIRS THAT HAVE ALSO BEEN OBSERVED IN THE OCTA HEME HYDROXYLAMINE OXIDO REDUCTASE )GARASHI ET AL )NDEED THERE IS ALSO STRUCTURAL SIMILARITY IN PROTEIN FOLDING TO (!/ THAT REVEALS A CLEAR EVOLUTIONARY RELATEDNESS BETWEEN THE TWO ENZYMES WHICH IS INTRIGUING GIVEN THAT .RF CAN REDUCE NITRITE TO A BOUND HYDROXYLAMINE INTERMEDI ATE AND (!/ CAN OXIDIZE HYDROXYLAMINE TO NITRITE (OWEVER BOTH ENZYMES APPEAR TO BE ESSENTIALLY UNIDIRECTIONAL )N % COLI .RF! THE ACTIVE SITE HEME DISPLAYS A DISTAL LYSINE LIGAND AND PROXIMAL OXYGEN LIGAND THAT IS LIKELY TO ARISE FROM WATER "AMFORD ET AL )N THE CASE OF THE 7 SUCCINOGENES ENZYME .RF! STRUCTURES WITH WATER SULFATE NITRITE HYDROX YLAMINE AND AZIDE BOUND IN THE ACTIVE SITE HAVE BEEN REPORTED %INSLE ET AL ! NUMBER OF COMMON FEATURES OF THE CYTOCHROME C NITRITE REDUCTASES ARE APPARENT IN THE ACTIVE SITES OF ALL OF THE 7 SUCCINOGENES 3 DELEYIANUM AND % COLI STRUCTURES 4HESE INCLUDE THE CATALYTIC HEME WITH THE LYSINE PRIMARY AMINE NITROGEN AS THE PROXIMAL LIGAND A PUTATIVE SUBSTRATE INLET CHANNEL WITH POSITIVELY CHARGED ELECTROSTATIC POTENTIAL AND A PUTATIVE PRODUCT EFmUX CHANNEL THAT EXHIBITS A MORE NEGATIVE ELECTRO STATIC POTENTIAL )N ADDITION AN ACTIVE SITE CALCIUM ION IS CONSERVED ACROSS ALL THREE STRUCTURES BOTH IN TERMS OF THE IDENTITY OF LIGATING RESIDUES AND IN THE PRESENCE OF TWO CONSERVED WATER LIGANDS &IG 4HE
CRYSTAL STRUCTURE OF THE 7 SUCCINOGENES ENZYME WITH BOUND NITRITE SHOWS THAT THE NITROGEN IS COORDINATED TO THE HEME IRON WITH THE TWO OXYGEN ATOMS BEING ( BONDED BY (IS AND !RG % COLI NUMBER ING )N THE HYDROXYLAMINE COORDINATED DERIVATIVE THE SINGLE OXYGEN IS ( BONDED BY !RG 0ROTON DELIVERY TO THE ACTIVE SITE MAY INVOLVE CONSERVED TYROSINE RESIDUES ! POSSIBLE SCHEME FOR REDUCTION OF NITRITE AT THE ACTIVE SITE HAS BEEN PROPOSED %INSLE ET AL 4HE STRUCTURE OF THE % COLI ENZYME HAS RECENTLY BEEN ANALYZED IN COMBINATION WITH AN -#$ AND SPEC TRO POTENTIOMETRIC %02 "AMFORD ET AL 4HIS HAS ALLOWED THE ASSIGNMENT OF SPECTROSCOPIC SIGNALS AND EQUILIBRIUM REDOX POTENTIALS TO INDIVIDUAL HEMES IN THE CRYSTAL STRUCTURE -#$ ANALYSIS CONlRMED THE PRESENCE OF FOUR BIS (IS COORDINATED &E))) HEMES IN A .RF! SOLUTION AT P( 4HE -#$ ANALYSIS ALSO CONlRMED THE PRESENCE OF A HIGH SPIN HEME &E))) WITH . AND / LIGATION IN WHICH THE OXYGEN LIGAND AROSE PREDOMINANTLY FROM WATER %02 SPECTRA WERE COLLECTED FROM .RF! SAMPLES POISED ELECTROCHEMI CALLY AT NUMBER OF DIFFERENT POTENTIALS "ROAD PER PENDICULAR MODE 8 BAND %02 SIGNALS WITH POSITIVE FEATURES AT G AND THAT ARE CHARACTERISTIC OF WEAKLY SPIN COUPLED 3 b 3 PARAMAGNETS TITRATED WITH AN APPARENT %M n M6 AND ARE LIKELY TO ARISE FROM THE ACTIVE SITE ,YS /( COORDINATED HEME HEME AND THE NEARBY BIS (IS COORDINATED HEME HEME 4HESE HEMES HAVE A CLOSEST RING DISTANCE OF ¯ AND INTER PLANE ANGLE OF $ ! RHOMBIC HEME &E))) %02 SIGNAL AT GZ GY GX TITRATES AS A N CENTER WITH %M n M6 AND IS LIKELY TO ARISE FROM BIS (IS COORDINATED HEME HEME IN WHICH THE INTERPLANAR ANGLE OF THE IMIDAZOLE RINGS IS $ 4HE lNAL TWO BIS (IS COORDINATED HEMES HEMES AND HAVE IMIDAZOLE INTERPLANAR ANGLES OF $ AND $ %ITHER OR BOTH OF THESE HEMES COULD GIVE RISE TO A @,ARGE G MAX %02 SIGNAL AT GZ THAT TITRATES AT POTENTIALS BETWEEN n M6 AND n M6 4HE CATALYTIC CYCLE OF .RF! MAY PROCEED VIA BOUND ./ AND .(/( DERIVATIVES ALTHOUGH THE HIGH + +FOR .(/( DOES RAISE THE QUESTION OF WHETHER IT CAN BE A LEGITIMATE INTERMEDIATE &IG #ATALYSIS HAS ALSO BEEN PROBED WITH PROTEIN lLM ELECTROCHEMISTRY !NGOVE ET AL 4HE CATALYTIC CURRENT POTENTIAL PROlLES OBSERVED ON PROGRESSION FROM SUBSTRATE LIMITED TO ENZYME LIMITED NITRITE REDUCTION REVEALED A lNGERPRINT OF CATALYTIC BEHAVIOR DISTINCT FROM THAT OBSERVED DURING HYDROXYLAMINE REDUCTION 3UGGEST ING THAT THE CYTOCHROME C NITRITE REDUCTASE INTERACTS
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
&IG 4HE STRUCTURE OF % COLI CYTOCHROME C NITRITE REDUCTASE .RF! AND ITS ACTIVE SITE POCKET
DIFFERENTLY WITH THESE TWO SUBSTRATES A NOTION SUP PORTED BY THE CRYSTAL STRUCTURES (OWEVER A SIGMOIDAL CATALYTIC WAVE WITH AN N SLOPE WITH %M CA n M6 WAS A COMMON FEATURE OF THE DEVELOPMENT OF THE VOLTAMMETRIC RESPONSE WITH INCREASING NITRITE OR HY DROXYLAMINE CONCENTRATION 4HE MIDPOINT OF THIS CATA LYTIC WAVE IS SIMILAR TO THAT DETERMINED FOR THE ACTIVE SITE DI HEME PAIR AND THUS SUGGESTS THAT A RATE LIMITING COORDINATED TWO ELECTRON REDUCTION OF THE ACTIVE SITE FOR BOTH SUBSTRATES AND SUGGEST THAT THE MECHANISMS FOR REDUCTION OF BOTH SUBSTRATES ARE UNDERPINNED BY COMMON RATE DElNING PROCESSES /NE QUESTION THAT NOW NEEDS TO BE ADDRESSED IS THE SIGNIlCANCE OF THE DIMERIC ORGANIZATION OF .RF! (EMES AT THE DIMER INTERFACE ARE CLOSE ENOUGH FOR INTER DIMER ELECTRON TRANSFER AND THIS COULD PROVE TO BE A MECHANISM BY WHICH DIMERS OF THE PENTA HEME MONOMER STORE SUFlCIENT ELECTRONS TO CATALYZE THE RAPID ELECTRON REDUCTION OF NITRITE &IG )T IS THEN POSSIBLE THAT A DIFFERENT ROUTE OF ELECTRON TRANSFER OPERATES FOR THE ELECTRON REDUCTION OF HYDROXYLAMINE &MFDUSPO5SBOTGFSUP/SG" !NALYSIS OF THE ORGANIZATION OF NRF! GENE CLUSTERS FROM
A RANGE OF BACTERIA REVEALS THAT THEY CAN BE DIVIDED INTO TWO GROUPS )N ONE GROUP THAT INCLUDES 7 SUC CINOGENES AND 3 DELEYIANUM &IG THE NRF! CLUSTERS WITH AN ADJACENT GENE NRF( WHICH ENCODES A MEM (
BRANE ANCHORED TETRA HEME QUINOL DEHYDROGENASE OF THE .AP# FAMILY DISCUSSED IN 3ECTION ))" 3IMON ET AL !NALYSIS OF .RF(! RECONSTITUTED INTO PROTEOLIPOSOMES HAS CLEARLY SHOWN THE ELECTRON TRANSPORT FROM QUINOL NITRITE IS NOT PROTON MOTIVE )N THE SECOND GROUP WHICH INCLUDES % COLI &IG THE NRF! CLUSTERS WITH GENES ENCODING A PERIPLASMIC PENTA HEME CYTOCHROME NRF" A PERIPLASMIC = ;&E3= FERREDOXIN NRF# AND AN INTEGRAL MEMBRANE # PUTATIVE QUINOL DEHYDROGENASE NRF$ CONTAINING SEVEN TRANSMEMBRANE HELICES (USSAIN ET AL 0OTTER ET AL #LEARLY ELECTRON TRANSFER FROM QUINOL TO THE .RF! IN THE DIFFERENT GROUPS IS DISTINCT AND IT CAN NOT BE EXCLUDED THAT ELECTRON TRANSFER FROM QUINOL TO NITRITE VIA .RF$#!" IS PROTONMOTIVE 4HE DIFFERENT PROTEIN PROTEIN AND COFACTOR COFACTOR INTERACTIONS BETWEEN .RF( ! AND .RF" ! MAY BE REmECTED BY INSERTIONS AND DELETIONS IN LOOP REGIONS OF THE POLYPEPTIDE CHAIN IN THE TWO .RF! SUBGROUPS THAT CAN BE IDENTIlED IN PRIMARY STRUCTURE ANALYSIS 4HIS LEADS TO DIFFERENCES IN THE SOLVENT EXPOSURE OF HEME
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO
&IG ! CATALYTIC SCHEME FOR THE CYTOCHROME C NITRITE REDUCTASE
AND IN ELECTROSTATIC SURFACE OF THE PROTEIN AROUND THIS HEME "AMFORD ET AL #ONSIDERATION OF THE CRYSTAL STRUCTURE MAKES HEME THE MOST LIKELY ELECTRON INPUT SITE &IG #$ZUPDISPNFDE /JUSJUF3FEVDUBTF DE/JS
4HE NAME CYTOCHROME CD RECOGNIZES THE CONTENT OF ONE C TYPE CYTOCHROME CENTER AND ONE D HEME PER POLYPEPTIDE CHAIN 4HE FORMER CENTER IS DElNED BY THE COVALENT ATTACHMENT OF &E PROTOPORPHYRIN )8 TO THE POLYPEPTIDE CHAIN AS A CONSEQUENCE OF THIOETHER BOND FORMATION BETWEEN THE VINYL GROUPS OF THE PORPHYRIN AND TWO THIOLS OF CYSTEINE RESIDUES IN A #88#( MOTIF 4HE D HEME IS UNIQUE TO THIS TYPE OF ENZYME AND IS CHARACTERIZED BY PARTIAL SATURATION OF TWO OF THE RINGS AND THE PRESENCE OF CARBONYL GROUPS 3TRUCTURES OF CYTOCHROME CD HAVE BEEN SOLVED FOR THE PROTEINS FROM THE BACTERIA 0 PANTOTROPHUS AND 0 AERUGINOSA WITH THE SURPRISING OUTCOME THAT THERE ARE SIGNIlCANT DIFFERENCES BETWEEN THE TWO AT LEAST IN THE OXIDIZED STATES 4HUS TO AVOID POTENTIAL CONFUSION IN THE READERS MIND WE DISCUSS HERE SEQUENTIALLY SOME OF THE lNDINGS CONCERNING THE TWO ENZYMES BEFORE RETURNING TO COMPARE THE TWO ENZYMES AND SEEKING TO SYNTHESIZE AN OVERALL PICTURE OF CURRENT UNDERSTANDING OF THIS TYPE OF ENZYME SEE ALSO !LLEN ET AL FOR ANOTHER RECENT REVIEW
1BSBDPDDVTQBOUPUSPQIVT$ZUPDISPNFDE 4HE CRYSTAL STRUCTURE OF THE DIMERIC OXIDIZED CYTO CHROME CD FROM 0 PANTOTROPHUS SHOWED THAT THE D HEME WAS ENCLOSED IN AN EIGHT BLADED BETA PROPELLER STRUCTURE WITH THE IRON HAVING ONE EXPECTED AXIAL LIGAND A PROXIMAL HISTIDINE AND ONE UNEXPECTED LIGAND A DISTAL TYROSINE &ULOP ET AL &IG %VEN MORE UNEXPECTED WAS THAT THIS TYROSINE RESIDUE WAS PART OF THE ALPHA HELICAL DOMAIN OF THE PROTEIN THAT FORMED THE C TYPE CYTOCHROME SECTOR OF THE ENZYME 4HERE WAS ALSO A SURPRISE IN THIS PART OF THE PROTEIN BECAUSE THE HEME IRON HAD TWO AXIAL HISTIDINE LIGANDS ONE OF WHICH RESIDUE WAS CLOSE IN SEQUENCE TO THE TYROSINE LIGAND TO THE D HEME IRON 4HE OTHER HISTIDINE LIGAND WAS PROVIDED FROM THE #88#( MOTIF 3OLUTION SPECTROSCOPIC MEASUREMENTS WERE IN ACCORD WITH THESE HEME LIGATIONS #HEESMAN ET AL 2EDUCTION OF THIS CRYSTAL FORM RESULTED IN LOSS OF THE TYROSINE FROM THE D HEME AND THUS THE PROVISION OF A BINDING SITE ON THE IRON FOR THE SUBSTRATE &IG (ISTIDINE HAD ALSO DISSOCIATED FROM THE &E OF THE C TYPE CYTOCHROME DOMAIN AND BEEN REPLACED BY THE SULFUR OF METHIONINE WHICH HAD UNDERGONE A CONSIDERABLE SPATIAL DISPLACEMENT FOLLOWING REDUCTION OF THE ENZYME 7ILLIAMS ET AL 4HE lRST MECHANISTIC PROPOSAL FOR THE ENZYME ENVISAGED THAT THESE LIGAND SWITCHES WOULD OCCUR DURING THE CATALYTIC CYCLE 4HIS WAS AN ATTRACTIVE IDEA
AS THE REACTION PRODUCT NITRIC OXIDE CAN HAVE A HIGH AFlNITY FOR HEMES AND A TYROSINE REBINDING MECHANISM COULD BE ENVISAGED TO CONTRIBUTE TO THE DISPLACEMENT OF THE PRODUCT (OWEVER SUBSEQUENT EXPERIMENTS IN WHICH FULLY REDUCED ENZYME WAS OXIDIZED BY EITHER OXYGEN AN ALTERNATIVE SUBSTRATE OR NITRITE UNDER RAPID REACTION CONDITIONS SHOWED THAT THE C TYPE CYTOCHROME HEME HAD SPECTROSCOPIC PROPERTIES CONSISTENT WITH A FERRIC STATE HAVING HISTIDINE PLUS METHIONINE RATHER THAN BIS HISTIDINYL COORDINATION !LLEN ET AL A B 'EORGE ET AL +OPPENHOFER ET AL A 5SE OF EITHER OXYGEN OR NITRITE AS OXIDANT IN SUCH EXPERI MENTS HAS COMPLICATIONS BECAUSE PRODUCTS REMAIN BOUND TO THE ENZYME AND NEITHER OF THESE SUBSTRATES REQUIRING FOUR AND ONE ELECTRONS RESPECTIVELY CAN RE MOVE THE ELECTRON FROM EACH OF THE TWO HEME CENTERS ON ONE POLYPEPTIDE CONCOMITANTLY WITH TURNOVER OF
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO A SINGLE ACCEPTOR IONMOLECULE AT THE ACTIVE SITE )N CONTRAST A THIRD SUBSTRATE HYDROXYLAMINE REQUIRES TWO ELECTRONS FOR ITS REDUCTION TO AMMONIUM 7HEN FULLY REDUCED ENZYME IN THE ABSENCE OF ANY EXCESS REDUCTANT WAS OXIDIZED BY HYDROXYLAMINE IT WAS NOTED THAT THE VISIBLE ABSORPTION SPECTRUM OF THE RESULTING FULLY OXIDIZED ENZYME WAS DISTINCT FROM THE INITIAL AS PREPARED OXIDIZED ENZYME !LLEN ET AL A B %XAMINATION OF THIS FORM OF THE ENZYME BY BOTH %02 AND .)2 -#$ SHOWED THAT THE HEME OF THE C TYPE CYTOCHROME HAD HISTIDINEMETHIONINE !LLEN ET AL A B /VER A PERIOD OF APPROXIMATELY MINUTES THIS FORM OF THE OXIDIZED ENZYME REVERTED TO THE AS PREPARED STATE WITH THE TWO HISTIDINE LIGANDS TO THE C TYPE CYTOCHROME DOMAIN THAT HAVE ALREADY BEEN DESCRIBED 4HIS REVERSION PROCESS ALSO CORRELATED WITH A LOSS OF THE ABILITY OF THE CYTOCHROME CD TO CATALYZE
&IG 4HE STRUCTURE OF THE 0 PANTOTROPHUS CYTOCHROME CD NITRITE REDUCTASE ! OVERALL STRUCTURE OF OXIDIZED ENZYME " DETAIL OF HEME COORDINATION IN OXIDIZED ENZYME # DETAIL OF HEME COORDINATION IN REDUCED ENZYME
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO THE OXIDATION OF REDUCED PSEUDOAZURIN A PHYSIOLOGI CAL TYPE ) #U ELECTRON DONOR PROTEIN BY NITRITE 4HUS THE FORM OF OXIDIZED ENZYME OBTAINED IMMEDIATELY FOLLOWING A CYCLE OF OXIDATION BY HYDROXYLAMINE WAS CONSIDERABLY MORE CATALYTICALLY COMPETENT THAN THE AS PREPARED OXIDIZED ENZYME !LLEN ET AL A B 3UBSEQUENTLY IT HAS BEEN SHOWN THAT THE LATTER FORM OF THE ENZYME REQUIRES ACTIVATION BY A CYCLE OF REDUC TION AND REOXIDATION IN ORDER TO ELICIT HIGH RATES OF ACTIVITY TOWARDS THE OXIDATION OF EITHER PSEUDOAZURIN CYTOCHROME C FROM 0 DENITRIlCANS OR HORSE HEART MITOCHONDRIAL CYTOCHROME C WITH ANY OF THE THREE ELECTRON ACCEPTORS NITRITE OXYGEN OR HYDROXYLAMINE 2ICHTER ET AL 4HUS A CURRENT HYPOTHESIS IS THAT THE AS PREPARED FORM OF THE OXIDIZED ENZYME REPRESENTS A RESTING STATE OF THE ENZYME 4HE PURPOSE OF THIS FORM OF THE ENZYME AND ITS MECHANISM OF ACTIVATION IN VIVO ARE NOT KNOWN 4HE CONCLUSION FROM SPECTROSCOPIC AND ACTIVITY MEASUREMENTS THAT THE OXIDIZED CYTOCHROME CD MOL ECULE CAN ADOPT A CONFORMATION IN WHICH IT IS ACTIVATED MIGHT REASONABLY BE CORRELATED WITH THE STRUCTURE SEEN ORIGINALLY FOR THE REDUCED ENZYME WHICH RECALL HAS HISTIDINEMETHIONINE COORDINATION AT THE C TYPE HEME (OWEVER A NEW CRYSTALLOGRAPHIC STUDY ON THIS ENZYME HAS ADDED AN UNEXPECTED TWIST OF COMPLEXITY TO THE STORY #RYSTALLIZATION OF REDUCED PROTEIN UNDER ANAEROBIC CONDITIONS HAS DEMONSTRATED THE EXISTENCE OF A THIRD CONFORMATION FOR CYTOCHROME CD FROM 0 PANTOTROPHUS 3JOGREN AND (AJDU 4HIS DIFFERS FROM THAT ORIGINALLY DESCRIBED FOR REDUCED ENZYME NOT IN RESPECT OF THE LIGANDS TO THE IRON ATOMS BUT RATHER IN TERMS OF A ROTATION BY APPROXIMATELY O OF THE C HEME DOMAIN RELATIVE TO THE D HEME DOMAIN 4REATMENT OF THIS CRYSTAL FORM WITH OXYGEN GAVE AN OXIDIZED CRYSTAL IN WHICH THE STRUCTURE OF THE PROTEIN WAS UNCHANGED 4HUS IT IS TEMPTING TO CONCLUDE THAT THIS NEW CRYSTAL STRUCTURE REPRESENTS THE PROTEIN IN THE ACTIVATED CONFORMATION THAT IS OBSERVED IN SOLU TION AFTER A CYCLE OF OXIDATION AND REDUCTION )N THIS CONTEXT THEREFORE IT COULD BE CONSIDERED THAT BOTH THE ORIGINAL OXIDIZED AND REDUCED CRYSTAL STRUCTURES ARE NOT CATALYTICALLY RELEVANT FORMS (OWEVER THE @NEW REDUCED CRYSTAL COULD NOT BE REOXIDIZED BY NITRITE WHICH THEREFORE INJECTS A NOTE OF CAUTION ABOUT ACCEPTING THIS CONCLUSION TOO PRECIPITATELY 3JOGREN AND (AJDU )NSPECTION OF THE @OLD AND @NEW CRYSTAL FORMS SHOWS THAT THE TWO FORMS ARE RELATED BY A ROTATION OF THE CYTOCHROME DOMAIN AROUND WHAT APPEARS TO BE A HINGE REGION &UTURE WORK WILL NEED TO ADDRESS THE POSSIBILITY THAT SUCH ROTATION IS PART OF
THE CATALYTIC MECHANISM ESPECIALLY SINCE A SIMILAR MOVEMENT HAS BEEN SEEN UNDER CERTAIN CIRCUMSTANCES WITH THE 0 AERUGINOSA ENZYME SEE 3ECTION )))" 4HE VARIOUS CONFORMATIONAL CHANGES NOW SEEN FOR CYTOCHROME CD MAY WELL EXPLAIN THE REMARKABLE HYSTERETIC REDOX TITRATION THAT IS SEEN FOR THE ENZYME FROM 0 PANTOTROPHUS +OPPENHOFER ET AL B /NE OF THE ENDURING ISSUES CONCERNING CYTOCHROME CD IS THE PURPOSE OF THE SPECIALIZED D HEME )T DIFFERS FROM THE USUAL HEME IN SEVERAL RESPECTS AMONGST WHICH IS THE PARTIAL SATURATION AND PRESENCE OF ELECTRON WITHDRAWING CARBONYL GROUPS ! PLAUSIBLE MECHANISM FOR THE ENZYME INVOLVES THE BINDING OF THE ANIONIC NITRITE TO THIS HEME IN ITS REDUCED STATE (OWEVER THIS WOULD BE AT VARIANCE WITH THE USUAL EX PECTATION THAT IT IS THE FERRIC STATE OF A HEME THAT BINDS AN ANION %VIDENCE THAT THE FERROUS D HEME IS TAILORED TO BIND AN ANION HAS COME FROM A CRYSTALLOGRAPHIC STUDY OF CYANIDE BINDING TO THE CYTOCHROME CD FROM 0 PANTOTROPHUS ! STRUCTURE OF CYANIDE BOUND TO THE FERROUS ENZYME WAS OBTAINED BUT WHEN THE CRYSTAL WAS OXIDIZED THE CYANIDE WAS LOST AND THE TYROSINE LIGAND RETURNED TO THE D HEME IRON COORDINATION SPHERE *AFFERJI ET AL 4HE DISSOCIATION CONSTANT FOR CYANIDE FROM THE FERROUS D HEME IN SOLUTION COULD BE SHOWN TO BE IN THE MICROMOLAR RANGE A LOW VALUE FOR A FERROUS HEME 4HESE OBSERVATIONS SHOW THAT THE DRIVING FORCE FOR TYROSINE LIGATION TO THE D HEME IRON WHATEVER ITS PURPOSE IS SUBSTANTIAL AND THAT THE D HEME IS TUNED IN ITS FERROUS STATE TO BIND ANIONS )T IS NOTABLE THAT ANOTHER VARIATION OF THE STANDARD HEME STRUCTURE SIROHEME ALSO BINDS CYANIDE IN ITS FERROUS STATE AND PARTICIPATES IN ANION REDUCTION REACTIONS 3TUDY OF THE BINDING OF CYANIDE TO THE REDUCED 0 AERUGINOSA ENZYME AND A MUTANT LACKING ONE OF THE ACTIVE SITE HISTIDINES HAS EMPHASIZED THE IMPORTANCE OF THIS SIDE CHAIN RESIDUE IN CONTROLLING THE BINDING OF ANIONIC LIGANDS 3UN ET AL 4HE BINDING OF NITRITE TO THE D HEME HAS ALSO BEEN INVESTIGATED RECENTLY &OR THE ENZYME FROM 0 PANTOTROPHUS IT WAS FOUND THAT ADDITION OF NITRITE TO THE OXIDIZED ENZYME PREVENTED UNLIKE CYANIDE THE RETURN OF TYROSINE TO THE HEME IRON .ITRITE WAS THE ONLY ION WHICH WAS SIGNIlCANTLY EFFECT IN THIS RESPECT !LLEN ET AL !N APPROXIMATE DISSOCIATION CONSTANT FOR THE OXIDIZED ENZYME NITRITE COMPLEX OF M- COULD BE OBTAINED 4HIS OBSERVATION SUGGESTS THAT THE ACTIVE SITE OF CYTOCHROME CD IS TAILORED TO BIND NITRITE RATHER THAN ANY OTHER ANION /NE CAN EXPECT THAT THE REDUCED FORM OF THE ENZYME WILL HAVE EVEN HIGHER AFlNITY FOR NITRITE BUT THIS IS DIFlCULT TO MEASURE
OWING TO ITS BINDING BEING FOLLOWED BY FORMATION OF NITRIC OXIDE ! CURRENT ESTIMATE FOR THE DISSOCIATION CONSTANT FOR NITRITE FROM THE REDUCED ENZYME IS OF THE ORDER OF +- #UTRUZZOLA ET AL (OWEVER THIS MAY BE AN OVERESTIMATE SINCE CYANIDE BINDS WITH THIS AFlNITY TO THE REDUCED ENZYME AND NITRITE APPEARS TO BIND MORE STRONGLY THAN CYANIDE TO THE ACTIVE SITE &INALLY WE NOTE THAT NITRITE IS A ` ACCEPTING LIGAND .ASRI ET AL A FEATURE THAT COULD PROMOTE ITS BINDING TO AN &E)) PORPHYRIN RATHER THAN THE LESS ELECTRON RICH &E))) SPECIES 3UCH BACKBONDING CAN STRENGTHEN THE &E . BOND WHILE CONTRIBUTING TO THE WEAKENING OF AN . / BOND IN NITRITE 4HIS IS WHAT IS REQUIRED FOR FORMATION OF NITRIC OXIDE FROM NITRITE BUT WHETHER THE SPECIAL FEATURES OF THE D HEME CONTRIBUTE TO THIS EFFECT IS NOT KNOWN )T MAY IN FACT BE THE CASE THAT THE D HEME IS LESS EFFECTIVE AT SUCH BACK BONDING THAN THE NORMAL PORPHYRIN OWING TO AN INVERSION OF ENERGY LEVELS SEE BELOW !S WE HAVE ALREADY DISCUSSED CYTOCHROME CD IS ABLE TO REDUCE NOT ONLY NITRITE TO NITRIC OXIDE BUT ALSO HYDROXYLAMINE TO AMMONIA A REACTION THAT IT SHARES WITH THE .RF! TYPE OF NITRITE REDUCTASE (OWEVER UNLIKE .RF! IT IS NOT ABLE TO REDUCE NITRIC OXIDE TO HYDROXYLAMINE 2EDUCTION OF NITRIC OXIDE TO HYDROX YLAMINE REQUIRES AN ACTIVE SITE THAT CAN OPERATE AT A POTENTIAL OF AROUND n M6 WHICH IS THE CASE FOR .RF! SEE SECTION )))! WHEREAS THE NITRITE TO NITRIC OXIDE AND HYDROXYLAMINE TO AMMONIA REACTIONS ARE COMPATIBLE WITH @OPERATING POTENTIALS IN THE RANGE OF n M6 !LTHOUGH DETERMINATION OF THE POTENTIAL OF THE D HEME IN CYTOCHROME CD HAS PROVED PROBLEMATIC IT CAN SAFELY BE TAKEN TO BE APPROXI MATELY M6 CONSISTENT WITH THE ENZYME RECEIVING ELECTRONS FROM THE CYTOCHROME CCUPREDOXIN LEVEL OF THE ELECTRON TRANSFER SYSTEM 4HIS IN TURN MEANS THAT ELECTRONS DESTINED FOR CYTOCHROME CD NITRITE REDUCTASE CAN PASS THROUGH THE PROTON TRANSLOCATING CYTOCHROME BC COMPLEX EN ROUTE FROM UBIQUINOL &IG )N CONTRAST THE OPERATING POTENTIALS OF THE .RF! HEMES HAVE TO HAVE LESS POSITIVE VALUES THAN THAT FOR D WITH THE RESULT THAT ELECTRONS MUST REACH .RF! UBIQUINOL OR MENAQUINOL WITHOUT PASSING THROUGH A CYTOCHROME BC COMPLEX 4HUS THE STOICHIOMETRY OF PROTON TRANS LOCATION PER ELECTRON AND THUS OF !40 SYNTHESIS IS HIGHER WHEN NITRITE IS REDUCED BY CYTOCHROME CD OR THE COPPER TYPE NITRITE REDUCTASE RATHER THAN BY .RF! !VOIDANCE OF REDUCTION OF ./ TO AMMONIUM VIA HYDROXYLAMINE BY CYTOCHROME CD ALSO AVOIDS SHORT CIRCUITING OF THE NITROGEN CYCLE &IG $ETAILED SPECTROSCOPIC ANALYSIS OF THE D HEME
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO CENTER SUGGESTS THAT THE RELATIVE ENERGY LEVELS OF THE D ORBITALS ASSOCIATED WITH THE D HEME IRON DIFFER FROM THE USUAL PATTERN IN THAT FOR THE LOW SPIN FERRIC STATE THE ELECTRON REDUCED HEME D MACROCYCLE STABILIZES D DXZ D DYZ D DXY RELATIVE TO THE MORE USUAL DXZ DDYZ DDXY ELECTRONIC CONlGURATION #HEESMAN ET AL 4HIS @INVERTED GROUND STATE REPRESENTS A RELOCATION OF UNPAIRED SPIN DENSITY FROM ORBITALS EXTENDING ABOVE AND BELOW THE HEME PLANE TO AN ORBITAL LYING IN THAT PLANE AND THIS WILL HAVE IMPORTANT CONSEQUENCES FOR THE BINDING OF / UNSATURATED LIGANDS SUCH AS NITRIC OXIDE IN THIS CASE THE PRODUCT MOLECULE 3PECIlCALLY ./ WOULD BE LESS STRONGLY BOUND TO THE D HEME THEN TO THE USUAL B TYPE HEME 2ECENTLY SPECTROSCOPIC WORK WITH THE ENZYME FROM 0 AERUGINOSA HAS ALSO ADDRESSED THE UNUSUAL ELECTRONIC PROPERTIES OF THE HEME D RING AND HOW THESE INmUENCE THE BINDING OF NITRIC OXIDE $AS ET AL 4HUS THESE PROPERTIES MAY BE A SIGNIlCANT REASON FOR THE DEVELOPMENT OF THE SPECIALIZED D HEME !NOTHER UNCERTAINTY SURROUNDING D HEME IS ITS ROUTE OF BIOSYNTHESIS AND HOW IT IS INCORPORATED INTO THE APO ENZYME IN THE PERIPLASM 4HE POLYPEPTIDE FOR CYTOCHROME CD IS TRANSLOCATED AS AN UNFOLDED APO PROTEIN BY THE 3EC SYSTEM AND ACQUIRES ITS C TYPE CYTOCHROME CENTER IN THE PERIPLASM AS A RESULT OF THE ACTION OF THE #CM BIOGENEISIS APPARATUS (EIKKILA ET AL AND SEE ARTICLE BY 4HONY -EYER IN THIS VOLUME 4HE .IR$ PROTEIN OF UNKNOWN FUNCTION BUT BELIEVED TO BE INVOLVED IN THE ASSEMBLY OF THE D PART OF THE ENZYME HAS A SIGNAL SEQUENCE SUGGESTING IT IS CARRIED BY THE 4AT SYSTEM (EIKKILA ET AL !S LOSS OF THE 4AT SYSTEM PREVENTS LOSS OF AN ACTIVE CYTOCHROME CD IT MAY BE TENTATIVELY CONCLUDED THAT .IR$ TRANSLOCATES SOME KEY COMPONENT NEEDED FOR ENZYME ASSEMBLY IN THE PERIPLASM 1TFVEPNPOBTBFSVHJOPTB$ZUPDISPNFDE 4HE CYTOCHROME CD THAT HAS RECEIVED MOST ATTENTION OVER THE YEARS IS THAT FROM 0 AERUGINOSA $ESPITE THE FACT THAT CYTOCHROME CD IS A SPECIALIZED ENZYME IT IS REMARKABLE THAT THE STRUCTURE OF THE 0 AERUGINOSA ENZYME IS NOT IDENTICAL IN SEVERAL CRITICAL RESPECTS TO THE ENZYME FROM 0 PANTOTROPHUS MOST NOTABLY IN TERMS OF THE COORDINATION OF THE C HEME AND D HEME IRON CENTERS 4HE DIFFERENCE IS REmECTED IN THE VISIBLE ABSORPTION SPECTRA OF THE OXIDIZED ENZYMES )N THE OXIDIZED STRUCTURE OF THE 0 AERUGINOSA ENZYME ONE SEES THAT THE OVERALL STRUCTURE OF THE DIMER IS THE SAME IN THE SENSE THAT THE C TYPE CYTOCHROME DOMAIN
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO AS FOR 0 PANTOTROPHUS IS HELICAL AND SEPARATED FROM THE BLADED ` PROPELLER DOMAIN THAT BINDS THE D HEME CENTER .URIZZO ET AL (OWEVER THERE ARE INTRIGUING DIFFERENCES &IRST THE FOLD OF THE CY TOCHROME C DOMAIN OF THE OXIDIZED 0 AERUGINOSA ENZYME IS ESSENTIALLY THE SAME AS IN THE REDUCED 0 PANTOTROPHUS &IG # ENZYME AND THE HEME IRON COORDINATION IS (IS-ET 4HE IRON OF THE D HEME DOES NOT HAVE A TYROSINE LIGAND IN CONTRAST TO THE ENZYME FROM 0 PANTOTROPHUS THE SIXTH LIGAND IS HYDROXIDE BUT THIS IN TURN IS COORDINATED TO 4YR (OWEVER THE LATTER RESIDUE IS IN NO SENSE EQUIVALENT TO THE 4YR OF THE 0 PANTOTROPHUS ENZYME 4HE 4YR WHICH IS NOT AN ESSENTIAL RESIDUE #UTRUZZOLA ET AL IS PROVIDED BY THE OTHER SUBUNIT TO THAT IN WHICH IT IS POSITIONED CLOSE TO THE D HEME IRON )N OTHER WORDS THERE IS A CROSSING OVER OF THE DOMAINS ! REDUCED STATE STRUCTURE OF THE 0 AERUGINOSA ENZYME HAS ONLY BEEN OBTAINED WITH NITRIC OXIDE BOUND TO THE D HEME IRON .URIZZO ET AL !S EXPECTED THE HEME C DOMAIN IS UNALTERED BY THE REDUCTION BUT THE 4YR HAS MOVED AWAY FROM THE HEME D IRON AND CLEARLY THE HYDROXIDE LIGAND TO THE D HEME HAS DISSOCIATED SO AS TO ALLOW THE BINDING OF THE NITRIC OXIDE 4HIS FORM OF THE ENZYME WAS PREPARED BY lRST REDUCING WITH ASCORBATE AND THEN ADDING NITRITE !LTHOUGH THE OBSERVED CONFORMATIONAL CHANGES IN THE WILD TYPE 0 AERUGINOSA ENZYME ARE CLEARLY LESS PRONOUNCED THAN THOSE IN THE 0 PANTOTROPHUS ENZYME THE DRIVING FORCES FOR THE CHANGES STILL REQUIRE TO BE UNDERSTOOD )T HAD BEEN POSTULATED .URIZZO ET AL THAT REDUCTION OF THE C TYPE CYTOCHROME DO MAIN MIGHT LEAD TO CONFORMATIONAL CHANGES LEADING TO THE RELEASE OF THE HYDROXIDE FROM THE D HEME &E (OWEVER A RECENT STUDY CONTRADICTS THIS VIEW )T HAS BEEN POSSIBLE TO OBTAIN CRYSTALS OF THE 0 AERUGINOSA ENZYME IN WHICH THE C HEME IS REDUCED BUT D HEME IS OXIDIZED AND THIS CONDITION PERSISTS FOR SUFlCIENT TIME FOR THE STRUCTURE TO BE OBTAINED 4HIS SHOWS THAT HYDROXIDE IS STILL BOUND TO THE D HEME AND THAT THEREFORE REDUCTION OF THE D HEME EITHER ALONE OR IN COMBINATION OF REDUCTION OF THE C TYPE HEME IS RESPONSIBLE FOR THE CONFORMATIONAL CHANGE AT THE D HEME IRON .URIZZO ET AL 2EDUCTION OF THE D HEME IRON ALONE SEEMS LIKELY TO BE THE ESSENTIAL FACTOR NOT LEAST BECAUSE NO CHANGE IN THE C HEME DOMAIN OCCURS UPON REDUCTION AND THEREFORE IT IS DIFlCULT TO SEE HOW THERE COULD BE ANY EFFECT OF REDUCTION OF THE C HEME CENTER RELAYED TO THE D HEME 2ECENTLY PULSE RADIOLYSIS WORK WITH THE 0 AERUGINOSA ENZYME HAS SHOWN THAT IN THE ABSENCE OF NITRITE THE ELECTRON
TRANSFER FROM THE C HEME TO THE D HEME IS VERY SLOW ORDER OF SECONDS +OBAYASHI ET AL 4HIS WOULD BE CONSISTENT WITH THE IDEA THAT IT IS ARRIVAL OF AN ELECTRON AT THE D HEME THAT TRIGGERS THE CONFOR MATIONAL CHANGE 4HIS CHANGE COULD EASILY LIMIT THE ELECTRON TRANSFER RATE 4HERE IS CLEARLY A PARALLEL HERE WITH THE 0 PANTOTROPHUS ENZYME WHERE THE REDUC TION OF THE D HEME HAS BEEN ARGUED TO TRIGGER THE CONFORMATIONAL CHANGE 4HE PREPARATION OF SITE DIRECTED MUTANTS OF CY TOCHROME CD IS NOT EASY AS THE USUAL EXPRESSION SYSTEMS DO NOT MAKE THE D HEME )N THE CASE OF THE CYTOCHROME CD FROM 0 AERUGINOSA THIS PROBLEM HAS BEEN OVERCOME BY EXPRESSION OF A SEMI APO FORM OF THE ENZYME IE POSSESSING THE C BUT NOT THE D HEME IN 0SEUDOMONAS PUTIDA AND THEN ADDING BACK D HEME THAT HAD BEEN EXTRACTED FROM THE WILD TYPE ENZYME 4HE lRST MUTANT TO BE STUDIES WAS 9& WHICH SHOWED THAT LOSS OF THE HYDROXYL GROUP HAD NO EFFECT ON THE ACTIVITY OF THE ENZYME #UTRUZZOLA ET AL 4HE lRST STRUCTURE OF THE ENZYME FROM 0 PANTOTROPHUS SHOWED THAT THE ACTIVE SITE CONTAINED TWO HISTIDINES THAT APPEARED TO BE WELL PLACED TO ACT AS PROTON DONORS TO THE OXYGEN OF THE NITRITE DESTINED TO BECOME WATER -UTAGENESIS OF THE CORRESPONDING RESIDUES IN 0 AERUGINOSA HAS SHOWN THAT INDEED THAT THESE TWO RESIDUES ARE IMPORTANT FOR NITRATE REDUCTION ALTHOUGH CURIOUSLY NOT FOR THE OXIDASE REACTION ALSO CATALYSED BY THE ENZYME (OWEVER THE REPLACEMENT OF ONE OF THESE HISTIDINES BY ALANINE RESULTED IN A LARGE CONFORMATIONAL CHANGE #UTRUZZOLA ET AL "ROWN ET AL ANALOGOUS TO THAT SEEN RECENTLY IN A NEW STRUCTURAL FORM OF THE 0 PANTOTROPHUS ENZYME 3JOGREN AND (AJDU 4HESE lNDINGS REINFORCE THE NEED FOR FURTHER STUDY OF THE CONFORMATIONAL CHANGES WITHIN THE CYTOCHROME CD MOLECULE !S NOTED EARLIER STUDIES BY 8 RAY CRYSTALLOGRAPHY OF THE BINDING OF CYANIDE TO THE ENZYME ALSO IMPLICATED ONE OF THE ACTIVE SITE HISTIDINES IN THE 0 AERUGINOSA SEQUENCE AS CRITICAL FOR THE BINDING OF ANIONIC LIGANDS 3UN ET AL *OUSBNPMFDVMBS&MFDUSPO5SBOTGFSBOEUIF .FDIBOJTNPG/JUSJUF3FEVDUJPO )T HAS LONG BEEN KNOWN THAT UNDER SOME CONDITIONS AT LEAST ELECTRON TRANSFER BETWEEN THE C AND D HEMES OF THE 0 AERUGINOSA ENZYME IS SLOW IN THE ORDER OF SECONDS #UTRUZZOLA +OBAYASHI ET AL 7HAT DOES THIS MEAN )T IS NOT NECESSARILY RELATED TO THE LOSS OF THE HYDROXIDE LIGAND FROM THE D HEME IRON
BECAUSE UNDER SOME EXPERIMENTAL CONDITIONS USED THE ENZYME WAS REDUCED AT THE OUTSET WITH AZURIN PRESENT AND IT IS THE MOVEMENT OF AN ELECTRON FROM THE C TO THE D HEME THAT IS SLOW DESPITE THE PRESENCE OF A LIGAND NITRIC OXIDE ON THE D HEME 4HIS RAISES A PROBLEM BECAUSE THE NITRIC OXIDE GETS TRAPPED AND THUS WHAT SEEMS TO BE A DEAD END FERROUS D HEME NITRIC OXIDE COMPLEX IS FORMED !N EXPECTED GATING MECHANISM THAT MIGHT BE ANTICIPATED TO PREVENT THE FORMATION OF AN &E)) D HEME NITRIC OXIDE COMPLEX APPEARS NOT TO OPERATE (OWEVER IT IS CLEAR THAT UNDER SOME CONDITIONS THE RATE OF INTERHEME ELECTRON TRANSFER CAN BE MUCH FASTER ! RECENT EXAMPLE IS PROVIDED BY A STUDY IN WHICH ELECTRON TRANSFER WAS TRIGGERED BY PHOTODISSOCIATION OF CARBON MONOXIDE FROM A MIXED VALENCE FORM OF THE ENZYME 7ILSON ET AL %LECTRONS MOVED FROM THE D TO THE C HEME AT A RATE OF THOUSANDS PER SECOND 4HE DISSOCIATION OF CARBON MONOXIDE AND STUDY OF THE KINETICS OF ITS REBINDING ALSO GAVE INSIGHT INTO THE DYNAMICS OF THE D HEME POCKET !GAIN THE MEANING OF THESE OBSERVATIONS FOR THE FUNCTIONING OF THE ENZYME IS NOT CLEAR )NFORMATION ABOUT THE KINETICS OF ELECTRON TRANSFER BETWEEN THE HEMES AND OF CHEMICAL EVENTS AT THE HEMES IS NOW INCREASING FOR THE ENZYME FROM 0 PANTOTROPHUS 4HE lRST REPORTED KINETIC STUDY ON THE 0 PANTOTROPHUS ENZYME UTILIZED PULSE RADIOLYSIS +OBAYASHI ET AL )N THIS WORK EXCEEDINGLY RAPID REDUCTION OF THE C TYPE CYTOCHROME CENTER IN THE ENZYME WAS FOLLOWED BY ELECTRON TRANSFER TO THE D HEME ON THE MILLISECOND TIMESCALE 4HIS METHOD INVOLVES ONE ELECTRON PROCESSES ONLY AND SO THE C HEME ONCE REOXIDIZED BY THE D HEME REMAINED OXIDIZED )NTERPRETATION OF THIS MILLISECOND RATE OF ELECTRON TRANSFER IS NOT STRAIGHTFORWARD BECAUSE WE DO NOT KNOW THE DRIVING FORCE IE THE REDOX POTEN TIALS OF THE C AND D TYPE HEMES (OWEVER UNDER THE CONDITIONS OF THE PULSE RADIOLYSIS EXPERIMENT THE ELECTRON TRANSFER FROM THE C TYPE CENTER TO THE D HEME OCCURS ESSENTIALLY TO COMPLETION 4HIS IMPLIES THAT AT UNDER THESE CONDITIONS AT LEAST THE REDOX PO TENTIAL DIFFERENCE IS OF THE ORDER OF AT LEAST M6 ! DIFFERENCE OF M6 AND AN EDGE TO EDGE HEME DISTANCE OF ¯ WOULD SUGGEST THAT ELECTRON TRANSFER MIGHT BE FASTER THAN IS OBSERVED AS JUDGED BY CURRENT THEORIES 0AGE ET AL (OWEVER THESE THEORIES SUPPOSE THAT NO CHEMICAL BOND REARRANGEMENTS AC COMPANY THE ELECTRON TRANSFER EVENT )N THE CASE OF CYTOCHROME CD FROM 0 PANTOTROPHUS AT LEAST TWO CHEMICAL BOND REARRANGEMENTS MIGHT ACCOMPANY THE OXIDATIONREDUCTION PROCESSES 4HESE ARE THE LIGAND SWITCHING AT THE C HEME AND THE DISSOCIATION OF 4YR
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO FROM THE D HEME IRON &OR THE FOLLOWING REASONS IT IS LIKELY IN THE PULSE RADIOLYSIS EXPERIMENT THAT THE LIGANDS DID NOT CHANGE AT THE C TYPE CENTER BUT DID SO AT THE D HEME 4HE SPECTRUM IN THE 3ORET REGION INDICATIVE OF THE C TYPE CENTER OF THE ENZYME IMMEDIATELY FOLLOWING THE REDUCTION IS NOT IDENTICAL TO THAT OBTAINED WHEN THE ENZYME IS FULLY REDUCED UNDER STEADY STATEEQUILIBRIUM CONDITIONS 4HIS SUGGESTS THAT THE REDUCED C TYPE CYTOCHROME CENTER FORMED UNDER THE CONDITIONS OF THE PULSE RADIOLYSIS EXPERI MENT RETAINED THE (IS(IS COORDINATION 4HE SPEED OF THE REDUCTION BY THE SOLUTION RADI CAL GENERATED IN THE PULSE RADIOLYSIS EXPERIMENT IS ALSO CONSISTENT WITH THIS PROPOSAL CONFORMATIONAL REARRANGEMENTS ARE HIGHLY UNLIKELY ON THE MICRO SECOND TIMESCALE 4HE MIDPOINT REDOX POTENTIAL OF A (IS(IS CO ORDINATED HEME IS LIKELY TO BE LESS THAN M6 'IVEN THAT THE D HEME IS CATALYZING A REACTION WITH A MID POINT POTENTIAL OF APPROXIMATELY M6 IT CAN BE EXPECTED TO HAVE A POTENTIAL CONSIDERABLY MORE POSITIVE THAN M6 4HIS WOULD ACCOUNT FOR THE STOICHIOMETRIC TRANSFER OF ELECTRONS FROM THE C HEME TO THE D HEME UNDER THE CONDITIONS OF THE PULSE RADIOLYSIS EXPERIMENT 4HE D HEME PROBABLY LOSES ITS 4YR LIGAND UNDER THESE CONDITIONS BECAUSE IF NITRITE IS PRES ENT DURING THE PULSE RADIOLYSIS EXPERIMENT THEN ALTHOUGH THE RATE OF ELECTRON TRANSFER BETWEEN THE HEMES IS ESSENTIALLY UNALTERED THERE IS EVIDENCE FOR A CHEMICAL PROCESS TAKING PLACE AT THE D HEME CENTER +OBAYASHI ET AL 4HIS IS SUG GESTIVE THAT AT LEAST IN THE PRESENCE OF NITRITE THE ARRIVAL OF AN ELECTRON AT THE D HEME TRIGGERS THE DISSOCIATION OF THE 4YR LIGAND !LL THESE OBSERVATIONS PROMPT THE QUESTION OF WHAT VALUES ARE OBTAINED FOR THE REDOX POTENTIALS OF THE C AND D HEMES UNDER EQUILIBRIUM CONDITIONS !S EXPLAINED EARLIER THIS CANNOT BE ANSWERED OWING TO THE HYSTERETIC REDOX TITRATION %LECTRON TRANSPORT FROM THE C TO THE D HEME ON THE MSECS TIMESCALE HAS ALSO NOW BEEN REPORTED WHEN THE REDUCED 0 PANTOTROPHUS ENZYME IS REOXIDIZED BY OXYGEN NITRITE OR HYDROX YLAMINE !LLEN ET AL A 'EORGE ET AL +OPPENHOFER ET AL A
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO 5IF.FDIBOJTNPG/JUSJUF3FEVDUJPO )T IS GENERALLY ACCEPTED THAT THE REACTION CATALYSED BY CYTOCHROME CD BEGINS BY NITRITE BINDING THROUGH ITS NITROGEN ATOM TO THE FERROUS D HEME IRON &IG &OR 0 PANTOTROPHUS THIS IS SUPPORTED BY CRYS TALLOGRAPHIC DATA 7ILLIAMS ET AL AND IS CON SISTENT WITH SOME CHEMICAL CONSIDERATIONS .ASRI ET AL (OWEVER STRICTLY SPEAKING IT CANNOT BE DElNITELY EXCLUDED THAT DURING STEADY STATE TURNOVER NITRITE MIGHT BIND TO THE FERRIC STATE OF THE CD HEME 7HEN IN RAPID REACTION STUDIES NITRITE BINDS TO THE REDUCED CD HEME THERE IS EVIDENCE FOR VERY RAPID DEHYDRATION OF NITRITE PROBABLY INVOLVING DONATION OF TWO PROTONS FROM ACTIVE SITE RESIDUES TO THE OXYGEN DESTINED TO FORM WATER (ETEROLYSIS OF THE . / BOND AND OXIDATION OF THE CD HEME IRON WOULD RESULT IN A SPECIES THAT CAN FORMALLY BE REGARDED AS &E ./ OR &E ./ 5NDER SOME CONDITIONS THE LATTER SPECIES MUST BE RELATIVELY LONG LIVED BECAUSE IT CAN UNDERGO REHYDRATION IN / CONTAINING WATER TO GIVE / LA BELED NITRITE WHICH EVENTUALLY YIELDS / LABELED NITRIC OXIDE 4HE &E ./ SPECIES CAN ALSO BE TRAPPED BY NUCLEOPHILES SUCH AS AZIDE HYDROXYLAMINE OR ANILINE 4HIS SPECIES HAS NOT SO FAR BEEN DIRECTLY DETECTED BY A SPECTROSCOPIC METHOD DURING STEADY STATE CATALYSIS (OWEVER IT HAS BEEN DETECTED BY ADDING NITRIC OXIDE TO THE OXIDIZED FORM OF 0 STUTZERI CYTOCHROME CD AND MORE RECENTLY IN A STOPPED mOW KINETIC STUDY OF THE 0 PANTOTROPHUS ENZYME 'EORGE ET AL 4HE METHOD USED WAS &4)2 1UANTUM MECHANICAL CALCULATIONS ALSO SUPPORT THE IMPORTANCE OF PROTON DONATION TO NITRITE BOUND TO A FERROUS D HEME MOIETY 2ANGHINO ET AL )T IS EXPECTED THAT THE &E ./ SPECIES WILL DECOM POSE TO RELEASE PRODUCT NITRIC OXIDE AND GENERATE A FERRIC FORM OF THE D HEME IRON CENTER 4HE IRON ATOM MAY THEN BE lVE OR SIX COORDINATED 3IX COORDINATION WOULD ARISE IN THE MECHANISM PROPOSED BY &àLÚP ET AL IN WHICH THE 4YR LIGAND WAS PREDICTED TO BECOME A D HEME IRON LIGAND FOLLOWING THE DE PARTURE OF NITRIC OXIDE )N PRINCIPLE AN ALTERNATIVE MECHANISM WOULD BE THAT THE FERRIC D HEME ./ COMPLEX MIGHT BECOME REDUCED BEFORE RELEASING NITRIC OXIDE 3UCH REDUCTION MIGHT BE RELATIVELY FACILE BECAUSE AN ELECTRON CAN BE AVAILABLE ON THE C HEME WHICH IS ONLY APPROXIMATELY ¯ AWAY AND FERROUS HEME ./ COMPLEXES ARE USUALLY MUCH MORE STABILE THEN THEIR FERRIC COUNTERPARTS WHICH WOULD PROVIDE A DRIVING FORCE FOR THE ELECTRON TRANSFER FROM THE C HEME CENTER (OWEVER THE GREAT STABILITY OF FERROUS
HEME ./ COMPLEXES SUGGESTS THAT FORMATION OF SUCH A SPECIES DURING CATALYSIS WOULD LEAD TO AN INHIBITED ENZYME FROM WHICH ./ RELEASE WOULD BE VERY SLOW 4HUS WE MIGHT EXPECT CYTOCHROME CD TO HAVE AN IMPORTANT DESIGN FEATURE FOR AVOIDING THE FORMATION OF FERROUS D HEME ./ COMPLEX 4HE SLOW RATE OF INTER HEME ELECTRON TRANSFER HAS SUGGESTED SUCH A MECHANISM "UT THE OFTEN OBSERVED FORMATION OF THE FERROUS D HEME ./ COMPLEX SUGGEST THAT EXPERIMEN TAL CONDITIONS WHICH WOULD PROMOTE THE RELEASE OF ./ HAVE NOT YET BEEN IDENTIlED ! ROLE FOR TYROSINE IN DISPLACING NITRIC OXIDE NOW SEEMS LESS LIKELY AS THE 9 VARIANT ENZYME IS FULLY ACTIVE 'ORDON ET AL )N ORIGINAL STOPPED mOW RAPID REACTION STUDIES ON CYTOCHROME CD FROM 0 AERUGINOSA THE FERROUS D HEME ./ SPECIES WAS FORMED DESPITE THE ELECTRON TRANSFER FROM THE C TO THE D HEME BEING RELATIVELY SLOW RATE CONSTANT APPROXIMATELY Sn GIVEN THE RELA TIVELY SHORT DISTANCE BETWEEN THE TWO HEMES 3UCH A DISTANCE WOULD NORMALLY PREDICT MUCH FASTER ELECTRON TRANSFER RATES 4HIS RELATIVELY SLOW INTER HEME ELECTRON TRANSFER RATE HAS BEEN FREQUENTLY OBSERVED AND BEFORE THE STRUCTURE OF THE PROTEIN WAS KNOWN WAS THOUGHT TO REmECT RELATIVELY LARGE INTER HEME SEPARATION DISTANCE ANDOR RELATIVE ORIENTATION OF THE TWO HEMES THAT WAS NOT CONDUCIVE TO RAPID ELECTRON TRANSFER -AKINEN ET AL 4HE CRYSTAL STRUCTURES PROVIDE NO EVIDENCE FOR EITHER OF THESE PROPOSALS THERE IS NOTHING UNUSUAL ABOUT THE RELATIVE ORIENTATION OF THE C AND D HEME GROUPS ! SLOW ELECTRON TRANSFER RATE MUST THEREFORE BE REGARDED AS DIAGNOSTIC OF RATE LIMITING CHEMICAL STEPS AT THE D HEME CENTER &URTHER RAPID REACTION ANALYSIS OF THE 0 AERUGINOSA ENZYME HAS RECENTLY BEEN REPORTED #UTRUZZOLA ET AL -IXING OF THE FULLY REDUCED ENZYME WITH BETWEEN AND +- NITRITE WAS FOLLOWED JUDGED BY VISIBLE ABSORPTION SPECTRA BY NITRITE BINDING TO THE ACTIVE SITE ON THE MSEC TIMESCALE 4HIS WAS FOLLOWED ON THE TO SEC TIMESCALE BY OXIDATION OF THE C HEME TO GIVE ENZYME THAT HAD NITRIC OXIDE BOUND TO THE D HEME 4HIS IS RATHER SLOW IF THE ENZYME IN STEADY STATE IS TO TURNOVER MANY TIMES PER SECOND 3UBSEQUENTLY AT TIMES BETWEEN S AND MIN THE ENZYME WAS FULLY REDUCED BY THE ASCORBATE PRESENT TO GIVE ESSENTIALLY A DEAD END COMPLEX WITH NITRIC OXIDE BOUND TO THE FERROUS HEME 4HE CHANGES IN THE RATES OF THESE STEPS WERE ALSO REPORTED FOR MUTANT FORMS OF THE ENZYME THAT EACH LACKED ONE OF THE TWO ACTIVE SITE HISTIDINE LIGANDS 4HE lRST RAPID REACTION STUDY OF THE REDUCTION
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
&IG 0OSSIBLE CATALYTIC CYCLES FOR THE CYTOCHROME CD NITRITE REDUCTASES OF 0ARACOCCUS PANTOTROPHUS AND 0SEUDOMONAS AERUGINOSA
OF NITRITE BY THE ENZYME FROM 0 PANTOTROPHUS WAS RECENTLY REPORTED 'EORGE ET AL 4HE FULLY RE DUCED ENZYME NOTABLY IN THE ABSENCE OF ANY EXCESS REDUCTANT WAS MIXED WITH NITRITE AND THE TIMECOURSE OF THE SUBSEQUENT REACTION FOLLOWED BY BOTH STOPPED mOW &4)2 AND ABSORBANCE SPECTROSCOPY /N THE TENS TO HUNDREDS OF MILLISECOND TIMESCALE ONLY ONE OF THE TWO ELECTRONS ON EACH POLYPEPTIDE CHAIN WAS USED TO REDUCE NITRITE TO NITRIC OXIDE BUT THE LATTER REMAINED BOUND TO THE D HEME ! FRACTION OF THE ENZYME MOL ECULES HAD A FERRIC D HEME WITH ./ BOUND READILY DETECTABLE BY &4)2 WITH THE C HEME IN THE REDUCED STATE WHILE THE REMAINDER HAD AN OXIDIZED C TYPE CY TOCHROME CENTER AND A FERROUS D HEME ./ COMPLEX AT THE ACTIVE SITE %02 ANALYSIS CLEARLY SHOWED THAT THE OXIDIZED C HEME IRON HAD METHIONINE AND HISTIDINE COORDINATION THUS REINFORCING THE EVIDENCE THAT THE BISHISTINDINYL COORDINATION SEEN IN THE AS PREPARED ENZYME IS NOT NECESSARILY SIGNIlCANT DURING TURNOVER SEE ABOVE 4HE PUZZLING OUTCOME FROM THIS RAPID REACTION STUDY IS WHY THE ENZYME DID NOT GO INTO TURNOVER AND CATALYZE SYNTHESIS OF TWO MOLECULES OF NITRIC OXIDE WITH REGENERATION OF THE FULLY OXIDIZED ENZYME
)N SUMMARY THERE IS STILL MUCH TO UNDERSTAND ABOUT THE NITRITE REDUCTION REACTION 4HE CRYSTAL STRUCTURES HAVE SHOWN HOW NITRITE CAN BIND TO THE D HEME IRON AND PROTONS CAN BE PROVIDED TO ONE OF ITS OXYGEN ATOMS FROM TWO HISTIDINE RESIDUES (OWEVER AS YET NO RAPID REACTION STUDY HAS DETECTED THE RELEASE OF PRODUCT NITRIC OXIDE RATHER THAT THE FORMATION OF THE INHIBITORY DEAD END FERROUS D HEME ./ COMPLEX )T IS ALSO NOT CLEAR WHY THE RATE OF INTER HEME ELECTRON TRANSFER IS SO SLOW OVER ¯ WHEN NITRITE OR NITRIC OXIDE IS THE LIGAND TO THE D HEME 5NDERSTANDING THESE ISSUES AND ALSO THE ROLE OF THE mEXIBILITY OF THE C HEME DOMAIN REMAIN MAJOR CHALLENGES FOR THE FUTURE &MFDUSPO%POPSTUP$ZUPDISPNFDE/JUSJUF 3FEVDUBTF !S MENTIONED IN THE )NTRODUCTION NITRITE REDUCTASES FOR WHICH NITRIC OXIDE IS THE REACTION PRODUCT ARE LINKED TO THE UNDERLYING RESPIRATORY CHAIN AT THE LEVEL OF C TYPE CYTOCHROMES ANDOR CUPREDOXINS 4HUS THE ELECTRONS DESTINED FOR CYTOCHROME CD PASS THROUGH THE CYTOCHROME BC COMPLEX (OW THEY PASS ON FROM THIS COMPLEX TO CYTOCHROME CD IS STILL A MATTER OF
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO UNCERTAINTY )T HAD LONG BEEN ASSUMED THAT AZURIN IS AN IN VIVO ELECTRON DONOR TO CYTOCHROME CD OF 0 AERUGINOSA 4HE CONSTRUCTION OF MUTANTS OF 0 AERU GINOSA IN WHICH ONE OR BOTH OF THE GENES FOR AZURIN AND CYTOCHROME C HAVE BEEN DELETED HAS LED TO THE CONCLUSION THAT IN VIVO CYTOCHROME C IS ESSENTIAL FOR THE DONATION OF ELECTRONS TO THE NITRITE REDUCTASE AND THAT AZURIN WAS INEFFECTIVE 6IJGENBOOM ET AL 4HE DISCREPANCY BETWEEN IN VIVO AND IN VITRO OBSERVATIONS COULD BE RECONCILED IF IT IS THE FAILURE OF AZURIN TO EXCEPT ELECTRONS FROM THE CYTOCHROME BC COMPLEX OR OTHER DONOR THAT IS RESPONSIBLE FOR ITS INEFFECTIVENESS IN VIVO )N THE CASE OF THE ENZYME FROM 0 PANTOTROPHUS IT HAS BEEN PROPOSED THAT EITHER PSEUDOAZURIN OR CYTO CHROME C ARE THE ELECTRON CARRIERS BETWEEN THE CYTO CHROME BC COMPLEX AND CYTOCHROME CD 4HE ORIGINAL INDICATIONS FOR THIS VIEW WERE THAT NITRITE REDUCTION WAS UNAFFECTED IN A CYTOCHROME C DElCIENT MUTANT OF THE RELATED ORGANISM 0 DENITRIlCANS AND THAT A COPPER CHELATOR INHIBITED NITRITE REDUCTION IN THE SAME MUTANT -OIR AND &ERGUSON 4HE LATTER OBSERVATION NATURALLY IMPLICATED A CUPREDOXIN 3UBSEQUENTLY THIS VIEW HAS BEEN SUBSTANTIATED BY THE OBSERVATION THAT A MUTANT OF 0 DENITRIlCANS CONSTRUCTED TO BE DElCIENT IN BOTH PSEUDOAZURIN AND CYTOCHROME C IS UNABLE TO REDUCE NITRITE 0EARSON ET AL )N ANTICIPATION OF DEMONSTRATING THAT CYTOCHROME C AND PSEUDOAZURIN WERE THE TWO ALTERNATE ELECTRON DONORS TO CYTOCHROME CD IT HAS BEEN DISCUSSED HOW TWO PROTEINS OF SUCH DIFFERENT STRUCTURES COULD EACH INTERACT WITH CYTOCHROME CD 7ILLIAMS ET AL )T WAS PROPOSED THAT HYDROPHOBIC PATCHES STUDDED WITH POSITIVE CHARGES ON THE SURFACES OF EITHER PSEUDOAZURIN OR CYTOCHROME C RECOGNIZE A SIMILAR PATCH BUT OF COMPLEMENTARY CHARGE ON THE CYTOCHROME CD 4HIS WAS TERMED PSEUDOSPECIlCITY 4HE SUPPLY OF ELECTRONS TO THE CYTOCHROME CD OF 0 STUTZERI RAISES AN INTERESTING QUESTION 3EVERAL YEARS AGO :UMFT AND COLLEAGUES DEMONSTRATED THAT DISRUP TION OF THE NIR4 4 GENE WHICH OCCURRED IN A CLUSTER WITH OTHER GENES CODING FOR COMPONENTS OF THE CYTOCHROME CD SYSTEM PREVENTED ELECTRON TRANSFER TO CYTOCHROME CD *UNGST ET AL .IR4 IS NOW RECOGNIZED AS A MEMBER OF THE .AP# FAMILY OF PROTEINS 3ECTION ))! THAT ARE INVOLVED IN CYTOCHROME BC INDE PENDENT ELECTRON TRANSFER TO OR FROM PERIPLASMIC REDUCTASES OR DEHYDROGENASES 4HUS THE REQUIREMENT OF .IR4 FOR NITRITE REDUCTION IN 0 STUTZERI WOULD IMPLY THAT THE PATHWAY OF ELECTRON mOW IS CYTOCHROME BC COMPLEX INDEPENDENT (OWEVER IT HAS BEEN SHOWN
THAT ELECTRON TRANSFER TO CYTOCHROME CD IN 0 STUTZERI WAS AS FULLY SENSITIVE TO THE BC INHIBITORS ANTIMYCIN AND MUCIDIN AS IN 0 DENITRIlCANS +UCERA ET AL /N THIS BASIS ANOTHER ROLE FOR .IR4 IN NITRITE REDUCTASE OF 0 STUTZERI NEEDS TO BE SOUGHT $$PQQFSDPOUBJOJOH/JUSJUF3FEVDUBTFT $V/JS
4HE COPPER CONTAINING NITRITE REDUCTASE #U.IR IS A PRODUCT OF THE NIR+ + GENE AND IS WIDELY DISTRIBUTED AMONGST "ACTERIA "RAKER ET AL AND HAS ALSO BEEN FOUND IN !RCHAEA AND THE FUNGUS &USARIUM OXYSPORUM (OWEVER MOST BIOCHEMICAL STUDIES HAVE FOCUSED ON THE ENZYMES FROM !CHROMOBACTER CYCLOCLASTES AND !LCALIGENES XYLOSOXIDANS 4HE #U.)2S ARE HOMOTRIMERS WHICH BIND THREE TYPE ) AND THREE TYPE )) COPPER CENTERS &IG 4HE TYPE ) COPPER IS COORDINATED BY TWO #YS ONE -ET AND ONE (IS RESIDUE &IG AND HAS INTENSE VISIBLE ABSORPTION BANDS AT CA NM AND NM WHICH ARISE FROM #YS3 #U)) CHARGE TRANSFER TRANSITIONS #U.IRS CAN BE CLASSIlED INTO TWO SPECTRALLY DISTINCT CLASSES @"LUE AND @'REEN DEPENDING ON THE RELATIVE INTENSITIES OF THE NM AND NM BANDS WHICH MAY BE INmUENCED BY THE ANGLE OF THE (IS #U -ET BONDS $ODD ET AL 4HE TYPE )) COPPERS LIE AT THE SUBUNIT SUBUNIT INTERFACES AND ARE LIGATED BY THREE HISTIDINE RESIDUES TWO BEING PROVIDED BY ONE SUBUNIT THE THIRD FROM THE ADJACENT SUBUNIT 'OD DEN ET AL +UKIMOTO ET AL &IG 4HE TETRAHEDRAL COORDINATION SPHERE OF THE #U)) IS COMPLETED BY A WATER OR HYDROXIDE MOLECULE BUT THIS IS ABSENT IN THE REDUCED #U) SPECIES 3PECTRO SCOPIC AND STRUCTURAL STUDIES OF THE OXIDIZED ENZYME IN THE PRESENCE AND ABSENCE OF NITRITE SUGGEST THAT THE TYPE )) COPPER CENTERS ARE THE SITES OF SUBSTRATE BIND ING 4HE TYPE ) COPPER THEN PLAYS A ROLE IN ELECTRON TRANSFER FROM SOLUBLE ELECTRON DONORS TO THE ACTIVE SITE +UKIMOTO ET AL )T IS GENERALLY ASSUMED THAT THE TYPE ) COPPER CENTER OF A COPPER NITRITE REDUCTASE RECEIVES ELECTRONS FROM A CUPREDOXIN PSEUDOAZURIN OR AZURIN DEPENDING UPON THE ORGANISM ! (ISA!LA MUTATION OF THE TYPE COPPER (IS LIGAND OF THE ! XYLOSOXIDANS #U.IR LEADS TO A CA M6 INCREASE IN THE %O| WHICH PREVENTS ELECTRON TRANSFER VIA THIS ROUTE 0RUDENCIO ET AL )N VITRO STUDIES COM BINED WITH SITE SPECIlC MUTAGENESIS HAVE IMPLICATED BROADLY THE SAME POSITIVELY CHARGED DOCKING PATCH ON PSEUDOAZURIN AS DISCUSSED ABOVE FOR THE DOCKING OF THIS CLASS OF PROTEIN ON CYTOCHROME CD -URPHY ET
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
AL !T PRESENT IT IS NOT CLEAR WHETHER ANY C TYPE CYTOCHROME CAN SUBSTITUTE FOR PSEUDOAZURIN IN THIS RESPECT ALTHOUGH IT IS NOTABLE THAT THE 0SEUDOMONAS AUREOFACIENS #U.IR IS ACTIVE IN VIVO IN MUTANTS OF 0S STUTZERI DElCIENT IN THE CYTOCHROME CD AND IN THIS CASE ELECTRON TRANSFER MAY PROCEED VIA C TYPE CYTOCHROMES 'LOCKNER ET AL %LECTRON mOW TO THE COPPER NITRITE REDUCTASE CAN BE DEDUCED TO BE VIA THE CYTOCHROME BC COMPLEX AT LEAST IN ! XYLOSOXI DANS BECAUSE IT IS FULLY INHIBITED IN INTACT CELLS BY ANTIMYCIN OR MUCIDIN +UCERA ET AL 3TUDIES ON THE MECHANISM OF #U.IR HAVE BEEN GREATLY AIDED BY CRYSTAL STRUCTURES OF NITRITE BOUND FORMS 4HESE REVEAL THAT UNLIKE THE CDDL.)2S WHICH BIND SUBSTRATE AT THE D HEME VIA THE NITROGEN THE OXIDIZED TYPE )) COPPER CENTERS OF #U.)2 COORDINATE NITRITE IN A BIDENTATE FASHION VIA THE OXYGENS !D MAN ET AL -URPHY ET AL /NE OF THESE OXYGENS IS FURTHER STABILIZED BY A HYDROGEN BOND TO A CONSERVED ASPARTATE RESIDUE THAT TOGETHER WITH A CONSERVED HISTIDINE ALSO ( BONDS A WATER MOLECULE %02 %.$/2 AND 8 RAY ABSORPTION ANALYSIS HAS ALSO CONlRMED THAT NITRITE BINDS TO THE OXIDIZED CUPRIC TYPE )) CENTER (OWES ET AL 3TRANGE ET AL "Y CONTRAST STUDIES ON THE REDUCED CUPROUS TYPE )) CENTER REVEAL THAT NITRITE AND OTHER LIGANDS EG AZIDE BIND POORLY 3TRANGE ET AL 4HUS A MECHANISM IN WHICH NITRITE BINDS TO AN OXIDIZED TYPE )) COPPER CENTER DISPLACING A HYDROXIDE ION HAS BEEN PROPOSED -URPHY ET AL 4HIS CENTER IS IN TURN REDUCED BY TRANSFER OF AN ELECTRON FROM THE TYPE ) CENTER .ITRITE DECOMPOSES AFTER PROTONATION AT THE NOW REDUCED TYPE )) CENTER TO GIVE A TRANSIENT
INTERMEDIATE WHICH HAS ./ AND HYDROXIDE ION BOUND 4HE CATALYTIC CYCLE IS COMPLETED WHEN THE BOUND ./ IS RELEASED 5NLIKE THE MECHANISMS PROPOSED FOR CDDL.)2 NEITHER SUBSTRATE NOR PRODUCT IS BOUND THROUGH THE NITROGEN DURING THE CATALYTIC CYCLE (OWEVER BASED BOTH ON THE STRUCTURE OF THE NITRITE BOUND FORM OF THE ! XYLOSOXIDANS ENZYME AND AN EARLIER %8!&3 STUDY %ADY AND CO WORKERS $ODD ET AL HAVE PROPOSED AN ALTERNATIVE MECHANISM WHICH INVOLVES REORIENTATION OF THE SUBSTRATE AT THE COPPER AFTER BINDING AND THE PARTICIPATION OF A BOUND NITROSYL #U) ./ INTERMEDIATE )T IS NOTABLE THAT IN THE LIGAND FREE STATE THE REDOX POTENTIALS OF THE TYPE ) %| 5 M6 AND TYPE )) %| M6 CENTERS DO NOT FAVOR RAPID ELECTRON TRANSFER (OWEVER %.$/2 AND %8!&3 ANALYSIS OF NITRITE BOUND TYPE CENTERS SUGGEST THAT THE %| WILL INCREASE SO MAK ING INTRAMOLECULAR ELECTRON TRANSFER MORE FAVORABLE 3TRANGE ET AL 6ESELOV ET AL (ENCE IN THE PRESENCE OF AN APPROPRIATE ELECTRON DONOR &IG " THE #U.)2 WILL BE IN A MIXED VALENCE STATE 4YPE ) #U) 4YPE )) #U)) AND SUBSTRATE BINDING MAY TRIGGER ELECTRON TRANSFER 4HE IMPORTANCE OF THE CONSERVED ACTIVE SITE AS PARTATE AND HISTIDINE HAS BEEN INVESTIGATED THOUGH SITE DIRECTED MUTAGENESIS -UTATION OF !SP IN ! XYLOSOXIDANS TO EITHER 'LU OR !SN LED ENZYMES THAT RETAINED TYPE ) #U CENTER WITH WILD TYPE PROPERTIES BUT WHICH HAD ALTERED ACTIVITIES AND SPECTROSCOPIC PROPERTIES OF THE TYPE )) CENTERS 4HE $% MUTANT WAS NO LONGER CAPABLE OF BINDING NITRITE AS MONITORED BY %02 SPECTROSCOPY AND HAD VERY LOW ENZYME ACTIVITY WITH THE ARTIlCIAL ELECTRON DONORS REDUCED -ETHYL
&IG 4HE STRUCTURE COPPER NITRITE REDUCTASE AND DETAIL OF THE 4YPE ) AND 4YPE )) COPPER COORDINATION
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO VIOLOGEN AND SODIUM DITHIONITE 4HE $. ALSO HAD VERY LOW ENZYME ACTIVITIES WITH THESE DONORS (OW EVER WHEN THE PHYSIOLOGICAL ELECTRON DONOR REDUCED AZURIN ) WAS USED BOTH MUTANT PROTEINS EXHIBITED SOME RESTORATION OF ENZYME ACTIVITY WHICH SUGGESTED THAT ON FORMATION OF AN ELECTRON TRANSFER COMPLEX WITH AZURIN A CONFORMATIONAL CHANGE IN .I2 OCCURS THAT RETURNS THE CATALYTIC #U CENTER TO A FUNCTIONALLY ACTIVE STATE CAPABLE OF BINDING AND REDUCING NITRITE )T IS INTRIGUING TO NOTE THAT ENZYME CARRYING A (IS& MUTATION HAS :N RATHER THAN COPPER AT THE ACTIVE SITE AND IS INACTIVE %LLIS ET AL 4HIS THEN MAKES THE ACTIVE SITE VERY SIMILAR TO THAT OF :N DEPENDENT CARBONIC ANHYDRASE %LLIS ET AL !NOTHER SITE DIRECTED MUTAGENESIS STUDY ON THE SAME RESIDUES BUT WITH ENZYME FROM ! FAECALIS 3 HAS BEEN REPORTED RECENTLY )N THE OXIDIZED $. NITRITE SOAKED STRUCTURES NITRITE IS COORDINATED TO THE TYPE )) COPPER VIA ITS OXYGEN ATOMS IN AN ASYMMETRIC BIDENTATE MANNER HOWEVER ELEVATED " FACTORS AND WEAK ELECTRON DENSITY INDICATED THAT BOTH NITRITE AND !SN ARE LESS ORDERED THAN IN THE NATIVE ENZYME 4HIS DISORDER LIKELY RESULTS FROM THE INABILITY OF THE . DELTA ATOM OF !SN TO FORM A HYDROGEN BOND WITH THE BOUND PROTONATED NITRITE INDICATING THAT THE HYDROGEN BOND BETWEEN !SP AND NITRITE IN THE NATIVE .)2 STRUCTURE IS ESSENTIAL FOR ANCHORING NITRITE IN THE ACTIVE SITE FOR CATALYSIS )N THE OXIDIZED NITRITE SOAKED (. CRYSTAL STRUCTURE NITRITE DID NOT DIS PLACE THE LIGAND WATER AND IS INSTEAD COORDINATED IN AN ALTERNATIVE MODE VIA A SINGLE OXYGEN TO THE TYPE )) COPPER (IS IS CLEARLY ESSENTIAL IN DElNING THE NITRITE BINDING SITE DESPITE THE LACK OF DIRECT INTERACTION WITH THE SUBSTRATE IN THE NATIVE ENZYME 4HE RESULTING PENTACOORDINATE COPPER SITE IN THE (. STRUCTURE MAY ALSO SERVE AS A MODEL FOR A PROPOSED TRANSIENT INTERMEDIATE IN THE CATALYTIC MECHANISM CONSISTING OF A HYDROXYL AND NITRIC OXIDE MOLECULE COORDINATED TO THE COPPER )NTERESTINGLY AN UNUSUAL DINUCLEAR TYPE ) COPPER SITE FORMED IN THE REDUCED NITRITE SOAKED $. AND (. CRYSTAL STRUCTURES 4HIS HAD SIMI LAR STRUCTURAL PROPERTIES TO THE DINUCLEAR #U! SITES OF NITROUS OXIDE REDUCTASE SEE BELOW AND CYTOCHROME AA OXIDASE AND MAY REPRESENT AN EVOLUTIONARY LINK BETWEEN THIS CENTER AND THE MONONUCLEAR TYPE ) COPPER CENTERS "OULANGER AND -URPHY *7/JUSJD0YJEF3FEVDUBTFT /PS
"ACTERIAL .ITRIC OXIDE REDUCTASES .OR CATALYZE THE
TWO ELECTRON REDUCTION OF ./ TO ./ WHICH INVOLVES FORMATION OF AN . . BOND ./ ( E A ./ (/
4HREE SUB CLASSES OF BACTERIAL .OR CAN BE IDENTIlED THE CYTOCHROME C DEPENDENT .OR#" ENZYMES 7AT MOUGH ET AL (ENDRIKS ET AL THE QUINOL OXIDIZING .OR: ENZYMES "USCH ET AL AND THE QUINOL OXIDIZING #U! CONTAINING .OR 3UHARTI ET AL &IG -OST WORK TO DATE ON BACTERIAL .ORS HAS BEEN CARRIED OUT ON THE .OR#" CLASS OF ENZYME AND THIS WILL BE DISCUSSED lRST "5IF$ZUPDISPNFD%FQFOEFOU/PS .OR#" ENZYMES HAVE BEEN PURIlED FROM 0 DENI TRIlCANS 0SEUDOMONAS STUTZERI AND 0ARACOCCUS HALODENITRIlCANS 'IRSCH AND DE 6RIES 3AKURAI AND 3AKURAI #HEESMAN ET AL (ENDRIKS ET AL 'RONBERG ET AL BUT SEQUENCE INFORMATION IS AVAILABLE FROM GENOME ANALYSIS OF A LARGE NUMBER OF BACTERIA INCLUDING 2HODOBACTER SPHAEROIDES AND 0SEUDOMONAS AERUGINOSA (ENDRIKS ET AL 4HE .OR# SUBUNIT IS A MONO HEME C TYPE CYTOCHROME THAT POSSESSES AN . TERMINAL TRANSMEMBRANE HELIX THAT ANCHORS THE HEME DOMAIN TO THE PERIPLASMIC FACE OF THE CYTOPLASMIC MEMBRANE 4HE .OR" SUBUNIT IS A CLOSE RELATIVE OF THE CATALYTIC SUBUNIT OF RESPIRATORY HEME COPPER OXIDASES )NDEED 0 DENITRIlCANS .OR#" POSSESSES OXIDASE AS WELL AS ./ REDUCTASE ACTIVITY 4HE CATALYTIC SUBUNIT OF HEME COPPER OXIDASES EG CYTOCHROME AA OXIDASE CHARACTERISTICALLY FOLDS INTO TRANSMEMBRANE HELICES THAT BINDS TWO METAL CENTERS I A BIS HISTIDINE COOR DINATED MAGNETICALLY ISOLATED LOW SPIN HEME AND II A MAGNETICALLY COUPLED DINUCLEAR CENTER FORMED BY A HIGH SPIN HEME AND A COPPER ION #U" WHICH WILL BIND AND REDUCE DIOXYGEN 3ECONDARY STRUCTURE MODELING OF .OR" SUGGESTS A SIMILAR HELICAL ARRANGEMENT AND COFACTOR BINDING PROPERTY 3ARASTE AND #ASTRESANA VAN DER /OST ET AL 3EVEN CONSERVED HISTIDINE RESIDUES RESPONSIBLE FOR LIGATING THE THREE REDOX ACTIVE METAL CENTERS CAN BE IDENTIlED IN HELICES )) 6) 6)) AND 8 4HE KEY DIFFERENCE BETWEEN .OR" AND OTHER HEME COPPER OXIDASES IS THE COMPOSITION OF THE DINUCLEAR CENTER WHICH CONTAINS NON HEME IRON &E" RATHER THAN COPPER #U" 4HE THREE HISTIDINE RESIDUES RESPONSIBLE FOR LIGATING #U" IN OXIDASES ARE COMPLETELY CONSERVED IN .OR" WHERE THEY ARE LIKELY TO BE &E" LIGANDS )T IS WELL
ESTABLISHED THAT &E))) PREFERS DIFFERENT COORDINATION GEOMETRY EG OCTAHEDRAL AND PENTA OR HEXA DENTATE TO #U)) EG DISTORTED TETRAHEDRAL AND TETRA DENTATE 0RIMARY SEQUENCE ANALYSIS OF .OR" REVEALS THREE CONSERVED GLUTAMIC ACID RESIDUES % % % 0 DENITRIlCANS NUMBERING LOCATED IN POTENTIAL TRANSMEMBRANE HELICES THAT ARE ABSENT FROM HEME COPPER OXIDASES "UTLAND ET AL 'IVEN THAT GLUTAMATE IS FREQUENTLY FOUND IN BIOLOGICAL SYSTEMS AS A NON HEME &E LIGAND IT IS PLAUSIBLE THAT ONE OR MORE OF THESE CONSERVED GLUTAMATES IS INVOLVED IN COORDINATING &E" ALONG WITH THE THREE CONSERVED @#U" HISTIDINE LIGANDS )N ADDITION THEY MAY SERVE TO MODULATE THE PROPERTIES OF THE CATALYTIC CENTER LEADING TO DIFFERENCES IN THE REDOX POTENTIALS OF THE HEME THAT MAY IN TURN INmUENCE THE CATALYTIC CYCLE ! RECENT STUDY OF THE INTERACTION OF THE SUBSTRATE ANA LOGUE CARBON MONOXIDE #/ WITH REDUCED .OR USED RESONANCE 2AMAN SPECTROSCOPY TO STUDY THE RESULTANT &E)) #/ HEME 4HE RESULTS SUGGESTED THAT IN .OR &E)) D/ A #/ / BACKBONDING IS MINIMIZED POS SIBLY AS A RESULT OF A NEGATIVELY CHARGED HEME DISTAL POCKET 4HIS IS PERTINENT BECAUSE PRIMARY SEQUENCE ANALYSIS SHOWS THAT A REGION OF HELIX 6) OF .OR THAT IS CLOSE TO THE DINUCLEAR CENTER CONTAINS TWO CONSERVED GLUTAMATE RESIDUES THAT ARE NOT FOUND IN HEME COPPER OXIDASES ONE OF WHICH % IS ESSENTIAL FOR ACTIVITY "UTLAND ET AL %LECTRON TRANSFER BETWEEN THE REDOX CENTERS IN .OR HAS BEEN STUDIED AND SUPPORTS THE VIEW THAT THE ARRANGEMENT OF THE REDOX CENTERS IS SIMILAR TO HEME COPPER OXIDASES )NTRAMOLECULAR ELECTRON REDISTRIBU TION AFTER PHOTOLYSIS OF THE PARTIALLY REDUCED #/ BOUND ENZYME SHOWED THAT ELECTRON TRANSFER IN .OR PROCEEDS FROM HEME C VIA THE LOW SPIN HEME B TO THE HEME B &E" ACTIVE SITE (ENDRIKS ET AL 4HE ELECTRON TRANSFER RATE BETWEEN HEMES C AND B WAS CA = Sn WHILE THE RATE OF ELECTRON TRANSFER BETWEEN HEMES B AND B WAS Sn %LECTRON TRANSFER BETWEEN HEME C AND HEME B WAS COUPLED TO THE GENERATION OF AN ELECTRIC POTENTIAL CONSISTENT WITH AN ELECTRON MOVING FROM THE OUTER FACE OF THEM MEMBRANE INTO THE MEMBRANE INTERIOR 4QFDUSPTDPQJD"OBMZTJTPG/PS$# )N THE ABSENCE OF A CRYSTAL STRUCTURE MUCH OF WHAT WE HAVE RECENTLY LEARNED ABOUT THE ACTICE SITE OF .OR HAS COME FROM SPECTRO POTENTIOMETRIC STUDIES 0 PANTOTROPHUS .OR#" IS NORMALLY PURIlED IN A FORM THAT EXHITS AN ABSORPTION SHOULDER AT NM IN THE
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO FULLY OXIDIZED SPECIES 2ESONANCE 2AMAN STUDIES ON .OR#" AND MODEL COMPOUNDS HAS SUGGESTED THAT THIS @ NM BAND IS A LIGAND TO METAL CHARGE TRANSFER ,-#4 BAND OF ACTIVE SITE HIGH SPIN FERRIC HEME B WHICH HAS NO PROXIMAL IRON LIGAND AND FORMS A + OXO BRIDGE WITH THE NON HEME IRON 0INAKOULAKI ET AL &IG )T IS PERTINENT TO NOTE THAT THIS + OXO BRIDGED FORM OF .OR" DOES NOT READILY BIND EXOGNOUS LIGANDS SUCH AS #. OR PROTONS AND SO MAY NOT BE A RELEVANT STATE FOR ./ BINDING 2EDOX POTENTIOMETRY HAS REVEALED THAT THE NON HEME IRON OF THE DINUCLEAR CENTER HAS AN %MP( OF M6 WHILE THE HIGH SPIN HEME B HAS A SUR PRISINGLY LOW MIDPOINT REDOX POTENTIAL %M P( M6 'RONBERG ET AL )N THE ABSENCE OF BOUND SUBSTRATE THIS IMPOSES A LARGE THERMODYNAMIC BARRIER TO REDUCTION BY THE LOW SPIN ELECTRON TRANSFER RING HEME C %M P( M6 AND HEME B %M P( M6 4HIS RAISES THE POSSIBILITY THAT ONE ELECTRON REDUCED MIXED VALENCE ACTIVE SITE &E HEME&E IS THE RELEVANT SUBSTRATE BINDING STATE FOR THE CATALYTIC CYCLE 2EDUCTION OF THE &E" IN THE DINUCLEAR CENTER RESULTS IN A SHIFT IN THE ABSORP TION MAXIMUM OF THE FERRIC HEME B ,-#4 BAND TO LONGER WAVELENGTHS P( THE ,-#4 BAND hMAX IS NM WHILE AT P( IT IS NM &IELD ET AL -AGNETIC #IRCULAR $ICHROISM SPECTROSCOPY SUGGESTS THAT AT ALL P(S EXAMINED THE PROXIMAL LIGAND TO THE FERRIC HEME B IN THE THREE ELECTRON REDUCED FORM IS HISTIDINE BUT THAT AT P( THE DISTAL LIGAND IS HYDROXIDE WHILE AT P( WHEN THE ENZYME IS MOST ACTIVE IT IS WATER &IG )T IS ALSO NOTABLE THAT THE RECOMINANT .OR#" EXPRESSED IN % COLI PURIlES WITH A NM BAND IN THE FULLY OXIDIZED STATE SUGGESTING THAT IN THIS SPECIES THE HEME B OF DI NUCLEAR CENTER IS NOT + OXO BRIDGED BUT BINDS A HYDROXIDE LIGAND "UTLAND ET AL 4HIS ENZYME IS STILL FULLY AC TIVE ADDING TO THE VIEW THAT THE + OXO BRIDGED FORM OF .OR" MAY NOT BE NOT RELEVANT TO THE CATALYTIC CYCLE BUT IS A RESTING STATE !T PRESENT THERE IS LITTLE AGREEMENT ON THE CATALYTIC CYCLE OF .OR !N EARLY STUDY ON ./ REDUCTION IN THE CYTOCHROME AA OXIDASE SUGGESTED THE INVOLVEMENT OF A ;HEME ./ ./ #U"=INTERMEDIATE !N ANALOGOUS ;HEME ./ ./ &E"=INTERMEDIATE HAS BEEN FORWARDED FOR .OR THE TRANS MECHANISM WHICH THEN LEADS TO THE FORMATION AND LOSS OF ./ LEAVING AN OXO BRIDGED SPECIES 'IRSCH AND DE 6RIES (ENDRIKS ET AL !N ALTERNATIVE MECHANISM IS THE BINDING OF TWO ./ MOLECULES TO A COORDINATELY UNSATURATED &E" CIS &E" MECHANISM 4HIS HAS BEEN OBSERVED
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO ON THE #U" OF THE CYTOCHROME BO OXIDASE "UTLER ET AL 7ATMOUGH ET AL AND HAS THE ADVANTAGE OF PREVENTING THE FORMATION OF A FERROUS HEME NITROSYL SPECIES WHICH IN MYOGLOBINS BIND ./ VERY TIGHTLY 7ATMOUGH ET AL /NE MEANS BY WHICH THIS MAY BE AVOIDED IS THE LOW REDOX POTENTIAL OF THE HEME OF THE DINCULEAR CENTER WHICH IS AROUND M6 LOWER THAN THAT OF CYTOCHROME AA OXIDASE 'RONBERG ET AL ! lNAL MECHANISM COULD BE ONE IN WHICH ALL OF THE CHEMISTRY TAKES PLACE ON THE HIGH SPIN HEME B CIS HEME MECHANISM IN A MODIlCATION OF THE MECHANISMS PROPOSED FOR THE FUNGAL 0 NITRIC OX IDE REDUCTASE OR mAVOHEMOGLOBIN 0ARK ET AL (AUSLADEN ET AL 0INAKOULAKI ET AL (ERE ONE ./ MOLECULE WOULD BIND TO FERRIC HEME B AND INTRAMOLECULAR ELECTRON TRANSFER WOULD YIELD THE TWO ELECTRON REDUCED &E ./ ! SECOND ./ MOLECULE WOULD THEN BIND IN THE ACTIVE SITE TO FORM HYPONITRITE (/../ AND ACHIEVE . . BOND FORMATION #LEAV AGE OF THE . / BOND WOULD THEN RELEASE ./ AND A FERRIC HEME B 4HIS MECHANISM HAS THE ADVANTAGE OF ACCOMODATING A MIXED VALENCE FORM AS THE STARTING POINT )NDEED IT ALSO ALLOWS FOR A CATALYTIC MECHANISM THAT DOES NOT INCLUDE THE @LOCKED OXO BRIDGED FULLY OXIDIZED ACTIVE SITE 2ECENT ELECTROMETRIC STUDIES ON 0 DENITRIlCANS .OR#" HAVE BEGUN TO PROVIDE SOME INSIGHT INTO POS SIBLE CATALYTIC MECHANISMS (ENDRIKS ET AL $URING TURNOVER OF THE FULLY REDUCED ENZYME WITH ./ A NUMBER OF PHASES COULD BE RESOLVED 4HE lRST PHASE K = Sn WAS ELECTRICALLY SILENT AND CHARACTERIZED BY THE DISAPPEARANCE OF ABSORBANCE AT NM AND THE APPEARANCE OF A BROAD PEAK AT NM AND WAS ASSINGED TO THE FORMATION OF A FERROUS ./ ADDUCT OF HEME B 3UCH AN INTERMEDIATE WOULD BE EXPECTED IN A TRANS MECHANISM INVOLVING BOTH HEME B AND &E" OR WITH CIS HEME MECHANISM BUT NOT WITH A CIS &E" MECHANISM (OWEVER IT SHOULD BE NOTED THAT THESE STUDIES BEGIN WITH THE FULLY REDUCED ENZYME RATHER THAN A MIXED VALANCE STATE
HAVE LEAD TO THE IDENTIlCATION OF AMINO ACID RESIDUES THAT FORM THE SO CALLED $ AND + CHANNELS WHICH ARE IMPORTANT IN THE DELIVERY OF PROTONS FROM THE CYTOPLASM TO THE DINUCLEAR CENTER 6YGODINA ET AL (OWEVER THESE RESIDUES ARE ABSENT FROM .OR AND FURTHERMORE IT HAS BEEN DEMONSTRATED THAT IN CHROMATOPHORES OF 2B CAPSULATUS THERE IS NO GEN ERATION OF MEMBRANE POTENTIAL WHEN ELECTRONS WERE FED INTO THE ELECTRON TRANSFER SYSTEM AT THE LEVEL OF PERIPLASMIC C TYPE CYTOCHROME "ELL ET AL )N ADDITION IN LIPOSOMES 0 DENITRIlCANS .OR DOES NOT SHOW ANY GENERATION OF AN MEMBRANE POTENTIAL DURING STEADY STATE TURNOVER (ENDRIKS ET AL 4OGETHER THESE RESULTS RULE OUT BOTH A CYTOPLASMIC SITE OF NITRIC OXIDE REDUCTION IE WITH PROTONS TAKEN FROM THE CYTOPLASM AND A PROTON PUMPING ACTIVITY OF THE NITRIC OXIDE REDUCTASE AND IMPLY THAT PROTONS ARE TAKEN UP FROM THE SAME SIDE THE PERIPLASMIC SIDE OF THE MEMBRANE AS ELECTRONS DURING CATALYSIS )T SEEMS PROBABLE THAT THE SOME OF CONSERVED GLU TAMIC ACID RESIDUES THAT ARE PRESENT IN PERIPLASMIC LOOP REGIONS AND PUTATIVE TRANSMEMBRANE HELICES IN .OR BUT ABSENT IN OTHER #C/S PLAY A ROLE IN PROTON MOVEMENTS FORMING AN @% CHANNEL )NVESTIGATIONS INTO THIS POSSIBILITY HAVE SHOWN THAT % WHICH LIES AT PERIPLASMIC SIDE OF HELIX )6 AND % WHICH LIES IN THE MIDDLE OF HELIX 6) ARE ESSENTIAL FOR ACTIVITY BUT NOT ASSEMBLY OF 0 DENITRIlCANS .OR#" "UTLAND ET AL &URTHER SUPPORT FOR A ROUTE OF PROTON UPTAKE FORM THE PERIPLASM HAS RECENTLY COME FROM ELECTROMETRIC STUDIES ON .OR#" (ENDRIKS ET AL ,ITTLE IS CURRENTLY KNOWN ABOUT THE MECHANISM OF PROTON OUTPUT FROM CYTOCHROME AA AND CYTOCHROME BO OXIDASES AND IT IS POSSIBLE THAT THE PROTON INPUT CHANNEL USED TO MOVE PROTONS FROM THE PERIPLASM TO THE DI NUCLEAR CENTER IN .OR" EVOLVED INTO THE PROTON OUTPUT CHANNEL IN HEME COPPER OXIDASES #OMPARITIVE STUDIES ON PROTON INPUT IN .OR AND PROTON OUTPUT IN CYTOCHROME AA OXIDASE MAY PROVE INFORMATIVE IN THIS RESPECT
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#5IF5ISFF$MBTTFTPG/JUSJD0YJEF3FEVD UBTF
0ROTON UPTAKE IS INTEGRAL TO ANY OF THE MODELS FOR THE CATALYTIC CYCLE OF .OR )N THE CASE OF CYTOCHROME AA OXIDASE IN ADDITION TO PROTON UPTAKE FOR CATALYSIS THE ENZYME ALSO PUMPS FOUR PROTONS ACROSS THE MEMBRANE FOR EVERY / THAT IS REDUCED TO WATER 2ECENT HIGH RESOLUTION 8 RAY CRYSTAL STRUCTURES OF CYTOCHROME AA OXIDASE TOGETHER WITH SITE SPECIlC MUTAGENESIS
!NALYSIS OF THE AMINO ACID SEQUENCES OF .ORS IN THE CURRENT DATABASES REVEALS THAT THERE THREE .OR SUB GROUPS )N THE .OR#" SUBGROUP THE NOR# AND NOR" GENES ARE FOUND ADJACENT TO EACH OTHER 4HE .OR# MEDIATES ELECTRON TRANSFER BETWEEN THE PROTONMOTIVE CYTOCHROME BC COMPLEX PERIPLASMIC CYTOCHROME OR CUPREDOXINS AND .OR" )N THE SECOND CLASS OF
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
&IG 3PECTRAL PROPERTIES AND MODELS FOR THE DINUCLEAR ACTIVE SITE OF THE NITRIC OXIDE REDUCTASE IN DIFFERENT REDOX AND PROTONATED STATES !MINO ACID LIGENDS TO &E" ARE OMMITTED SEE TEXT FOR DETAILS 3PECTRA ARE DERIVED FROM &IELD ET AL
.ORS FOR EXAMPLE IN THE DENITRIFYER 2ALSTONIA EU TROPHA #RAMM ET AL THE NOR" GENE IS ALWAYS FOUND IN ISOLATION NOR# IS NOT PRESENT )N # THESE CASES THE .OR" PROTEIN IS PREDICTED TO HAVE AN # TERMINAL EXTENSION WHICH FOLDS TO GIVE TWO EXTRA TRANSMEMBRANE HELICES LINKED BY A GLOBULAR REGION IN THE PERIPLASM )T HAS BEEN SUGGESTED THAT THIS EXTEN SION SERVES AS A FUNCTIONAL SUBSTITUTE FOR .OR# WITH A POSSIBILITY BEING THAT IT SERVES AS A DIRECT QUINOL DEHYDROGENASE #RAMM ET AL SO THAT BY ANAL OGY TO OXIDASES THERE ARE CYTOCHROME C DEPENDENT AND QUINOL DEPENDENT SYSTEMS 4HIS WOULD LEAD TO A CYTOCHROME BC COMPLEX INDEPENDENT ROUTE FOR ELECTRON TRANSPORT AND SO THE .OR# DEPENDENT AND QUINOL DEPENDENT .ORS WOULD HAVE DIFFERENT COU PLING EFlCIENCIES 4HE Q En FOR THE .OR# DEPENDENT SYSTEM WOULD BE AND WITH .!$( AND SUCCINATE AS ELECTRON DONOR RESPECTIVELY BUT IT WOULD BE AND FOR THE QUINOL DEPENDENT .OR 4HESE DIFFERENCES IN ENERGY COUPLING THEN RESEMBLE THE SITUATION DESCRIBED EARLIER FOR THE MEMBRANE BOUND AND PERIPLASMIC NITRATE REDUCTASES !N UNUSUAL QUINOL OXIDIZING .OR HAS BEEN PURIlED
FROM THE 'RAM POSITIVE BACTERIUM "ACILLUS AZOTO FORMANS 3UHARTI ET AL 4HE ENZYME CONSISTS OF TWO SUBUNITS WITH -WS OF AND K$A !S FOR 0 DENITRIlCANS .OR THE " AZOTOFORMANS ENZYME COMPLEX BINDS ONE LOW SPIN B TYPE HEME AND ONE HIGH SPIN B HEME THAT IS MAGNETICALLY COUPLED TO A NON HEME IRON PRESUMABLY FORMING A BINUCLEAR CENTER WHERE REDUCTION OF ./ OCCURS (EME C IS ABSENT BUT UNIQUELY THE ENZYME BINDS TWO COPPER ATOMS IN THE FORM OF A #U! CENTER THAT IS CHARACTER ISTIC OF CYTOCHROME AA OXIDASES AND NITROUS OXIDE REDUCTASES 4HE ENZYME USES MENAQUINOL AS ELECTRON DONOR WHEREAS CYTOCHROME C WHICH IS THE SUBSTRATE OF OTHER ./ REDUCTASES IS NOT USED #OPPER ! AND BOTH HEMES ARE REDUCIBLE BY MENAQUINOL 4HUS THE " AZOTOFORMANS ./ REDUCTASE IS A HYBRID BETWEEN COPPER ! CONTAINING CYTOCHROME OXIDASES AND ./ REDUCTASES PRESENT IN 'RAM NEGATIVE BACTERIA )N THIS SENSE IT MAY REPRESENT AN ANCIENT PROGENITOR OF THE FAMILY OF HEME COPPER OXIDASES #OMPARISON OF THE GENE CLUSTERS FOR CYTOCHROME C DEPENDENT .OR AND Q .OR REVEAL A NUMBER OF EX TRA GENES IN THE FORMER THAT ARE ABSENT IN THE LATTER
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO 4HUS IN 0 DENITRIlCANS THE NOR#" GENES ARE PART OF A NOR#"1$%& & OPERON WHERE AS IN 3YNECHOCYSTIS AND 2 EUTROPHUS THE ONLY NOR" GENE IS PRESENT IN A SINGLE GENE OPERON #RAMM ET AL .OR1 IS PREDICTED TO BE A CYTOSOLIC PROTEIN THAT BINDS !40 .OR$ IS PREDICTED ALSO PREDICTED TO BE A CYTOSOLIC PROTEIN BUT HAS NOT FEATURES THAT GIVE A CLUE AS TO ITS FUNCTION .OR% IS PREDICTED TO BE AN INTEGRAL MEMBRANE PROTEIN WITH lVE TRANSMEMBRANE HELICES THAT HAS HOMOLOGY TO THE SUBUNIT ))) OF CYTOCHROME AA OXIDASE AND .OR& IS PREDICTED TO ENCODE A SMALL INTEGRAL MEMBRANE PROTEIN WITH TRANSMEMBRANE HE LICES DE "OER ET AL 4HE ABSENCE OF NOR1$%& IN THE GENE CLUSTERS OF ENCODING THE SINGLE SUBUNIT Q .OR SUGGEST THAT THEY PLAY A ROLE IN REGULATION AS SEMBLY OR STABILITY OF THE .OR#" COMPLEX !LTHOUGH .OR#" IS NORMALLY PURIlED AS A TWO SUBUNIT COMPLEX IT CANNOT BE EXCLUDED THAT ADDITIONAL SUBUNITS PAR TICULARLY .OR% OR .OR& ARE LOST DURING PURIlCATION )NDEED EARLY PURIlCATION PROTOCOLS OF .OR DID RESULT IN PREPARATIONS THAT HAD ADDITIONAL POLYPEPTIDES PRES ENT #ARR AND &ERGUSON 7/JUSPVT0YJEF3FEVDUBTF .ITROUS OXIDE REDUCTASE .OS CATALYSES THE REDUCTION OF NITROUS OXIDE TO DINITROGEN ./ En ( A ./ (/
)N ENVIRONMENTAL TERMS THIS IS A VERY IMPORTANT REACTION AS ./ IS A GREENHOUSE GAS THE EMISSION OF WHICH FROM BIOLOGICAL SOURCES MAKES A SIGNIlCANT CONTRIBUTION TO GLOBAL WARMING .OS IS A HOMO DIMER OF A K$A COPPER CONTAINING SUBUNIT THAT BINDS A DI NUCLEAR #U! ELECTRON ENTRY SITE SIMILAR TO THAT FOUND IN THE CYTOCHROME AA OXIDASE AND A TETRA NUCLEAR #U: CATALYTIC CENTER %ACH MONOMER IS MADE UP OF TWO DOMAINS THE @#U! DOMAIN THAT HAS A CUPREDOXIN FOLD AND THE @#UZ DOMAIN WHICH IS A SEVEN BLADED PROPELLER OF ` SHEETS 4HE DIMER ORGANIZATION IS SUCH THAT INTER DIMER ELECTRON TRANSFER MUST TAKE PLACE BETWEEN THE #U! CENTER OF ONE MONOMER AND THE #U: CENTER OF THE SECOND MONOMER "ROWN ET AL A B %LECTRON INPUT INTO #U! IS USUALLY VIA MONO HAEM C TYPE CYTOCHROMES OR CUPREDOXINS %M VALUES AROUND M6 4HE MOLECULAR NATURE OF THE #U: CENTER HAS ONLY EMERGED RECENTLY THROUGH STRUCTURAL STUDIES ON THE ENZYMES 0SEUDOMONAS NAUTICA AND 0 DENITRIlCANS
"ROWN ET AL A B 4HESE STRUCTURES REVEALED THAT THE #U: CENTER BELONGS TO A NEW TYPE OF METAL CLUSTER IN WHICH FOUR COPPER IONS ARE LIGANDED BY SEVEN HISTIDINE RESIDUES TWO HYDROXIDE MOLECULES AND MOST NOTABLY A BRIDGING INORGANIC SULlDE &IG 4HE PRESENCE OF A ;#U3= CENTER IS SUPPORTED BY ELEMENTAL ANALYSES AND RESONANCE 2AMAN SPECTROSCOPY 2ASMUSSEN ET AL )T HAS BEEN SUGGESTED THAT ./ BINDS TO THE #U: CENTER VIA A SINGLE COPPER ION WITH THE REMAINING COPPER IONS ACTING AS AN ELECTRON RESERVOIR "ROWN ET AL A B 4HIS WOULD THEN ALLOW FOR FAST ELECTRON TRANSFER AVOIDING THE FORMATION OF DEAD END PRODUCTS (OWEVER IT IS ALSO POSSIBLE THAT THE INORGANIC SULlDE IS THE BINDING SITE FOR ./ WITH THE OXYGEN ATOM THEN BEING TRANSFERRED TO FORM SULFOXIDE AND . BEING RELEASED 4HE BOUND OXYGEN WOULD THEN BE PROTONATED AND RELEASED AS WATER 4HE ;#U3= CENTER CAN IN PRINCIPLE EXIST IN lVE DIFFERENT OXIDATION STATES &IG WITH ALL BUT THE FULLY REDUCED ALL #U) STATE BEING EXPECTED TO GIVE RISE TO STRONG OPTICAL BANDS IN THE VISIBLE ABSORPTION SPECTRUM AS A CONSEQUENCE OF 3 TO #U)) CHARGE TRANSFER AND INTERVALENCE TRANSITIONS BETWEEN #U) AND #U)) SEE 2ASMUSSEN ET AL /NLY TWO STATES ;#U3= AND ;#U3= ARE EXPECTED TO GIVE RISE TO STRONG 3 %02 SIGNALS &IG 4HE RANGE OF REDOX STATES CLEARLY COULD ALLOW FOR GATING OF ELECTRON TRANSFER FROM THE #U! WHICH IS ONE ELECTRON DONOR TO FACILITATE COORDINATED ELECTRON REDUCTION OF ./ ! RECENT SPECTROSCOPIC STUDY ON A DITHIONITE REDUCED FORM OF NITROUS OXIDE REDUCTASE FROM 0 NAUTICA HAS SHOWN THAT ALTHOUGH THE #U! CENTER IS REDUCED THE #U: CENTER HAS A 3 STATE #HEN ET AL 4HIS IS ATTRIBUTED MAINLY TO ONE OF THE FOUR COPPERS IN THE #UZ CENTER KNOWN AS #U) WITH SIGNIlCANT CONTRIBUTION FROM #U)) AND THE BRIDGING SULlDE 4HE PRESUMED SUBSTRATE BINDING EDGE OF THE TETRANUCLEAR CENTER WOULD THUS BIND NITROUS OXIDE VIA THE OXIDIZED #U) AND REDUCED #U)66 "INDING MIGHT BE FOLLOWED BY ELECTRON TRANSFER FROM #U)6 DIRECTLY AND FROM #U)) VIA THE BRIDGING SULFUR ON A SUPEREXCHANGE PATHWAY #HEN ET AL )T HAS LONG BEEN RECOGNIZED THAT .OS CAN BE PURIlED IN DIFFERENT SPECTROSCOPIC FORMS WHICH ARE CHARACTERIZED BY DIFFERENT COLOURS SEE 2ASMUSSEN ET AL 4HESE COLOUR CHANGES ARISE FROM DIFFERENCES IN THE #UZ CENTER $ETAILED STUDIES ON THE 0 PANTOTROPHUS .OS HAVE RECENTLY ESTABLISHED THAT EXPOSURE OF THE ;#U3= TO AIR LEADS TO A CHANGE IN ITS REDOX PROPERTIES 2ASMUSSEN ET AL 4HUS IN ANAEROBICALLY PURIlED .OS THE #U U: CENTER EXHIBITS N
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO
&IG 4HE STRUCTURE OF THE NITROUS OXIDE REDUCTASE AND DETAIL OF ACTIVE SITE
REDOX CHARACTER WITH %O M6 BUT THE AIR EXPOSED CENTER EXHIBITS NO DETECTABLE REDOX CHANGE UNDER SIMILAR EXPERIMENTAL CONDITIONS SUGGESTING A STRONG POSITIVE SHIFT IN THE REDOX POTENTIAL 3PECTROSCOPIC PROPERTIES OF THIS @REDOX lXED CENTER TERMED #U: ARE SIMILAR BUT NOT IDENTICAL TO SPECTRA OF THE REDUCED @REDOX ACTIVE FORM OF #U: 4HE STRUCTURAL RELATIONSHIP BETWEEN THESE TWO FORMS OF THE CATALYTIC SITE IS UNCLEAR AS IS THEIR CATALYTIC RELEVANCE (OWEVER %02 AND MAGNETIC CIRCULAR DICHROISM SPECTRA SUGGEST THAT THE BASIC ;#U3= STRUCTURE IS COMMON TO BOTH AND IT IS NOTABLE THAT THE REDUCED METHYL VIOLOGEN LINKED STEADY STATE ACTIVITY OF THE ENZYMES WITH EACH FORM OF THE CENTER IS COMPARABLE !CTIVITY WAS NOT ASSESSED WITH PHYSIOLOGICAL ELECTRON DONORS AND IT SHOULD BE NOTED THAT ELECTRON TRANSPORT TO THE #U: SITE FROM ELECTRON DONORS SUCH AS CYTOCHROME C WOULD BE THERMODYNAMICALLY UNFAVOURABLE (OWEVER IT HAS BEEN SHOWN THAT CHANGES IN THE COORDINATION ENVIRONMENT OF THE #U! SITE CAN EFFECT THE SPECTRAL PROPERTIES OF THE #U: SITE #HARNOCK ET AL AND
IT IS CONCEIVABLE THAT STRUCTURAL CHANGES IN THE #U! DOMAIN MAY OCCUR ON BINDING OF A REDOX PARTNER THAT MODULATE THE PROPERTIES OF THE #U: DOMAIN AND CREATE A DRIVING FORCE FOR ELECTRON TRANSFER 4HE ASSEMBLY OF THE NOVEL ;#U3 = IS INTRIGUING AS .OS IS A PERIPLASMIC ENZYME .OS IS A SUBSTRATE FOR THE 4AT EXPORT SYSTEM THAT TRANSLOCATES FOLDED PROTEINS AND THIS SUGGESTS THAT THE TETRA NUCLEAR COPPER CENTER IS ASSEMBLED IN THE CYTOPLASM (OWEVER A RECENT STUDY SHOWED THAT A MUTANT OF 0 STUTZERI WITH A DEFECTIVE 4AT SYSTEM ACCUMULATED AN APO PROTEIN IN THE CYTOPLASM SUGGESTING THAT THE COPPER CENTERS ARE INCORPORATED IN THE PERIPLASM (EIKKILA ET AL /N THE OTHER HAND DISTURBANCE BY MUTATION OF THE #U! SITE IN THE # TERMINAL REGION OF THE PROTEIN WAS DELETERIOUS FOR PROTEIN EXPORT TO THE PERIPLASM (EIKKILA ET AL SUGGESTING THAT THE 4AT SYSTEM MAY ACCOMMODATE A SUBSTANTIALLY FOLDED APOPROTEIN IN THIS CASE RATHER THAN A HOLO PROTEIN CONTAINING A REDOX COFACTOR AS IS GENERALLY THOUGHT TO BE THE CASE FOR THIS PROTEIN TRANSPORT SYSTEM 4HE NITROUS OXIDE
$IBQUFS /0YJEFBOE/0YZBOJPO3FTQJSBUJPO REDUCTASE SUBUNIT IS ENCODED BY THE NOS: GENE 4HIS USUALLY CLUSTERS WITH A NUMBER OF OTHER .OS GENES WHICH INCLUDE NOS$&9, THAT ARE REQUIRED FOR ASSEMBLY OF THE ACTIVE SITE .OS$&9 MAY FORM AN !40 DEPENDENT COPPER TRANSPORTER AND .OS, MAY BE A COPPER BINDING CHAPERONE .OS, HAS RECENTLY BEEN CHARACTERIZED AND IS A MONOMERIC K$A PROTEIN THAT SPECIlCALLY AND STOICHIOMETRICALLY BINDS #U) WHICH IS LIGATED BY A #YS RESIDUE AND PROBABLY ONE -ET AND ONE (IS ARE THOUGHT TO SERVE AS THE OTHER LIGANDS -C'UIRL ET AL 7*$PODMVEJOH3FNBSLT )N RECENT YEARS THE IMPACT OF STRUCTURAL BIOLOGY ON THE ENZYMOLOGY OF THE . CYCLE HAS BEEN IMMENSE .OT ONLY ARE STRUCTURES OF MANY KEY ENZYMES NOW AVAILABLE BUT HIGH RESOLUTION STRUCTURES OF SYSTEMS CLOSELY RELATED TO THE MEMBRANE BOUND NITRATE RE DUCTASE AND NITRIC OXIDE REDUCTASE PROVIDE MODELS WHICH CAN FORM THE BASIS OF MECHANISTIC STUDIES #OUPLING THIS STRUCTURAL INFORMATION TO SPECTROSCOPIC AND ELECTROCHEMICAL ANALYSES IS NOW PROVIDING GREAT INSIGHT INTO THE ENZYMATIC MECHANISMS THAT UNDERLIE THE INTERCONVERSION OF NITROGEN SPECIES AND THE WAY IN WHICH THESE REACTIONS ARE COUPLED TO ENERGY CON SERVING ELECTRON TRANSPORT PATHWAYS "DLOPXMFEHNFOUT 7E WOULD LIKE TO THANK 'ARY 3AWERS "EN "ERKS .ICK 7ATMOUGH AND 4IM 2ASMUSSEN FOR HELPFUL CONTRIBUTIONS TO THIS MANUSCRIPT AND SOME OF THE lGURES AND THE ""32# FOR SUPPORTING WORK ARISING FROM OUR OWN LABORATORIES 3FGFSFODFT !DMAN %4 'ODDEN *7 AND 4URLEY 3 4HE STRUCTURE OF COP PER NITRITE REDUCTASE FROM !CHROMOBACTER CYCLOCLASTES AT lVE P( VALUES WITH ./n BOUND AND WITH TYPE )) COPPER DEPLETED * "IOL #HEM n !LEFOUNDER 02 AND &ERGUSON 3* 4HE LOCATION OF DISSIMILA TORY NITRITE REDUCTASE AND THE CONTROL OF DISSIMILATORY NITRATE REDUCTASE BY OXYGEN IN 0ARACOCCUS DENITRIlCANS "IOCHEM * n !LEFOUNDER 02 -C#ARTHY *%' AND &ERGUSON 3* 4HE BASIS FOR THE CONTROL OF NITRATE REDUCTION BY OXYGEN IN 0ARACOCCUS DENITRIlCANS &%-3 -ICROBIOL ,ETT n !LEFOUNDER 02 'REENlELD !* -C#ARTHY *%' AND &ERGUSON 3*
3ELECTION AND ORGANIZATION OF DENITRIFYING ELECTRON TRANSFER PATHWAYS IN 0ARACOCCUS DENITRIlCANS "IOCHIM "IOPHYS !CTA n !LLEN *7! #HEESMAN -2 (IGHAM #7 &ERGUSON 3* AND 7AT MOUGH .* A ! NOVEL CONFORMER OF OXIDIZED 0ARACOC CUS PANTOTROPHUS CYTOCHROME CD D OBSERVED BY FREEZE QUENCH .)2 -#$ SPECTROSCOPY "IOCHEM "IOPHYS 2ES #OMMUN n !LLEN *7! 7ATMOUGH .* AND &ERGUSON 3* B ! SWITCH IN HEME AXIAL LIGATION PREPARES 0ARACOCCUS PANTOTROPHUS CYTO CHROME CD FOR CATALYSIS .AT 3TRUCT "IOL n !LLEN *7! &ERGUSON 3* AND &ULOP 6 #YTOCHROME CD NITRITE REDUCTASE )N -ESSERSCHMIDT ! (UBER 2 0OULOS 4 AND 7IEGHARDT + EDS (ANDBOOK OF -ETALLOPROTEINSK PP n *OHN 7ILEY 3ONS .EW 9ORK !LLEN *7! (IGHAM #7 :AJICEK 23 7ATMOUGH .* AND &ER GUSON 3* ! NOVEL KINETICALLY STABLE CATALYTICALLY ACTIVE ALL FERRIC NITRITE BOUND COMPLEX OF 0ARACOCCUS PANTOTROPHUS CYTOCHROME CD "IOCHEM * n !NDERSON ,* 2ICHARDSON $* AND "UTT *. #ATALYTIC PROTEIN lLM VOLTAMMETRY FROM A RESPIRATORY NITRATE REDUCTASE PROVIDES EVIDENCE FOR COMPLEX ELECTROCHEMICAL MODULATION OF ENZYME ACTIVITY "IOCHEMISTRY n !NGOVE (! #OLE *! 2ICHARDSON $* AND "UTT *. 0ROTEIN lLM VOLTAMMETRY REVEALS DISTINCTIVE lNGERPRINTS OF NITRITE AND HYDROXYLAMINE REDUCTION BY A CYTOCHROME C NITRITE REDUCTASE * "IOL #HEM !RNOUX 0 3ABATY - !LRIC * &RANGIONI " 'UIGLIARELLI " !DRIANO *- AND 0IGNOL $ 3TRUCTURAL AND REDOX PLASTICITY IN THE HETERODIMERIC PERIPLASMIC NITRATE REDUCTASE .AT 3TRUCT "IOL n "ALLARD !, AND &ERGUSON 3* 2ESPIRATORY NITRATE REDUCTASE FROM 0ARACOCCUS DENITRIlCANS %VIDENCE FOR TWO B TYPE HAEMS IN THE GAMMA SUBUNIT AND PROPERTIES OF A WATER SOLUBLE ACTIVE ENZYME CONTAINING ALPHA AND BETA SUBUNITS %UR * "IOCHEM n "AMFORD 6! !NGOVE (# 3EWARD (% 4HOMSON !* #OLE *! "UTT *. (EMMINGS !- AND 2ICHARDSON $* 3TRUCTURE AND SPECTROSCOPY OF THE PERIPLASMIC CYTOCHROME C NITRITE REDUCTASE FROM %SCHERICHIA COLI "IOCHEMISTRY n "ARKER 0$ AND &ERGUSON 3* 3TILL A PUZZLE 7HY IS HAEM COVALENTLY ATTACHED IN C TYPE CYTOCHROMES 3TRUCT &OLD $ES 2n "ELL ,# 2ICHARDSON $* AND &ERGUSON 3* )DENTIlCATION OF NITRIC OXIDE REDUCTASE ACTIVITY IN 2HODOBACTER CAPSULATUS THE ELECTRON TRANSPORT PATHWAY CAN EITHER USE OR BYPASS BOTH CYTOCHROME C AND THE CYTOCHROME BC COMPLEX * 'EN -ICRO BIOL n "ENNETT " "ERKS "# &ERGUSON 3* 4HOMSON !* AND 2ICHARDSON $* -O6 ELECTRON PARAMAGNETIC RESONANCE SIGNALS FROM THE PERIPLASMIC NITRATE REDUCTASE OF 4HIOSPHAERA PANTOTROPHA %UR * "IOCHEM n "ENNETT " #HARNOCK *- 3EARS (* "ERKS "# 4HOMSON !* &ERGUSON 3* 'ARNER #$ AND 2ICHARDSON $* 3TRUCTURAL INVESTIGATION OF THE MOLYBDENUM SITE OF THE PERIPLASMIC NITRATE REDUCTASE FROM 4HIOSPHAERA PANTOTROPHA BY 8 RAY ABSORPTION SPECTROSCOPY "IOCHEM * n "ERKS "# 2ICHARDSON $* 2OBINSON # 2EILLY ! !PLIN 24 AND &ERGUSON 3* 0URIlCATION AND CHARACTERIZATION OF THE PERIPLASMIC NITRATE REDUCTASE FROM 4HIOSPHAERA PANTOTROPHA %UR * "IOCHEM n
"ERKS "# &ERGUSON 3* -OIR *7 AND 2ICHARDSON $* A %NZYMES AND ASSOCIATED ELECTRON TRANSPORT SYSTEMS THAT CATALYZE THE RESPIRATORY REDUCTION OF NITROGEN OXIDES AND OXYANIONS "IOCHIM "IOPHYS !CTA n "ERKS "# 0AGE -$ 2ICHARDSON $* 2EILLY ! #AVILL ! /UTEN & AND &ERGUSON 3* B 3EQUENCE ANALYSIS OF SUBUNITS OF THE MEMBRANE BOUND NITRATE REDUCTASE FROM A DENITRIFYING BACTERIUM THE INTEGRAL MEMBRANE SUBUNIT PROVIDES A PROTOTYPE FOR THE DIHAEM ELECTRON CARRYING ARM OF A REDOX LOOP -OL -ICROBIOL n "ERKS "# 2ICHARDSON $* 2EILLY ! 7ILLIS !# AND &ERGUSON 3* C 4HE NAP%$!"# GENE CLUSTER ENCODING THE PERIPLASMIC NITRATE REDUCTASE SYSTEM OF 4HIOSPHAERA PANTOTROPHA "IOCHEM * n "ERTERO -' 2OTHERY 2! 0ALAK - (OU # ,IM $ "LASCO & 7EINER *( AND 3TRYNADKA .# )NSIGHTS INTO THE RESPI RATORY ELECTRON TRANSFER PATHWAY FROM THE STRUCTURE OF NITRATE REDUCTASE ! .AT 3TRUCT "IOL n "LASCO & 'UIGLIARELLI " -AGALON ! !SSO - 'IORDANO ' AND 2OTHERY 2! 4HE COORDINATION AND FUNCTION OF THE REDOX CENTERS OF THE MEMBRANE BOUND NITRATE REDUCTASES #ELL -OL ,IFE 3CI n "OOGERD &# 6AN 6ERSEVELD (7 AND 3TOUTHAMER !( 2ESPIRATION DRIVEN PROTON TRANSLOCATION WITH NITRITE AND NITROUS OXIDE IN 0ARACOCCUS DENITRIlCANS "IOCHIM "IOPHYS !CTA n "OULANGER -* AND -URPHY -% !LTERNATE SUBSTRATE BINDING MODES TO TWO MUTANT $. AND (. FORMS OF NITRITE REDUCTASE FROM !LCALIGENES FAECALIS 3 3TRUCTURAL MODEL OF A TRANSIENT CATALYTIC INTERMEDIATE "IOCHEMISTRY n "OYINGTON *# 'LADYSHEV 6. +HANGULOV 36 3TADTMAN 4# AND 3UN 0$ #RYSTAL STRUCTURE OF FORMATE DEHYDROGENASE ( #ATALYSIS INVOLVING -O MOLYBDOPTERIN SELENOCYSTEINE AND AN &E3 CLUSTER 3CIENCE n "RAKER ' &ESEFELDT ! AND 7ITZEL +0 $EVELOPMENT OF 0#2 PRIMER SYSTEMS FOR AMPLIlCATION OF NITRITE REDUCTASE GENES NIR+ + AND NIR3 TO DETECT DENITRIFYING BACTERIA IN ENVIRONMENTAL 3 SAMPLES !PPL %NVIRON -ICROBIOL n "RETON * "ERKS "# 2EILLY ! 4HOMSON !* &ERGUSON 3* AND 2ICHARDSON $* #HARACTERIZATION OF THE PARAMAGNETIC IRON CONTAINING REDOX CENTERS OF 4HIOSPHAERA PANTOTROPHA PERIPLASMIC NITRATE REDUCTASE &%"3 ,ETT n "RIGE ! #OLE *! (AGEN 72 'UISEZ 9 AND 6AN "EEUMEN ** /VERPRODUCTION PURIlCATION AND NOVEL REDOX PROPER TIES OF THE DIHAEM CYTOCHROME C .AP" FROM (AEMOPHILUS INmUENZAE "IOCHEM * n "RIGE ! ,EYS $ -EYER 4% #USANOVICH -! AND 6AN "EEUMEN ** 4HE ¯ RESOLUTION STRUCTURE OF THE DIHEME .AP" SUBUNIT OF SOLUBLE NITRATE REDUCTASE REVEALS A NOVEL CYTOCHROME C FOLD WITH A STACKED HEME ARRANGEMENT "IOCHEMISTRY n "RONDIJK 4( &IEGEN $ 2ICHARDSON $* AND #OLE *! 2OLES OF .AP& .AP' AND .AP( SUBUNITS OF THE %SCHERICHIA COLI PERIPLASMIC NITRATE REDUCTASE IN UBIQUINOL OXIDATION -OL -ICROBIOL n "ROWN + $JINOVIC #ARUGO + (ALTIA 4 #ABRITO ) 3ARASTE - -OURA ** -OURA ) 4EGONI - AND #AMBILLAU # A 2EVISIT ING THE CATALYTIC #U: CLUSTER OF NITROUS OXIDE ./ REDUCTASE %VIDENCE OF A BRIDGING INORGANIC SULFUR * "IOL #HEM n
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3CHNEIDER & ,OWE * (UBER 2 3CHINDELIN ( +ISKER # AND +NABLEIN * #RYSTAL STRUCTURE OF DIMETHYL SULFOXIDE REDUCTASE FROM 2HODOBACTER CAPSULATUS AT ¯ RESOLUTION * -OL "IOL n 3EARS (* "ENNETT " 3PIRO 3 4HOMSON !* AND 2ICHARDSON $* )DENTIlCATION OF PERIPLASMIC NITRATE REDUCTASE -O6 %02 SIGNALS IN INTACT CELLS OF 0ARACOCCUS DENITRIlCANS "IOCHEM * n 3EARS (* 3AWERS ' "ERKS "# &ERGUSON 3* AND 2ICHARDSON $* #ONTROL OF PERIPLASMIC NITRATE REDUCTASE GENE EXPRESSION NAP%$!"# FROM # 0ARACOCCUS PANTOTROPHUS IN RESPONSE TO OXY GEN AND CARBON SUBSTRATES -ICROBIOLOGY n 3HAW !, (OCHKOEPPLER ! "ONORA 0 :ANNONI $ (ANSON '2 AND -C%WAN !' #HARACTERIZATION OF $OR# FROM 2HODOBACTER CAPSULATUS A C TYPE CYTOCHROME INVOLVED IN ELECTRON TRANSFER TO DIMETHYL SULFOXIDE REDUCTASE * "IOL #HEM n 3IMON * 'ROSS 2 %INSLE / +RONECK 0- +ROGER ! AND +LIM MEK / ! .AP#.IR4 TYPE CYTOCHROME C .RF( IS THE MEDIATOR BETWEEN THE QUINONE POOL AND THE CYTOCHROME C NITRITE REDUCTASE OF 7OLINELLA SUCCINOGENES -OL -ICROBIOL n 3IMON * 0ISA 2 3TEIN 4 %ICHLER 2 +LIMMEK / AND 'ROSS 2 4HE TETRAHEME CYTOCHROME C .RF( IS REQUIRED TO ANCHOR THE CYTOCHROME C NITRITE REDUCTASE .RF! IN THE MEMBRANE OF 7OLINELLA SUCCINOGENES %UR * "IOCHEM n 3JOGREN 4 AND (AJDU * 4HE 3TRUCTURE OF AN ALTERNATIVE FORM OF 0ARACOCCUS PANTOTROPHUS CYTOCHROME CD NITRITE REDUCTASE * "IOL #HEM n 3TRANGE 27 $ODD &% !BRAHAM :( 'ROSSMANN *' "RUSER 4 %ADY 22 3MITH "% AND (ASNAIN 33 4HE SUBSTRATE BIND ING SITE IN #U NITRITE REDUCTASE AND ITS SIMILARITY TO :N CARBONIC ANHYDRASE .AT 3TRUCT "IOL n 3TRANGE 27 -URPHY ,- $ODD &% !BRAHAM :( %ADY 22 3MITH "% AND (ASNAIN 33 3TRUCTURAL AND KINETIC EVI DENCE FOR AN ORDERED MECHANISM OF COPPER NITRITE REDUCTASE * -OL "IOL n 3UHARTI 3TRAMPRAAD -* 3CHRODER ) AND DE 6RIES 3 ! NOVEL COPPER ! CONTAINING MENAQUINOL ./ REDUCTASE FROM "ACILLUS AZOTOFORMANS "IOCHEMISTRY n 3UN 7 !RESE - "RUNORI - .URIZZO $ "ROWN + #AMBILLAU # 4EGONI - AND #UTRUZZOLA & #YANIDE BINDING TO CD NITRITE REDUCTASE FROM 0SEUDOMONAS AERUGINOSA 2OLE OF THE ACTIVE SITE (IS IN LIGAND STABILIZATION "IOCHEM "IOPHYS 2ES #OMMUN n VAN DER /OST * DE "OER !0 DE 'IER *7 :UMFT 7' 3TOUTHAMER !( AND VAN 3PANNING 2* 4HE HEME COPPER OXIDASE FAMILY CONSISTS OF THREE DISTINCT TYPES OF TERMINAL OXIDASES AND IS RELATED TO NITRIC OXIDE REDUCTASE &%-3 -ICROBIOL ,ETT n
4UVBSU+'FSHVTPOBOE%BWJE+3JDIBSETPO 6ESELOV ! /LESEN + 3IENKIEWICZ ! 3HAPLEIGH *0 3CHOLES #0 %LECTRONIC STRUCTURAL INFORMATION FROM 1 BAND %.$/2 ON THE TYPE AND TYPE COPPER LIGANDING ENVIRONMENT IN WILD TYPE AND MUTANT FORMS OF COPPER CONTAINING NITRITE REDUCTASE "IOCHEM n 6IJGENBOOM % "USCH *% AND #ANTERS '7 )N VIVO STUD IES DISPROVE AN OBLIGATORY ROLE OF AZURIN IN DENITRIlCATION IN 0SEUDOMONAS AERUGINOSA AND SHOW THAT AZU EXPRESSION IS UNDER CONTROL OF RPO33 AND !.2 -ICROBIOLOGY n 6YGODINA 46 #APITANIO . 0APA 3 AND +ONSTANTINOV !! 0ROTON PUMPING BY CYTOCHROME C OXIDASE IS COUPLED TO PEROXI DASE HALF OF ITS CATALYTIC CYCLE &%"3 ,ETT n 7ANG ( 4SENG #0 AND 'UNSALUS 20 4HE NAP& & AND NAR' NITRATE REDUCTASE OPERONS IN ' %SCHERICHIA COLI ARE DIFFER ENTIALLY EXPRESSED IN RESPONSE TO SUBMICROMOLAR CONCENTRATIONS OF NITRATE BUT NOT NITRITE * "ACTERIOL n 7ATMOUGH .* #HEESMAN -2 "UTLER #3 ,ITTLE 2( 'REENWOOD # AND 4HOMSON !* 4HE DINUCLEAR CENTER OF CYTOCHROME BO FROM %SCHERICHIA COLI * "IOENERG "IOMEMBR n 7ATMOUGH .* "UTLAND ' #HEESMAN -2 -OIR *7 2ICHARDSON $* AND 3PIRO 3 .ITRIC OXIDE IN BACTERIA 3YNTHESIS AND CONSUMPTION "IOCHIM "IOPHYS !CTA n 7ILLIAMS 0! &ULOP 6 ,EUNG 9# #HAN # -OIR *7 (OWLETT ' &ERGUSON 3* 2ADFORD 3% AND (AJDU * 0SEUDOSPECIlC DOCKING SURFACES ON ELECTRON TRANSFER PROTEINS AS ILLUSTRATED BY PSEUDOAZURIN CYTOCHROME C AND CYTOCHROME CD NITRITE REDUCTASE .AT 3TRUCT "IOL n 7ILLIAMS 0! &ULOP 6 'ARMAN %& 3AUNDERS .& &ERGUSON 3* AND (AJDU * (AEM LIGAND SWITCHING DURING CATALYSIS IN CRYSTALS OF A NITROGEN CYCLE ENZYME .ATURE n 7ILSON %+ "ELLELLI ! ,IBERTI 3 !RESE - 'RASSO 3 #UTRUZZOLA & "RUNORI - AND "RZEZINSKI 0 )NTERNAL ELECTRON TRANSFER AND STRUCTURAL DYNAMICS OF CD NITRITE REDUCTASE REVEALED BY LASER #/ PHOTODISSOCIATION "IOCHEMISTRY n 7OOD .* !LIZADEH 4 "ENNETT 3 0EARCE * &ERGUSON 3* 2ICHARDSON $* AND -OIR *7 -AXIMAL EXPRESSION OF MEMBRANE BOUND NITRATE REDUCTASE IN 0ARACOCCUS IS INDUCED BY NITRATE VIA A THIRD &.2 LIKE REGULATOR NAMED .AR2 * "ACTERIOL n 7OOD .* !LIZADEH 4 2ICHARDSON $* &ERGUSON 3* AND -OIR *7 4WO DOMAINS OF A DUAL FUNCTION .AR+ PROTEIN ARE REQUIRED FOR NITRATE UPTAKE THE lRST STEP OF DENITRIlCATION IN 0ARACOCCUS PANTOTROPHUS -OL -ICROBIOL n :AFRA / 2AMIREZ 3 #ASTAN 0 -ORENO 2 #AVA & 6ALLES # #ARO % AND "ERENGUER * ! CYTOCHROME C ENCODED BY THE NAR OPERON IS REQUIRED FOR THE SYNTHESIS OF ACTIVE RESPIRA TORY NITRATE REDUCTASE IN 4HERMUS THERMOPHILUS &%"3 ,ETT n
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4VNNBSZ 0OSSIBLE MECHANISMS BY WHICH ENERGY MAYBE CONSERVED FROM THE AEROBIC OXIDATION OF FERROUS IRON BY 7KLREDF FLOOXVIHUURR[LGDQV ARE DISCUSSED ! RATIONALE BASED ON A CONSIDERATION OF THE THERMODYNAMIC CONSTRAINTS OF THE SYSTEM AND AN ANALYSIS OF SEQUENCE INFORMATION OF THE RESPIRATORY CYTOCHROME OXIDASE IS APPLIED ,IMITATIONS ARE IMPOSED ON POSSIBLE MODELS AND IT IS SUGGESTED THAT 7EIHUURR[LGDQV CYTOCHROME OXIDASE MAY BE UNIQUELY PARTIALLY DECOUPLED FROM PROTON PUMPING
**OUSPEVDUJPO 4HE THIOBACILLI ARE PLACED IN THE 3URWHREDFWHULD DIVISION CLOSE TO THE JUNCTION BETWEEN THE ` AND _ SUB DIVISIONS ON THE BASIS OF THE SEQUENCE OF THEIR U 32.! 2AWLINGS 7KLREDFLOOXV 7E IHU URR[LGDQV CAN GROW SOLELY ON THE AEROBIC OXIDATION OF FERROUS IRON ;&E)) = 4HIS OCCURS IN A MEDIUM OF DILUTE SULFURIC ACID DOWN TO P( FOR REVIEWS SEE )NGLEDEW %HLRICH ET AL "LAKE ET AL 2AWLINGS 4HE REDOX POTENTIALS OF THE RESPIRATORY SUBSTRATE AND OXIDANT ARE BOTH HIGH AND THE REDOX POTENTIAL DIFFERENCE BETWEEN THEM RATHER SMALL 4HE BACTERIUM CAN GROW IN THE CHEMOSTAT AT A
&E)) ))) ELECTRODE POTENTIAL %H AS HIGH AS M6 AT P( !T P( THE MID POINT POTENTIAL %M FOR THE /(/ COUPLE IS M6 GIVING A REDOX POTENTIAL DIFFERENCE 6% OF APPROXIMATELY M6 ELECTRODE POTENTIAL VALUES ARE GIVEN WITH RESPECT TO THE HYDROGEN HALF CELL 4HE BACTERIUM HAS TO CONSERVE ENERGY AVAILABLE FROM THIS SMALL 6% TO LIVE INCLUDING lXING ITS OWN #/ VIA A #ALVIN #YCLE AND IF REQUIRED ITS OWN . TO lX ONE #/ VIA A #ALVIN #YCLE REQUIRES !40 AND .!$0( 4HIS THUS REPRESENTS ONE OF THE NARROWEST THERMODYNAMIC MARGINS CAPABLE OF SUSTAINING LIFE 4HESE PROCESSES ARE SCHEMATICALLY OUTLINED IN &IG WHERE THE DOWNHILL mOW OF REDUC ING EQUIVALENTS FROM &E)) TO / IS SHOWN COUPLED
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)LJ 2EDOX AND METABOLIC PROlLE OF ENERGY CONSERVATION DURING AEROBIC RESPIRATION ON FERROUS IRON 4HE DOMINANT ARROW INDICATES THE mOW OF REDUCING EQUIVALENTS FROM &E)) TO OXYGEN AND THEIR APPROXIMATE RESPECTIVE REDOX POTENTIALS 4HIS PROCESS GENERATES A TRANSMEMBRANE 6P WHICH IS GIVEN ITS OWN VOLTAGE SCALE 4HE 6P IS USED EITHER TO MAKE !40 OR TO DRIVE REVERSED ELECTRON TRANSFER UPHILL FROM &E)) TO .!$0 "OTH !40 AND .!$0( ARE USED TO lX #/ VIA A #ALVIN #YCLE
TO THE GENERATION OF A TRANSMEMBRANE 6P THIS 6P IS USED TO DRIVE !40 SYNTHESIS AND UPHILL ELECTRON TRANSFER FROM &E)) TO .!$0 ! REDOX POTENTIAL SCALE IS SHOWN ON THE LEFT A SCALE IS ALSO SHOWN FOR THE 6P 4HE LATTER SCALE IS ALSO IN VOLTS THE MAXIMAL VALUE SHOWN REPRESENTS THE MAXIMUM MEASURED 6P IN 7EIHUURR[LGDQV DURING &E)) OXIDATION #OX ET AL 4HESE SCALES SERVE TO ILLUSTRATE THE THER MODYNAMIC CONSTRAINTS UNDER WHICH 7EIHUURR[LGDQV GROWS ON &E)) OXIDATION 4HE 6% IS NOT MUCH GREATER THAN 6P THIS HAS IMPLICATIONS FOR THE STOICHIOMETRY WHICH LINKS THE PROCESSES !BBREVIATIONS 6s n THE MEMBRANE POTENTIAL 6%BULK n THE ELECTRODE POTENTIAL DIFFERENCE BETWEEN THE DONOR AND ACCEPTOR COUPLES IN THE BULK PHASE 6P n THE PROTON MOTIVE FORCE 6P( n THE P( DIFFERENCE BETWEEN THE INSIDE AND THE OUTSIDE OF THE CELL #/8 n THE CYTOCHROME C OXIDASE #YT n CYTOCHROME %H&E n THE ELECTRODE POTENTIAL OF THE &E)) ))) COUPLE P( RELATIVE TO THE HYDROGEN HALF CELL %H/P(X n THE ELECTRODE POTENTIAL OF THE /(/ COUPLE AT THE SPECIlED P( RELATIVE TO THE HYDROGEN HALF CELL %M n THE MIDPOINT POTENTIAL OF THE SPECIlED COUPLE AT THE SPECIlED P( RELATIVE TO THE HYDROGEN HALF CELL & n THE &ARADAY 2 n THE 'AS CONSTANT 37)33 02/4 n PROTEIN SEQUENCE DATABASE 4 n THE ABSOLUTE TEMPERATURE 4R%-", n GENE SEQUENCE DATABASE
!S THERE IS ONLY A SMALL 6% AVAILABLE FROM AEROBIC FERROUS IRON OXIDATION THE MECHANISM OF ENERGY CONSERVATION CAN APPEAR ENIGMATIC !NY PROPOSAL MUST ACCOUNT FOR THE GENERATION OF A PROTON MOTIVE FORCE 6P 4HIS 6P IS SUBSEQUENTLY USED TO DRIVE UPHILL ELECTRON TRANSFER TO .!$0 !40 SYNTHESIS AND ALL THE OTHER ENERGY REQUIRING PROCESSES OF THE CELL 4WENTY lVE YEARS AGO A SIMPLE SCHEME WAS PROPOSED TO EXPLAIN THIS PHENOMENON )NGLEDEW ET AL 4HIS PROPOSAL IS OUTLINED IN &IG ! AND IS REVISITED IN THIS PAPER )N THE MODEL &E)) IS OXIDIZED ON THE EXTERNAL SURFACE OF THE CELL THERE IS COMPELLING LOGIC AND STRONG EVIDENCE FOR THIS LOCATION )NGLEDEW THE REDUCING EQUIVALENTS PASS TO THE CYTOCHROME F OXIDASE #/8 FOR OXYGEN REDUCTION AND THE PROTONS FOR OXYGEN REDUCTION COME FROM THE CYTOPLASM 4HE SIDE OF ACCESS AND EGRESS OF THE / AND (/ DOES NOT NEED TO BE DElNED AS IT DOES NOT CONTRIBUTE TO THE TRANSMEMBRANE CHARGE SEPARATION )T IS THE USE OF CYTOPLASMIC PROTONS FOR / REDUCTION THAT WAS CONTROVERSIAL AS IS DISCUSSED BELOW 4HE SEPARATION OF THE TWO ELECTROCHEMICAL HALF REAC TIONS DElNED BY THE SIDEDNESS OF &E)) OXIDATION AND PROTON CONSUMPTION CAUSES THE GENERATION OF A TRANSMEMBRANE 6P -ISUNDERSTANDING AROSE BECAUSE THE 6% BETWEEN THE %H OF THE &E)) ))) COUPLE IN THE CHEMOSTAT M6 AND THE %H OF THE /(/ COUPLE IN THE CYTOSOL NOMINALLY THE %M AT P( M6 IS ONLY M6 WHEREAS IF THE / REDUC TION WAS LOCATED IN EQUILIBRIUM WITH THE EXTERNAL PHASE %M AT P( OF THE /(/ COUPLE M6 THE 6% IS MUCH LARGER M6 AND THEREFORE ABLE TO PUMP PROTONS AGAINST THE 6P 3O WHY WOULD THE ORGANISM REDUCE / IN THE CYTOPLASM WHERE THE 6% IS LESS 4HIS ARGUMENT OVERLOOKS THE FACT THAT IN THE FORMER CASE THE GENERATION OF A 6P IS INCLUDED IN THE THERMODYNAMICS IE THE SMALL 6% BEEN THE TWO COUPLES IS THE RESIDUAL 6% AFTER THE GENERATION OF A 6P )N FACT IF YOU WRITE THE SCHEME WITH THE SCALAR PROTONS FOR / REDUCTION COMING FROM THE PERIPLASM AND THE PROCESS LINKED TO THE PUMPING OF VECTORIAL PROTONS ACROSS THE MEMBRANE AS IN DIAGRAM &IG " THE NET RESULT IS THERMODYNAMICALLY IDENTICAL IN SCHEME ! ( ARE LOST FROM INSIDE AND En ARE LOST FROM OUTSIDE IN SCHEME " PRECISELY THE SAME CHANGES OCCUR 4HEREFORE THE TWO SCHEMES &IG ! AND " MUST BE THERMODYNAMICALLY EQUIVALENT 4HE GOVERNING THERMODYNAMICS ARE THE THERMODYNAMICS
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OF THE BULK PHASE AND THERE ARE A NUMBER OF WAYS OF PRESENTING THIS MORE FORMALLY /NE WAY IS TO DElNE 6%%4 AS THE REDOX POTENTIAL DIFFERENCE BETWEEN THE &E)) ))) COUPLE AND THE /(/ COUPLE %4 STANDS FOR ELECTRON TRANSFER )NGLEDEW )F THE PROTONS FOR / REDUCTION COME FROM THE INSIDE 6%%4 %&E n %/P( FOR THE SAKE OF ARGUMENT PUTTING P(IN AT AND % BULK %&E n %/P( 6%BULKK 6%%4 n %H/P(OUT n %H/P(IN
IE THE OVERALL REDOX POTENTIAL DIFFERENCE IS EQUIVA LENT TO THE REDOX POTENTIAL DIFFERENCE OF THE ELECTRON TRANSFER REACTION PLUS THE REDOX POTENTIAL DIFFERENCE @LOST THROUGH LOCATING THE / REACTION IN THE CYTOSOL @LOST DUE TO THE P( DEPENDENCE OF THAT COUPLE 4HIS GIVES 6% BULK %H%4 n %H/P( n 24& P(IN %H/P( n 24& P(OUT AS 6%%4 %H&E n %H/P(IN %Q REDUCES TO 6%BULK %H&E n %H/P( n 24& 6P(
5NDER NORMAL RESPIRING CONDITIONS THE EXTERNAL P( IS AND THE INTERNAL P( IS CLOSE TO NOMINAL THEREFORE THE 6P( IS CLOSE TO n #OX ET AL 4HE POTENTIAL OF THE /(/ COUPLE IN EACH PHASE IS RELATED TO THE P( AND THE ELECTRODE POTENTIAL AT P( )LJ 3CHEMES FOR ENERGY COUPLING ! 4HE COUPLING SCHEME PROPOSED BY )NGLEDEW ET AL 4HE EXTERNAL P( IS AND THE INTERNAL P( IS CLOSE TO NEUTRALITY &OUR ELECTRONS NEGATIVE CHARGES ARE MOVED FROM OUTSIDE TO INSIDE THIS RESULTS IN THE GENERATION OF FOUR POSITIVE CHARGES OUTSIDE AND THE LOSS OF FOUR POSITIVE CHARGES INSIDE 4HE LATTER ARE DUE TO THE REMOVAL OF THE FOUR PROTONS FOR / REDUCTION 4HIS GENERATES A 6P " !N ALTERNATIVE TO SCHEME ! WITH THE SAME COUPLING STOICHIOMETRY BUT WITH THE PROTONS FOR / REDUCTION ENTERING FROM THE OUTSIDE AND VECTORIAL PROTONS BEING PUMPED ACROSS THE MEMBRANE # 4HE NORMAL COUPLING SCHEME FOR CYTOCHROME OXIDASES WHEREBY IN ADDITION TO THE SEPARATION OF THE ELECTROCHEMICAL HALF REACTIONS 3CHEME ! ADDITIONAL PROTONS ARE TRANSLOCATED )F THIS WERE A MITOCHONDRIAL SYSTEM THE &E)) COULD BE REPRESENTING FERROCYANIDE OXIDATION OR FERRO CYTOCHROME F OXIDATION 4HE TWO PROTON ENTRY POINTS IN THE DIAGRAM ARE NOT MEANT TO INDICATE NECESSARILY SEPARATE CHAN NELS $ ! HYBRID PARTIAL COUPLING SCHEME IN WHICH WITH THE LOSS OF THE + CHANNEL SOME PROTONS MAY ACCESS THE / REDUCTION SITE FROM THE OUTSIDE SURFACE 4HIS GIVES A STOICHIOMETRY BETWEEN THAT OF SCHEME ! OR " AND # WHICH MAY BE ACCOMMODATED BY THE THERMODYNAMICS
%H/P( %H/P( n 24& § FOR P( AND %H/P( %H/P( n 24& § FOR P( 3UBSTITUTING FOR %H/P( AND 6P( INTO %Q 6%BULK %H&E n %H/P( n24& n 24 & n 7HICH REARRANGES TO 6%BULK %H&E n %H/P( n 24&
&ROM THE RELATIONSHIP BETWEEN P( AND ELECTRODE POTENTIAL FOR THE /(/ COUPLE THE LAST TERM CAN BECOME %H/P( THEREFORE
8+PIO*OHMFEFX
6%BULK %H&E n %H/P(
NOT SURPRISINGLY THE THERMODYNAMICS OF THE BULK PHASE &E)) OXIDATION HAS BEEN SHOWN TO GENERATE A 6P )NGLEDEW ET AL 4HE 6P WAS ESTIMATED TO BE AT M6 IN COUPLED RESPIRING CELLS IT WAS MAINLY A 6P( UNITS THE INTERNAL P( MEASURED AT P( THE 6s BEING SMALL n M6 4HE 6% UNDER WHICH THIS 6P WAS MEASURED IS NOT PRECISELY DElNED THE %H OF THE /(/ COUPLE IS 6 BUT THE POTENTIAL OF THE &E)) &E))) COUPLE DURING THE EXPERIMENT CAN ONLY BE CRUDELY ASSESSED ***5IF3FTQJSBUPSZ$IBJO$PNQPOFOUT 4HE COMPONENTS OF THE RESPIRATORY CHAIN OF &E)) GROWN 4B FERROOXIDANS HAVE BEEN QUITE WELL CHARAC TERIZED OVER THE YEARS ALTHOUGH SOME OF THE lNE DETAILS OF &E)) OXIDATION AND ELECTRON TRANSFER PATHWAYS IN THE PERIPLASM ARE NOT FULLY RESOLVED )NGLEDEW AND #OBLEY "ARR ET AL 2AWLINGS 9ARZABAL ET AL 2EDOX TITRATIONS MONITORED BY OPTICAL AND %02 SPECTROSCOPY REVEALED MULTIPLE C TYPE CYTOCHROMES #YT TWO A TYPE CYTOCHROMES ONE HIGH SPIN ONE LOW SPIN TWO B TYPE HEMES AND COPPER CENTERS )NGLEDEW AND #OBLEY ! NUM BER OF THE RESPIRATORY CHAIN COMPONENTS NOW APPEAR IN THE PROTEIN AND GENE SEQUENCE DATABASES (ALL ET AL !PPIA !YME ET AL 4HESE INCLUDE THE ( TRANSLOCATING !40ASE AN IRON OXIDASE AND RUSTICYANIN IN THE PROTEIN DATABASE ;3WISS 0ROT = AND IN THE GENE SEQUENCE DATABASE ;4R%-", = A PUTATIVE FOUR SUBUNIT #/8 ALL THE COMPONENTS OF A CYTOCHROME BC 2EISKE COMPLEX A HIGH POTENTIAL IRON PROTEIN (I0)0 PLUS CYTOCHROMES C ! RECENT REPORT ON SEQUENCES MINED IN THE 4B FERROOXIDANS !4## GENOME lNDS GENES ENCODING FOR PUTATIVE CYTOCHROMES C !T LEAST CYTOCHROMES C WERE DIFFERENTIATED ON GELS OF THESE ONE WAS SPECIlC FOR CELLS GROWN ON SULFUR AND THREE SPECIlC FOR CELLS GROWN ON &E)) !PPIA !YME ET AL 9ARZABAL ET AL ! DIHEME C TYPE CYTOCHROME HAS ALSO BEEN ISOLATED 'IUDICI /RTICONI ET AL 4HIS PLETHORA OF REDOX COMPONENTS IS RATIONAL IZED WHEN THEY ARE SORTED INTO RESPIRATORY CHAIN COMPLEXES THE CYTOCHROME OXIDASE CONTAINS TWO D TYPE CYTOCHROMES AND COPPER CENTERS )NGLEDEW AND #OBLEY !PPIA !YME ET AL ;4R%MBL = THE EF COMPLEX CONTAINS THE 2EISKE IRON
SULFUR CENTER AND CYTOCHROMES E AND F )NGLEDEW AND #OBLEY %LBEHTI ET AL ;4R%MBL = ,ITTLE IS KNOWN OF THE .!$( DEHYDROGENASE COMPLEX ,OCATED IN THE PERIPLASM ARE A NUMBER OF INDIVIDUAL ENZYMES AN &E)) #YT F OXIDOREDUC TASE +USANO ET AL #YT F !PPIA !YME ET AL THE COPPER PROTEIN RUSTICYANIN #OX AND "OXER AND SOME ADDITIONAL F TYPE CYTOCHROMES )NGLEDEW AND #OBLEY 'IUDICI /RTICONI ET AL ;4R%MBL = 9ARZABAL ET AL 3OME OF THESE COMPONENTS WILL FACILITATE THE TRANSFER OF REDUCING EQUIVALENTS FROM &E)) TO THE CYTOCHROME OXIDASE 4HE CYTOCHROME OXIDASE USES THE REDUCING EQUIVALENTS TO REDUCE OXYGEN TO WATER CONSERVING THE ENERGY AVAILABLE IN A 6P 4HE GENE SEQUENCE OF A PUTATIVE #/8 IS KNOWN AND IT IS CLEARLY A MEMBER OF THE #/8 SUPERFAMILY 4HIS FAMILY CONTAINS A BINUCLEAR COPPER &E)) HEME SITE AT WHICH OXYGEN REDUCTION OCCURS AND WHICH ALSO FUNCTIONS TO GENERATE 6P DUR ING TURNOVER 4HE OXIDASES OF THIS SUPERFAMILY PUMP PROTONS FROM INSIDE TO OUTSIDE AND REMOVE PROTONS FROM THE INTERNAL PHASE FOR THE REDUCTION OF OXYGEN TO WATER DURING TURNOVER THIS IS ILLUSTRATED IN &IG # 4HE 7EIHUURR[LGDQV ENZYME HAS SOME DIFFERENCES FROM OTHER MEMBERS OF THE SUPERFAMILY THE h MAX OF ITS REDUCED _ BAND IS AT NM INSTEAD OF THE NORMAL NM THIS IS WHY IT WAS ORIGINALLY REFERRED TO AS A #YT D A CLASS OF HEME WHICH HAS BEEN SHOWN TO BE EITHER AN ANOMALOUS DD TYPE OR A HIGH SPIN E TYPE HEME !LSO GIVEN THE THERMODYNAMIC CONSTRAINTS OF &E)) OXIDATION BY 7EIHUURR[LGDQV ) WILL POSE THE QUESTION @IS THE 7EIHUURR[LGDQV ENZYME DIFFER ENT FROM THE REST OF THE SUPERFAMILY WHEN IT COMES TO ( PUMPING "ECAUSE THERE MAY NOT BE ENOUGH REDOX POTENTIAL ENERGY AVAILABLE TO DRIVE THE ADDITIONAL PROTONS ACROSS THE MEMBRANE AGAINST THE 6P "5IJPCBDJMMVTGFSSPPYJEBOT$ZUPDISPNF 0YJEBTF $PY
4HE 7EIHUURR[LGDQV CYTOCHROME OXIDASE IS THE ONLY MEMBRANE BOUND COMPONENT OF THE PUTATIVE &E)) OXIDATION PATHWAY AND THE ONLY SECTION THAT CAN THEREFORE BE DIRECTLY COUPLED TO ENERGY CONSERVATION 4HE ENZYME IS A MEMBER OF THE WELL CHARACTERIZED #/8 SUPERFAMILY BUT THE 7EIHUURR[LGDQV ENZYME IS UNUSUAL IN A NUMBER OF RESPECTS ITS h MAX IN THE _ BAND OF THE REDUCED ENZYME IS AT NM INSTEAD OF AT NM IT COPES WITH AN ENVIRONMENT WHERE ITS EXTERNAL SURFACE IS IN AN ACIDIC MEDIUM AND ITS INTERNAL SURFACE IS IN A NEUTRAL MEDIUM IT IS UNIQUELY
$IBQUFS 5IF#JPFOFSHFUJDTPG'F ** 0YJEBUJPO THERMODYNAMICALLY CONSTRAINED AND IT HAS SOME INTERESTING AMINO ACID SEQUENCE DIFFERENCES FROM THE REST OF ITS SUPERFAMILY 4HE THERMODYNAMIC CON SIDERATIONS RAISE THE QUESTION IS THE 7EIHUURR[LGDQV CYTOCHROME OXIDASE AN @ORTHODOX MEMBER OF ITS SUPERFAMILY 4HIS QUESTION IS IMPORTANT BECAUSE IF THE ENZYME FUNCTIONS THE SAME AS OTHER MEMBERS OF ITS GROUP THE MECHANISM OF ENERGY CONSERVATION MUST BE AS SHOWN IN &IG # IE TRANSLOCATING TWICE THE NUMBER OF PROTONS AGAINST THE 6P THAN IN MODELS ! AND " )S THIS THERMODYNAMICALLY FEASIBLE 4HE 6P IN 7EIHUURR[LGDQV CELLS RESPIRING ON &E)) AT P( HAS BEEN MEASURED AT M6 #OX ET AL THUS TO MOVE THE @EQUIVALENT OF ( ACROSS THE MEMBRANE PER ELECTRON TRANSFERRED REQUIRES A 6% IN EXCESS OF = M6 M6 4HE THERMODYNAMIC REQUIREMENTS OF A PATHWAY INVOLVING A NORMAL CYTOCHROME OXIDASE MAY BE GREATER THAN THE AVAILABLE ENERGY &IG # 4O MOVE THE EQUIVALENT OF ( PER ELECTRON REQUIRES A 6% IN EXCESS OF M6 AS SHOWN IN &IG! AND " AND THE ENERGY AVAILABLE IN THIS CASE IS SUFlCIENT #AN THE REQUIREMENTS OF THE MODEL BE MORE AC CURATELY DElNED 5NFORTUNATELY THE THERMODYNAMIC PARAMETERS ARE NOT ALL ACCURATELY KNOWN 4O lX THER MODYNAMIC CONSTRAINTS ON THE MODELS WE NEED THREE VALUES THE %H FOR THE /(/ COUPLE IN THE BULK PHASE THE %H FOR THE &E)) ))) COUPLE IN THE BULK PHASE AND THE 6P UNDER CONDITIONS FOR WHICH THE PRECEDING TWO PARAMETERS ARE KNOWN 4HE 6P HAS BEEN ESTIMATED AS HIGH AS M6 DURING &E)) OXIDATION AT P( #OX ET AL !LEXANDER ET AL 4HE %M FOR THE /(/ COUPLE AT P( IS M6 THE %H FOR THIS COUPLE UNDER THE CONDITIONS USED FOR THE MEA SUREMENT OF 6P IS PROBABLY CLOSE TO THIS VALUE 4HE OPERATIONAL VALUE FOR THE &E)) ))) COUPLE IS MORE COMPLEX IN THE USUAL SULFATE MEDIA AT P( THE %M IS APPROXIMATELY M6 ALTHOUGH IN THE CHEMOSTAT IN WHICH THE CELLS ARE GROWING THE VALUE OF %H CAN RISE AS HIGH AS M6 4HE CONDITIONS IN WHICH THE 6P WAS MEASURED IN RESPIRING CELLS IS PROBABLY CLOSE TO THE M6 VALUE )F WE USE THAT VALUE THIS GIVES A 6% OF n M6 IE M6 0ROBABLY NOT ENOUGH TO SUPPORT THE MECHANISM IN &IG # BUT THERE IS ENOUGH UNCERTAINTY IN THE MEASUREMENTS AND ESTIMATES NOT TO RULE IT OUT COMPLETELY )N THE SCHEME OF &IG # THE &E)) COULD BE REPLACED BY FERROCYANIDE OR FERROCYTOCHROME F AND REPRESENT THE SITUATION IN MAMMALIAN MITOCHONDRIA AND OTHER SYSTEMS *ASAITIS ET AL (OWEVER THE PRECEDING ARGUMENTS INDICATE THAT THIS MECHA NISM MAY BE BEYOND THE THERMODYNAMIC CAPACITY
OF THE &E)) OXIDIZING SYSTEM )S IT POSSIBLE THAT THE 7EIHUURR[LGDQV #/8 DIFFERS IN ITS PROTON PUMPING FROM OTHER MEMBERS OF THE FAMILY 4HE STRUCTURES OF CYTOCHROME OXIDASES FROM 3DUDFRFFXVGHQLWUL¿FDQV )WATA ET AL AND FROM BOVINE HEART MITOCHON DRIA 4SUKIHARA ET AL HAVE BEEN DETERMINED BY 8 RAY CRYSTALLOGRAPHY AND HUNDREDS OF ESTABLISHED AND PUTATIVE PROTEIN AND GENE SEQUENCES ARE ALSO AVAILABLE IN DATABASES ! COMPARISON OF THESE WITH THE PUTATIVE OXIDASE SEQUENCE OF 7E IHUURR[LGDQV COULD BE INFORMATIVE 4HE PUMPING OF PROTONS BY ORTHODOX #/8 IS WELL ESTABLISHED ALTHOUGH THE MECHANISM BY WHICH THE PROTONS ARE PUMPED REMAINS HOTLY DEBATED 7HAT IS KNOWN IS THAT THE PROCESS INVOLVES TURNOVER AT THE CATALYTIC CORE OF THE ENZYME AND TWO PROTON CONDUCTING CHANNELS LEAD FROM THE CYTOSOLMATRIX TO THE CATALYTIC SITE AT WHICH / IS REDUCED 4HE STRUCTURES OF THESE TWO PROTON CONDUCTING CHANNELS ARE KNOWN THEY ARE CALLED THE @$ CHANNEL AND THE @+ CHANNEL FOR DETAILS ON THIS TOPIC SEE #HAPTERS AND BY !BRAMSON ET AL AND "RZEZINSKI ET AL 6OL OF THIS SERIES RESPECTIVELY 4HE RESPECTIVE ROLES OF THESE CHANNELS IN DELIVERING EITHER THE @VECTORIAL PROTONS FOR TRANSLOCATION OR THE @SCALAR PROTONS FOR THE CHEMICAL REACTION ARE NOT RESOLVED )N SUMMARY IT IS THOUGHT THAT THE $ CHANNEL DELIVERS THE VECTO RIAL PROTONS AND PERHAPS SOME OF THE PROTONS FOR / REDUCTION AND THE + CHANNEL DELIVERS PROTONS FOR / REDUCTION 4HE GENE SEQUENCE OF A PUTATIVE 4B FERROOXIDANS CYTOCHROME OXIDASE IS AVAILABLE IN DATABASES ;4R%MBL= 4HIS ALLOWS US TO EXPLORE STRUC TURE OF THE 4B FERROOXIDANS ENZYME BY COMPARISON WITH THE KNOWN STRUCTURES OF PARTICULAR INTEREST ARE THE STRUCTURES OF THESE PROTON CONDUCTING CHANNELS #OMPARING THE SEQUENCE TO OTHER SEQUENCES AND TO THE KNOWN STRUCTURES SHOWS SOME INTERESTING DIFFERENCES IN THE + CHANNEL AND IN THE CATALYTIC CORE REGIONS OF THE ENZYME 4HROUGHOUT THE LARGE FAMILY NEARLY SEQUENCES FROM DIFFERENCE ORGANISMS WERE COMPARED SOME RESIDUES ARE CONSERVED THROUGHOUT THE FAMILY OTHERS WERE CONSERVED WITH THE NOTABLE EXCEPTION OF 4B FERROOXIDANS *TUIF,$IBOOFM"CTFOU 4HE + CHANNEL TAKES ITS NAME FROM THE CONSERVED LYSINE + IN 3DUDFRFFXV 3 GHQLWUL¿FDQV NUM BERING )N THE 3DUDFRFFXV ENZYME A PROTON CHAN NEL LEADS FROM SERINE 3 WHICH IS IN CONTACT WITH EXTERNAL WATER MOLECULES VIA LYSINE + TO TYROSINE
8+PIO*OHMFEFX
9 4WO WATER MOLECULES ARE PLACED IN THIS CHANNEL 4HE LYSINE + IS CLOSE TO 3 AND MOST LIKELY NON PROTONATED 4HE CHANNEL CONSEQUENTLY HAS A GAP BETWEEN + AND 4 THAT IS NOT BRIDGED BY WATER MOLECULES )WATA ET AL 4SUKIHARA ET AL (OFACKER AND 3CHULTEN 3IMULATIONS OF THE 0ARACOCCUS ENZYME SHOW THAT + IS mEXIBLE ENOUGH TO BRIDGE THE GAP BY MOVEMENT OF ITS #` .¡ CHAIN /NCE PROTONATED THE LYSINE HEAD GROUP MOVED WITHIN PS BY ¯ TO A POSITION CLOSE TO 4 (OFACKER AND 3CHULTEN &IGURE SHOWS A SUMMARY OF THE RESULTS OF A SIMPLE ALIGNMENT CARRIED OUT ON SEQUENCES SELECTED BY A "LAST ON A FRAGMENT OF THE 7E IHUURR[LGDQV SEQUENCE !LTSCHUL ET AL 4HIS FOCUSES ON THE REGION OF THE PRINCIPLE RESIDUES OF THE + CHANNEL AND ALSO PICKS OUT SOME LIGANDS TO THE PROSTHETIC GROUPS OF THE CATALYTIC CORE 4HE PRINCIPLE RESIDUE OF THE + CHANNEL THE CONSERVED LYSINE + 3GHQLWUL¿FDQV IS UNIQUELY REPLACED IN 7EIHUURR[LGDQV WHICH HAS AN ISOLEUCINE IN THIS POSITION ) HAVE LOOKED AT NEARLY SEQUENCES AND THE CONSERVATION OF THE LYSINE IS REMARKABLE ONLY ONE OTHER CASE OF ITS ABSENCE IS FOUND IN THE LITERATURE THIS IS WITH GLUTAMINE IN THIS POSITION THERE CAN BE AS LITTLE ONE BASE DIFFERENT BETWEEN A 1 AND A + AND A PREVIOUS REPORT OF 1 IN THIS POSITION WAS SUBSEQUENTLY REVISED TO A + 4HIS REMAINING EXAMPLE IS THE PUTA TIVE OXIDASE FROM 5KRGREDFWHU 5E VSKDHURLGHV 1:
,$937*,.,)6:,$70:** 0 *
THE CONSENSUS MOTIF
__ 96,37*),
4B FERROOXIDANS
4HUS 4B FERROOXIDANS APPEARS TO BE THE ONLY EX AMPLE WITH A HYDROPHOBIC RESIDUE IN THIS POSITION 4HE + CHANNEL LEADING UP TO THE CRUCIAL LINKING LYSINE INCORPORATES OTHER HIGHLY CONSERVED RESIDUES A TRYPTOPHAN 7 AND A SERINE 3 BOTH OF THESE ARE ABSENT 0ROXIMAL TO THE LYSINE AND CLOSE TO THE REACTION CENTER THE THREONINE 4 AND TYROSINE 9 ARE STILL CONSERVED )N THE 4B FERROOXIDANS ENZYME THE 7 IS REPLACED BY AN ALANINE /UT OF NEARLY SEQUENCES PROBED THE 7 IS REPLACED IN CASES IN OF THESE IT IS REPLACED BY AN ARGININE )N ONLY TWO CASES $EINOCOCCUS RADIODURANS AND 4HERMUS THER R MOPHILUS IS IT REPLACED WITH A HYDROPHOBIC RESIDUE )N A QUICK ALIGNMENT OF THE CLOSEST SEQUENCES TO THE 4B FERROOXIDANS SEQUENCE 3 IS ONLY REPLACED IN THE 4B FERROOXIDANS ENZYME ITS REPLACEMENT IS THE
HYDROPHOBIC LEUCINE &IG 4HE ADJACENT HISTIDINE ( WHICH IS AT THE SURFACE OPENING OF THE PUTATIVE CHANNEL IS REPLACED BY A GLUTAMATE (OWEVER THIS HISTIDINE IS NOT SO HIGHLY CONSERVED AND THERE ARE OTHER EXAMPLES OF THIS SUBSTITUTION 4HERE ARE NO APPARENT ALTERNATIVE POTENTIAL PROTON TRANSPORTING RESIDUES IN THIS LOCATION 4HE 4B FERROOXIDANS ENZYME IS THUS UNIQUE IN LACKING MOST OF THE ESSENTIAL RESIDUES OF THE OUTER + CHANNEL INCLUDING THE MECHANISTICALLY CRUCIAL LYSINE + THESE ARE HIGHLIGHTED IN BOLD IN &IG 0RELIMINARY MOLECULAR MODELING STUDIES SHOW THAT AN ALTERNATIVE ( BONDING PROTON CHANNEL NETWORK CANNOT BE GENERATED SO IT MAYBE THAT THIS CHANNEL IS UNIQUELY INOPERATIVE IN THIS ENZYME 5IF%$IBOOFMJT1SFTFOU 4HE $ CHANNEL IS FORMED BY A CHAIN OF WATER MOL ECULES LEADING FROM ASPARTATE $ TO GLUTAMATE % 0ARACOCCUS NUMBERING THE POLAR RESIDUES LINING THE CHANNEL SERVE TO STABILIZE THE WATER MOLECULES 4HE $ CHANNEL IS SO CALLED BECAUSE OF THE ASPARTATE $ AT ITS ENTRANCE BUT THIS RESIDUE IS NOT HIGHLY CONSERVED ! KEY RESIDUE OF THE CHANNEL IS THE GLU TAMATE % NEAR THE CATALYTIC CENTER OF THE ENZYME LABELED $ IN &IG )WATA ET AL (OFACKER ET AL 4HE EQUIVALENT OF % IS PRESENT IN THE 4B FERROOXIDANS ENZYME /THER RESIDUES FORMING THE CHANNEL ARE ASPARTATE $ THREONINE 4 ASPARAGINE . AND SERINES 3 AND 3 )N THE 4B FERROOXIDANS OXIDASE THE $ IS REPLACED BY AN ASPARAGINE BUT THIS IS NOT A UNIQUE REPLACEMENT !LL OF THE OTHER RESIDUES ARE FOUND AND PRELIMINARY STRUCTURAL MODELING INVESTIGATION SHOWS THE CHANNEL TO BE INTACT 0UIFS.PEJàDBUJPOTJO5IJPCBDJMMVTGFSSPPYJ EBOT$ZUPDISPNF0YJEBTF !S STATED IN A PRECEDING SECTION THE 4B FERROOXIDANS CYTOCHROME OXIDASE NORMALLY HAS ONE EXPOSED SUR FACE IN A MEDIUM AT P( AND THE OTHER EXPOSED SURFACE IN A MEDIUM CLOSE TO NEUTRALITY IN BETWEEN IT IS BURIED IN THE MEMBRANE 4HE CONSEQUENCES OF THIS IN TERMS OF ITS AMINO ACID SEQUENCE MAY BE THAT SOME MODIlCATIONS WOULD BE EXPECTED ON THE ACID SURFACE TO COPE WITH THE ACIDITY THESE MODIlCATIONS WOULD NOT BE UNIQUE TO 4B FERROOXIDANS BUT WOULD ALSO BE PREDICTED IN OTHER ACIDOPHILES (OWEVER THE LOSS OF THE + CHANNEL MAY REQUIRE FURTHER ADAPTATIONS OF THE
$IBQUFS 5IF#JPFOFSHFUJDTPG'F ** 0YJEBUJPO
)LJ!LIGNMENT OF SELECTED SECTIONS OF SUBUNIT ) SEQUENCE 4HE THREE PRINCIPAL OUTER + CHANNEL RESIDUES ARE HIGHLIGHTED IN BOLD ADDITIONAL ONES ARE LABELED + 4HE MOST HOMOLOGOUS SEQUENCES TO THE PUTATIVE 7EIHUURR[LGDQV CYTOCHROME OXIDASE SEQUENCE WHERE SELECTED BY ",!34 ;BLASTP BLOSUM %-"NET #(3)" !LTSCHUL ET AL = TAILORED FOR RANGE AND LENGTH AND ALIGNED USING #,534!,7 %-"NET #( 'APS WERE ALLOWED ALTHOUGH NONE REMAIN IN THE RANGES SHOWN THE RANGES SHOWN ARE n n AND n + DENOTES RESIDUES INVOLVED IN THE @+ CHANNEL $ DENOTES A RESIDUE OF THE @$ CHANNEL NUMBERS n INDICATE RESIDUES DISCUSSED IN THE TEXT INDICATES A FULLY CONSERVED RESIDUE AND INDICATE STRENGTH OF CONSERVATION OF CLASS OF RESIDUE
8+PIO*OHMFEFX
4B FERROOXIDANS CYTOCHROME OXIDASE )N #/8 WITH THIS CHANNEL BLOCKED BY MUTATION OF ESSENTIAL RESI DUES THE ENZYME TURNOVER IS INHIBITED +ONSTANTINOV ET AL ! FURTHER MODIlCATION OF THE ENZYME WOULD BE REQUIRED ALLOWING PROTONS FOR / REDUCTION TO ACCESS THE REACTION SITE FROM THE EXTERNAL PHASE EITHER &IG " OR &IG $ !PART FROM THE PRINCIPAL PROSTHETIC GROUP LIGATING RESIDUES AND THE PROTON CHANNEL RESIDUES THERE ARE A NUMBER OF OTHER HIGHLY CONSERVED RESIDUES IN THE #/8 SUPERFAMILY WHICH ARE NOT RETAINED IN THE 4B FERROOXIDANS ENZYME )N THE CONSENSUS #U UB BINDING SEQUENCE 9:$ : ++0)7 __
_ 6*9+ + < ) 7
CONSENSUS MOTIF
REQUIRED TO AID / / BOND SCISSION BY THE ENZYME 3O THE OUTCOMES OF THE LOSS OF THE + CHANNEL ARE NOT CLEAR ALTHOUGH IN LINE WITH THE THERMODYNAMIC IMPERATIVES IT IS PROBABLY RELATED TO THE LOWERING OF THE PROTON TRANSLOCATING STOICHIOMETRY 4HE IMPLICATION IS THAT AT LEAST SOME OF THE PROTONS FOR / REDUCTION MUST BE ALLOWED TO ACCESS THE CATALYTIC SITE FROM THE EXTERNAL SURFACE 4HESE DO NOT NEED TO BE ONLY THE SPECIlC + CHANNEL PROTONS SOME PROTONS NORMALLY CARRIED BY THE $ CHANNEL MAY ACCESS FROM THE OUTER SURFACE IN THIS MODIlED OXIDASE AND A HYBRID SCHEME SUCH AS THAT SHOWN IN &IG $ MAY BE POSSIBLE AND WITHIN THE THERMODYNAMIC RESTRICTIONS FOR FURTHER DISCUSSION SEE #HAPTERS AND BY !BRAMSON ET AL AND BY "RZEZINSKI ET AL 6OL OF THIS SERIES
4B FERROOXIDANS
4HE 67X((- IS HIGHLY CONSERVED EXCEPT IN 4B FERROOXIDANS 4HE VALINE TRYTOPHAN 6 7 IS LABELED IN &IG THE METHIONINE - IS LABELED 4HE CONSERVED (( FORMS THE BIDENTATE HISTIDINE LIGAND TO THE COPPER CENTER #UUB IN ALL SEQUENCES ! QUICK "LAST PROBING THE MOST HOMOLOGOUS SEQUENCES SHOWS ARE NO OTHER CASES THE VALINE TRYTO PHAN 67 BEING REPLACED THE - IS LESS HIGHLY CONSERVED 4HESE REPLACEMENTS COULD IMPACT ON THE FUNCTIONING OF THE CATALYTIC SITE !NOTHER UNIQUE REPLACEMENT IS OF THE CONSERVED GLUTAMINEnHISTIDINE 1 ( FOR LEUCINE AND ASPARTATE LABELED IN &IG )NVESTIGATION OF THESE AND OTHER UNIQUE SUB STITUTIONS IN THE 4B FERROOXIDANS CYTOCHROME OXIDASE MAY PROVE INFORMATIVE TO MECHANISTIC STUDIES ON THE ENZYME SEE ALSO #HAPTERS AND BY !BRAMSON ET AL AND BY "RZEZINSKI ET AL 6OL OF THIS SERIES 5IF$POTFRVFODFTPGB.PEJàFE$ZUP DISPNF0YJEBTF 4HE POSSIBLE ABSENCE OF ONE OF THE TWO PROTON CON DUCTING CHANNELS WHICH ALLOW PROTONS TO PASS FROM THE CYTOPLASM TO THE CATALYTIC CORE MAY RESULT IN A MODIlCATION OF THE PROTON TRANSLOCATING STOICHIOM ETRY OF THE ENZYME (OWEVER IT IS NOT CLEAR WHAT THE CONSEQUENCES OF AN ABSENCE OF THE + CHANNEL MIGHT BE 4HE + CHANNEL IS THOUGHT TO PROVIDE ACCESS FOR THE @SCALAR PROTONS FOR / REDUCTION AND PERHAPS NOT EVEN ALL OF THESE 7IKSTRÚM AND CO WORKERS 7IKSTRÚM ET AL HAVE GONE AS FAR AS TO SUGGEST THAT ONLY ONE OF THE EIGHT PROTONS TAKEN UP BY THE ENZYME DURING ITS CATALYTIC CYCLE IS TRANSFERRED VIA THE + PATHWAY AND THAT THE UNIQUE + PATHWAY PROTON MAY BE SPECIlCALLY
*7$PODMVEJOH3FNBSLT 4B FERROOXIDANS IS A CHEMOLITHOTROPHIC AUTOTROPH IT CAN GROW ON &E)) OXIDATION ALONE UTILIZING THE ENERGY AVAILABLE FROM THE OXIDATION FOR ALL ITS GROWTH REQUIREMENTS 4HE ENERGY AVAILABLE IS SMALL SO THE TURNOVER IS HIGH 4HE TIGHT THERMODYNAMIC CONSTRAINTS MAY LIMIT THE PROTON TRANSLOCATING STOI CHIOMETRY OF THE TERMINAL RESPIRATORY OXIDASE 4HIS LEAD TO AN ANALYSIS OF THE THERMODYNAMICS OF THE SYSTEM A DISCUSSION OF POTENTIAL COUPLING MODELS AND AN INVESTIGATION INTO SOME UNIQUE FACETS OF THE 4B FERROOXIDANS CYTOCHROME OXIDASE 4HIS INVESTIGA TION RAISES THE POSSIBILITY THAT THE 4B FERROOXIDANS IS UNIQUELY PARTIALLY DECOUPLED FROM PROTON PUMPING -ORE EXTENSIVE INVESTIGATION OF THIS UNUSUAL CYTO CHROME OXIDASE MAY HELP RESOLVE THIS QUESTION AND PROVE HELPFUL IN RESOLVING OUTSTANDING QUESTIONS TO MECHANISTIC QUESTIONS ON THE ENZYME 3FGFSFODFT !LEXANDER " ,EACH 3 AND )NGLEDEW 7* 4HE RELATIONSHIP BETWEEN CHEMIOSMOTIC PARAMETERS AND SENSITIVITY TO ANIONS AND ORGANIC ACIDS IN THE ACIDOPHILE 4HIOBACILLUS FERROOXIDANS * 'EN -ICROBIOL n !LTSCHUL 3 & -ADDEN 4, !LEJANDRO ! 3CHAFFER !! :HANG * :HANG : -ILLER 7 AND ,IPMAN $* 'APPED ",!34 AND 03) ",!34 ! NEW GENERATION OF PROTEIN DATABASE SEARCH PROGRAMS .UCLEIC !CIDS 2ES n !PPIA !YME # "ENRINE ! #AVAZZA # 'UIDICI /RTICONI - 4 "RUSCHI - #HIPPAUX - AND "ONNEFOY 6 #HARACTER IZATION AND EXPRESSION OF THE CO TRANSCRIBED CYC AND CYC GENES ENCODING THE CYTOCHROME C C AND A HIGH MO LECULAR MASS CYTOCHROME C FROM 4HIOBACILLUS FERROOXIDANS
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4VNNBSZ #ERTAIN ANAEROBIC PROKARYOTES GROW BY OXIDATIVE PHOSPHORYLATION WITH ELEMENTAL SULFUR AS THE TERMINAL ELEC TRON ACCEPTOR INSTEAD OF / SULFUR RESPIRATION OR ANAEROBIC RESPIRATION WITH SULFUR 4HE MECHANISM OF SULFUR RESPIRATION HAS BEEN INVESTIGATED IN GREAT DETAIL ONLY IN 7OLINELLA 7 7 SUCCINOGENES 4HIS ¡ PROTEOBACTERIUM USES AS TERMINAL ELECTRON ACCEPTOR POLYSULlDE WHICH IS FORMED ABIOTICALLY FROM ELEMENTAL SULFUR AND SULlDE 0OLYSULlDE REDUCTION IS FACILITATED BY A PERIPLASMIC SULFUR BINDING PROTEIN WHOSE STRUCTURE WAS DETERMINED 0OLYSULlDE RESPIRATION WITH ( OR FORMATE AS ELECTRON DONOR IS CATALYZED BY THE MEMBRANE OF 7 SUCCINOGENES AND IS COUPLED TO APPARENT PROTON TRANSLOCATION ACROSS THE MEMBRANE 4HE ELECTROCHEMICAL PROTON POTENTIAL 6P THUS GENERATED AND THE CORRESPONDING ( En RATIO WERE DETERMINED TO BE M6 NEGATIVE INSIDE AND RESPECTIVELY 4HE ELECTRON TRANSPORT CHAIN CATALYZING POLYSULlDE RESPIRATION BY ( OR FORMATE CONSISTS OF POLYSULlDE REDUCTASE METHYL MENAQUINONE AND HYDROGENASE OR FORMATE DEHYDROGENASE %ACH ENZYME CONSISTS OF TWO DIFFERENT HYDROPHILIC AND A HYDROPHOBIC SUBUNIT WHICH ANCHORS THE ENZYME IN THE MEMBRANE 4HE MEM BRANE ANCHORS OF HYDROGENASE AND FORMATE DEHYDROGENASE ARE SIMILAR DI HEME CYTOCHROMES B WHICH CARRY THE SITE OF QUINONE REDUCTION -ETHYL MENAQUINONE IS THOUGHT TO BE BOUND TO THE MEMBRANE ANCHOR OF POLYSULlDE REDUCTASE AND TO ACCEPT ELECTRONS FROM THE CYTOCHROMES B OF THE DEHYDROGENASES 0ROTEOLIPOSOMES CONTAINING POLYSULlDE REDUCTASE METHYL MENAQUINONE AND FORMATE DEHYDROGENASE ISOLATED FROM 7 SUCCINOGENES CATALYZE POLYSULlDE REDUCTION BY FORMATE WHICH IS COUPLED TO 6P GENERATION 4HE COUPLING MECHANISM IS DISCUSSED
!UTHOR FOR CORRESPONDENCE EMAIL KLIMMEK EMUNI FRANKFURTDE o 3EE /BITUARY AT THE BEGINNING OF THIS VOLUME $AVIDE :ANNONI ED D 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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DRIVEN BY REACTION B WHERE THE SULFUR IN REACTION A IS REPLACED BY / 2ICHARDSON 4HE TWO REACTIONS DIFFER IN THE NUMBER OF PROTONS APPARENTLY TRANSLOCATED PER ELECTRON TRANSPORTED FROM ( TO THE ACCEPTOR SUBSTRATE ( En RATIO N( NE 4HE ( En RATIO IS ESTIMATED TO BE FOR REACTION A AND FOR REACTION B 4HESE NUMBERS WERE CALCULATED FROM THE REDOX POTENTIALS OF THE SUBSTRATES 4ABLE ACCORDING TO %Q WITH 6P 6OLT AND ASSUMING THAT OF N( NE Q q 6% 6P
THE FREE ENERGY OF THE REDOX REACTIONS IS CONSERVED Q 4HE RELATIVELY LOW ( En RATIO OF REACTION A IS DUE TO THE MUCH LOWER REDOX POTENTIAL OF THE COUPLE 3(3n AS COMPARED TO THAT OF /(/ !S SUMING THAT THREE PROTONS HAVE TO BE TRANSLOCATED FOR THE SYNTHESIS OF A MOLECULE OF !40 THE ( En RATIO OF REACTION A SUGGESTS THAT SIX ELECTRONS HAVE TO BE TRANSPORTED FROM ( TO SULFUR FOR THE SYNTHESIS OF ONE MOLECULE OF !40 4HIS IS A STRIKING EXAMPLE OF THE ADVANTAGE OF OXIDATIVE PHOSPHORYLATION AS COMPARED TO SUBSTRATE PHOSPHORYLATION )N CONTRAST TO SUBSTRATE PHOSPHORYLATION OXIDATIVE PHOSPHORYLATION ALLOWS !40 SYNTHESIS TO BE DRIVEN BY REACTIONS WHICH DO NOT ALLOW THE SYNTHESIS OF ONE MOL OF !40 PER MOL OF SUBSTRATE )N OXIDATIVE PHOSPHORYLATION THE NUMBER OF PROTONS TRANSLOCATED CAN BE ADJUSTED TO THE FREE ENERGY OF THE DRIVING REDOX REACTION 4HIS WOULD POSSIBLY EXPLAIN THE PREVALENCE OF OXIDATIVE PHOSPHORYLATION ESPECIALLY IN ANAEROBIC ORGANISMS WHERE THE FREE ENERGY CHANGE OF THE CATABOLIC REACTIONS IS USUALLY MUCH LOWER THAN IN AEROBIC ORGANISMS )N THIS CHAPTER SULFUR RESPIRATION OF 7 SUCCINO GENES WILL BE DESCRIBED AND THE COUPLING MECHANISM OF APPARENT PROTON TRANSLOCATION TO SULFUR REDUCTION WILL BE DISCUSSED 3ULFUR RESPIRATION AND ANAEROBIC RESPIRATION WITH OTHER ACCEPTORS HAVE BEEN REVIEWED PREVIOUSLY 3CHAUDER AND +RÚGER 5NDEN AND "ONGAERTS (EDDERICH ET AL 2ICHARDSON +RÚGER ET AL **1PMZTVMàEFBT*OUFSNFEJBUFJO4VMGVS 3FTQJSBUJPO !S ELEMENTAL SULFUR IS NEARLY INSOLUBLE IN WATER G L AT # "OULÏGUE IT IS UNLIKELY THAT ELEMENTAL SULFUR IS THE SUBSTRATE OF SULFUR RESPIRATION FOR MESOPHILIC BACTERIA %LEMENTAL SULFUR READILY DISSOLVES IN AQUEOUS SOLUTIONS CONTAINING SULlDE
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#ELLS OF 7 SUCCINOGENES CATALYZING REACTION E OR F WERE FOUND TO TAKE UP TETRAPHENYLPHOSPHONIUM F 400 FROM THE EXTERNAL MEDIUM 7LOCZYK ET AL +LIMMEK 400 UPTAKE WAS PREVENTED BY THE PRESENCE OF A PROTONOPHORE &ROM THE AMOUNT OF 400 TAKEN UP THE 6s 6OLT WAS CALCULATED TO BE APPROXIMATELY THE SAME AS THAT GENERATED BY FUMARATE RESPIRATION WITH FORMATE REACTION G 4ABLE 4HE VALUES OF 6s AND 6P WERE NEARLY THE SAME SINCE THE 6P(
4HE MEMBRANE FRACTION ISOLATED FROM 7 SUCCINOGENES CELLS GROWN WITH FORMATE AND EITHER POLYSULlDE OR FUMARATE CATALYZES POLYSULlDE REDUCTION BY ( OR FORMATE 3CHAUDER AND +RÚGER &AUQUE ET AL 4HE CORRESPONDING ELECTRON TRANSPORT CHAIN CONSISTS OF POLYSULlDE REDUCTASE METHYL MENAQUI NONE &IG AND EITHER HYDROGENASE OR FORMATE DEHYDROGENASE &IG (EDDERICH ET AL 4HIS WAS SHOWN BY RECONSTITUTING A FUNCTIONAL ELECTRON TRANSPORT CHAIN FROM THE ISOLATED ENZYMES 4ABLE 0ROTEOLIPOSOMES CONTAINING POLYSULlDE REDUCTASE METHYL MENAQUINONE AND EITHER HYDROGENASE OR FORMATE DEHYDROGENASE CATALYZED POLYSULlDE REDUC TION BY ( REACTION E OR FORMATE REACTION F 4HE F TURNOVER NUMBER OF POLYSULlDE REDUCTASE IN ELECTRON TRANSPORT WAS COMMENSURATE WITH THAT MEASURED IN THE MEMBRANE FRACTION OF 7 SUCCINOGENES 7HEN PREPARED ACCORDING TO THE METHOD DESIGNED BY 2IGAUD ET AL PROTEOLIPOSOMES CONTAINING POLYSULlDE REDUCTASE METHYL MENAQUINONE AND FORMATE DE HYDROGENASE CATALYZED POLYSULlDE REDUCTION BY FORMATE 4HIS REACTION WAS COUPLED TO THE UPTAKE OF 400 FROM THE EXTERNAL MEDIUM &IG 400 UPTAKE WAS PREVENTED BY A PROTONOPHORE 4HE 6s 6OLT CALCULATED FROM THE AMOUNT OF 400 TAKEN UP UNDER STEADY STATE ELECTRON TRANSPORT WAS NOT SIGNIlCANTLY LOWER THAN THAT GENERATED ACROSS THE MEMBRANE OF CELLS BY THE SAME REACTION 4ABLE 4HESE EXPERIMENTS DEMONSTRATE THAT THE ISOLATED ENZYMES CONTAIN ALL THE COMPONENTS REQUIRED FOR ELECTRON TRANSPORT AND FOR 6P GENERATION BY POLYSULlDE RESPIRATION %ACH OF THE THREE ENZYMES INVOLVED IN POLYSULlDE RESPIRATION CONSISTS OF TWO HYDROPHILIC AND ONE HYDRO PHOBIC MEMBRANE SUBUNIT &IG 4HE HYDROPHOBIC SUBUNIT OF POLYSULlDE REDUCTASE PROBABLY CARRIES METHYL MENAQUINONE --B 4HE HYDROPHOBIC SUB UNITS OF HYDROGENASE AND FORMATE DEHYDROGENASE ARE
(#/ &UMARATE (/ A (#/ 3UCCINATE G ACROSS THE MEMBRANE WAS NEGLIGIBLE 4HE ( E AND THE !40E RATIOS WERE DETERMINED FOR FUMARATE RESPIRA TION BUT NOT FOR POLYSULlDE RESPIRATION (OWEVER THE LATTER VALUES CAN BE ESTIMATED FROM THE GROWTH YIELDS 9 OF 7 SUCCINOGENES GROWING AT THE EXPENSE OF REACTIONS F F AND G 4HE GROWTH YIELD OF POLYSULlDE RESPIRATION WAS MEASURED TO BE APPROXIMATELY HALF THAT OF FUMARATE RESPIRATION SUGGESTING THAT THE ( E AND !40E RATIO OF POLYSULlDE RESPIRATION WAS ALSO HALF THAT OF FUMARATE RESPIRATION 3CHAUDER AND +RÚGER 4HIS VIEW IS CONlRMED BY THE REDOX POTENTIAL DIFFERENCES 6% BETWEEN FORMATE AND EACH OF THE TWO ELECTRON ACCEPTORS UNDER THE GROWTH CONDI TIONS OF THE BACTERIA 4HE 6% OF POLYSULlDE RESPIRATION IS APPROXIMATELY HALF THAT OF FUMARATE RESPIRATION 4HE FREE ENERGY REQUIRED FOR !40 SYNTHESIS CALCULATED FROM 6% AND THE !40E RATIOS ARE AND K* MOL IN ANAEROBIC RESPIRATION WITH POLYSULlDE AND FUMARATE RESPECTIVELY "OTH VALUES ARE CONSISTENT WITH THE GENERAL OBSERVATION THAT IN MOST INSTANCES PHOSPHORYLATION REQUIRES ABOUT K* PER MOL OF !40 IN GROWING BACTERIA
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&IG 3TRUCTURES OF THE QUINONES OF 7 SUCCINOGENES #OLLINS AND &ERNANDEZ 4HE SECOND METHYL GROUP IS BOUND EITHER IN POSI TION OR
&IG #OMPOSITION OF THE ELECTRON TRANSPORT CHAIN CATALYZING POLYSULlDE RESPIRATION WITH ( OR FORMATE IN 7 SUCCINOGENES (YD!"# HYDROGENASE 0SR!"# POLYSULlDE REDUCTASE &DH!"# FORMATE DEHYDROGENASE .I NICKEL IRON CENTER &E3 IRON SULFUR CENTERS -O MOLYBDENUM ION BOUND TO MOLYBDOPTERIN GUANINE DINUCLEOTIDE THE DARK SQUARES DESIGNATE HEME B GROUPS
RELATED DI HEME CYTOCHROMES B "IEL ET AL 4HE LARGER HYDROPHILIC CATALYTIC SUBUNITS OF THE ENZYMES CARRY THE SUBSTRATE SITES AND ARE ORIENTED TOWARDS THE PERIPLASMIC SIDE OF THE MEMBRANE 4HE SMALLER HYDRO PHILIC SUBUNITS CARRY THREE (YD! OR FOUR IRON SULFUR CENTERS 0SR" AND &DH" WHICH PROBABLY MEDIATE ELECTRON TRANSFER FROM THE CATALYTIC TO THE MEMBRANE INTEGRATED SUBUNIT OR VICE VERSA 0SR" 4HE GENES
ENCODING THE SUBUNITS OF EACH ENZYME ARE LOCATED IN OPERONS THE SEQUENCES OF WHICH WERE DETERMINED $ELETION MUTANTS WERE CONSTRUCTED WHICH LACK ONE OR MORE GENES OF EACH OPERON 4HE MUTANTS CAN BE COMPLEMENTED BY GENOMIC INTEGRATION OF THE RESPEC TIVE GENES 4HIS ALLOWS THE CONSTRUCTION OF VARIANTS OF THE ENZYMES BY SITE DIRECTED MUTAGENESIS
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&IG 2ECORDING OF THE EXTERNAL 400 CONCENTRATION IN A SUSPENSION OF PROTEOLIPOSOMES CATALYZING POLYSULlDE REDUCTION BY FORMATE 0ROTEOLIPOSOMES G PHOSPHOLIPIDE q Ln CONTAINING FORMATE DEHYDROGENASE NMOL q Gn PHOSPHOLIPID POLYSULlDE REDUCTASE NMOL q Gn PHOSPHOLIPID AND METHYL MENAQUINONE MOL q Gn PHOSPHOLIPID WERE SUSPENDED IN AN ANOXIC 4RIS CHLORIDE BUFFER P( # #ALIBRATION OF THE 400 ELECTRODE WAS DONE BY FOUR ADDITIONS OF - 400 THIN ARROWS %LECTRON TRANSPORT WAS STARTED BY THE ADDITION OF POLYSULlDE ;3= AND FORMATE
4ABLE !CTIVITIES OF POLYSULlDE RESPIRATION IN PROTEOLIPOSOMES CONTAINING DIFFERENT NAPHTHOQUINONES $IETRICH AND +LIMMEK 0OLYSULlDE RESPIRATION WITH ( ( A ;3= OR WITH FORMATE (#/ A ;3= WAS MEASURED IN PROTEOLIPOSOMES CONTAINING POLYSULlDE REDUCTASE AND EITHER HYDROGENASE OR FORMATE DE HYDROGENASE ISOLATED FROM 7 SUCCINOGENES &UMARATE RESPIRATION WITH FORMATE (#/ A &UMARATE WAS MEASURED IN PROTEOLIPOSOMES CONTAINING FUMARATE REDUCTASE AND FORMATE DEHYDROGENASE 4HE ACTIVITIES ARE GIVEN AS SUBSTRATE TURNOVERS OF POLYSULlDE REDUCTASE OR FUMARATE REDUCTASE AT # 1UINONE
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1PMZTVMàEF3FEVDUBTF )SOLATED POLYSULlDE REDUCTASE CONSISTS OF THE THREE SUBUNITS PREDICTED BY THE PSR!"# # OPERON AND CON TAINS A MOLYBDENUM ION COORDINATED BY MOLYBDOP TERIN GUANINE NUCLEOTIDE -'$ +RAFFT ET AL *ANKIELEWICZ ET AL &IG 4HESE COFACTORS ARE LIKELY TO BE BOUND TO THE CATALYTIC SUBUNIT 0SR! TOGETHER WITH A ;&E n 3= IRON SULFUR CENTER WHICH IS PREDICTED BY THE SEQUENCE OF 0SR! &OUR ADDITIONAL ;&E n 3= CENTERS ARE PREDICTED BY THE SEQUENCE OF
0SR" 4HE ISOLATED ENZYME CONTAINS APPROXIMATELY MOL OF FREE IRON AND SULlDE IN AGREEMENT WITH THE PRESENCE OF lVE ;&E n 3= CENTERS (EDDERICH ET AL 4HE ENZYME DOES NOT CONTAIN HEME mAVIN OR HEAVY METAL IONS IN ADDITION TO MOLYBDENUM !BOUT ONE MOL OF A MIXTURE OF METHYL MENAQUINONE AND MENAQUINONE WAS CONSISTENTLY FOUND TO BE BOUND TO THE ISOLATED ENZYME THE DATA WERE CORRECTED FOR THE AMOUNT OF QUINONE WHICH IS ASSOCIATED WITH THE PHOSPHOLIPID PRESENT IN THE PREPARATION "ECAUSE OF ITS LIPOPHILIC NATURE THE QUINONE IS LIKELY TO BE BOUND
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TO THE MEMBRANE ANCHOR 0SR# OF THE ENZYME 4HE ISOLATED ENZYME CATALYZES THE REDUCTION OF POLYSULlDE BY "( REACTION H AND THE OXIDATION OF SULlDE BY DIMETHYLNAPHTHOQUINONE $-. REACTION I AT 3Nn "( A 3n Nn (3 "(
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COMMENSURATE TURNOVER NUMBERS q Sn AND q Sn (EDDERICH ET AL 4HE APPARENT +- FOR POLYSULlDE SULFUR REACTION H IS - AND THAT FOR SULlDE REACTION I IS M- 0SR# IS NOT RE QUIRED FOR BOTH CATALYTIC ACTIVITIES ! MUTANT LACKING PSR# # STILL CATALYZED BOTH REACTIONS )N THE ABSENCE OF THE MEMBRANE ANCHOR 0SR# THE ENZYME WAS LOCATED IN THE PERIPLASMIC CELL FRACTION +RAFFT ET AL $IETRICH AND +LIMMEK &URTHERMORE NINETEEN MUTANTS WITH AN ALTERED RESIDUE OF 0SR# HAD WILD TYPE ACTIVITIES WITH RESPECT TO REACTION H AND I AND 0SR! WAS BOUND TO THE MEMBRANE $IETRICH AND +LIMMEK (OWEVER SIX OF THESE MUTANTS DID NOT CATALYZE ELECTRON TRANSPORT FROM ( OR FORMATE TO POLYSULlDE SUGGESTING THAT THE OXIDATION OF REDUCED METHYL MENAQUINONE BY POLYSULlDE WAS IMPAIRED IN THESE MUTANTS SEE &IG 4HEREFORE THE SITE OF $-. REDUCTION REACTION I APPEARS TO BE DIFFERENT FROM THAT OF METHYL MENAQUINONE INTERACTION %ACH OF THE FOUR IRON SULFUR CENTERS OF 0SR" APPEARS TO BE REQUIRED FOR THE ACTIVITIES OF REACTION H AND I AS F WELL AS FOR ELECTRON TRANSPORT REACTIONS E AND F 4HIS IS SUGGESTED BY MUTANTS IN WHICH EACH CYSTEINE RESIDUE OF THE FOUR CYSTEINE CLUSTERS COORDINATING THE FOUR IRON SULFUR CENTERS WAS REPLACED BY ALANINE / +LIM MEK UNPUBLISHED RESULTS 0SR! OF THESE MUTANTS WAS BOUND TO THE MEMBRANE SUGGESTING THAT THE STRUCTURE OF THE ENZYME WAS NOT DRASTICALLY IMPAIRED (OWEVER THE MUTANTS DID NOT CATALYZE REACTIONS H AND I OR ELECTRON TRANSPORT FROM ( OR FORMATE TO POLYSULlDE SUGGESTING THAT EACH OF THE FOUR IRON SULFUR CENTERS OF 0SR" WAS REQUIRED FOR ENZYMIC ACTIVITY !S SEEN FROM ITS AMINO ACID SEQUENCE THE CATALYTIC SUBUNIT 0SR! OF POLYSULlDE REDUCTASE BELONGS TO THE $-3/ REDUCTASE FAMILY OF MOLYBDO OXIDOREDUCTASES +RAFFT ET AL +ISKER ET AL SEE ALSO #HAPTER BY -C%WAN ET AL 6OL OF THIS SERIES 4HE STRUCTURE OF SEVERAL SINGLE SUBUNIT ENZYMES OF THIS FAMILY IS KNOWN !S A RULE THE MOLYBDENUM ION
COORDINATED BY TWO -'$ MOLECULES APPEARS TO BE THE ELECTRON DONOR TO OR ACCEPTOR FROM THE RESPECTIVE SUBSTRATE IN THESE ENZYMES ! CAVITY EXTENDING FROM THE SURFACE OF THE PROTEIN TO THE MOLYBDENUM CLOSE TO ITS CENTER IS SEEN IN ALL STRUCTURES 4HE SUBSTRATES PROBABLY REACH THE ACTIVE SITE NEAR THE MOLYBDENUM THROUGH THIS CAVITY AND THE PRODUCTS ARE RELEASED TO THE SURFACE THROUGH THE CAVITY 7ITH THE ASSUMPTION THAT 0SR! RESEMBLES THESE ENZYMES IT IS LIKELY THAT THE SUBSTRATES POLYSULlDE AND PROTONS REACH THE MOLYBDENUM THROUGH A SIMILAR CAVITY (ERE A SUL FUR ATOM IS REDUCTIVELY CLEAVED FROM THE END OF THE SULFUR CHAIN OF POLYSULlDE ACCORDING TO REACTION J &IG AND THE 3Nn En ( A3n Nn (3
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PRODUCTS ARE RELEASED THROUGH THE CAVITY 3INCE 0SR! IS ORIENTED TOWARDS THE PERIPLASMIC SIDE OF THE MEM BRANE THE SUBSTRATES ARE TAKEN UP FROM THE PERIPLASM AND THE PRODUCTS ARE RELEASED TO THE SAME SIDE 4HE ELECTRONS REACTION J ARE THOUGHT TO BE PROVIDED BY THE MOLYBDENUM ION IN THE REDUCED STATE )N THE OXIDIZED STATE MOLYBDENUM ION IS THOUGHT TO BE REDUCED VIA THE IRON SULFUR CENTERS OF 0SR! AND 0SR" BY REDUCED METHYL MENAQUINONE BOUND TO 0SR# 'PSNBUF%FIZESPHFOBTF 4HE TWO FDH OPERONS ON THE GENOME OF 7 SUCCINO GENES DIFFER IN THEIR PROMOTER REGIONS BUT ARE NEARLY IDENTICAL IN THEIR GENE SEQUENCES "OKRANZ ET AL ,ENGER ET AL $ELETION MUTANTS LACKING ONE OF THE OPERONS STILL GROW BY ANAEROBIC RESPIRA TION WITH POLYSULlDE OR FUMARATE USING FORMATE AS ELECTRON DONOR 4HE FORMATE DEHYDROGENASE SERVING IN POLYSULlDE RESPIRATION IS ALSO INVOLVED IN FUMARATE RESPIRATION WHERE IT CATALYZES THE REDUCTION BY FORMATE OF MENAQUINONE WHICH IS DISSOLVED IN THE MEMBRANE "IEL ET AL +RÚGER ET AL ,IKE % COLI FORMATE DEHYDROGENASE . THE ENZYME CONSISTS OF THREE DIFFERENT SUBUNITS WHOSE SEQUENCES ARE SIMILAR TO THOSE OF THE % COLI ENZYME 4HE STRUCTURE OF % COLI FORMATE DEHYDROGENASE . WAS DETERMINED RECENTLY AND THAT OF THE 7 SUCCINOGENES ENZYME IS THOUGHT TO BE SIMILAR *ORMAKKA ET AL 4HE TWO ENZYMES ARE ANCHORED IN THE MEMBRANE BY RELATED DI HEME CYTOCHROMES B WHICH CARRY THE SITE OF QUINONE REDUC TION 4HE TWO HEME GROUPS OF THE % COLI ENZYME ARE NEARLY ON TOP OF EACH OTHER WHEN VIEWED ALONG THE MEMBRANE NORMAL 4HE IRON SULFUR SUBUNIT IS SITUATED
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&IG (YPOTHETICAL MECHANISM OF POLYSULlDE REDUCTION AT THE SUBSTRATE SITE OF POLYSULlDE REDUCTASE ! SULFUR ATOM IS CLEAVED FROM THE END OF THE POLYSULlDE CHAIN n } n 3 n3 ³e } AND BOUND TO THE MOLYBDENUM ION -O WHICH IS THEREBY OXIDIZED !FTER THE UPTAKE OF A PROTON AND TWO ELECTRONS (3n IS RELEASED AND MOLYBDENUM ION RETURNS TO THE REDUCED STATE AGAIN
BETWEEN THE CYTOCHROME B AND THE CATALYTIC SUBUNIT WHICH CARRIES A MOLYBDENUM ION COORDINATED BY TWO -'$ MOLECULES AND A ;&E n 3= CENTER &IG 4HE PROSTHETIC GROUPS OF THE ENZYME FORM A PATHWAY FOR RAPID ELECTRON TRANSFER FROM THE MOLYBDENUM ION TO THE DISTAL HEME GROUP WHICH IS THOUGHT TO BE CLOSE TO THE SITE OF QUINONE REDUCTION SEE &IG !S IN OTHER MOLYBDENUM ENZYMES THE SUBSTRATE FORMATE REACHES THE ACTIVE SITE NEAR THE MOLYBDENUM THROUGH A CAV ITY EXTENDING FROM THE MOLYBDENUM TO THE SURFACE OF THE SUBUNIT !FTER OXIDATION OF FORMATE ACCORDING TO REACTION K K (#/ A #/ ( En
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THE PRODUCTS #/ AND ( LEAVE THE SUBSTRATE SITE THROUGH THE CAVITY 3INCE THE CATALYTIC SUBUNIT &DH! OF FORMATE DEHYDROGENASE IS EXPOSED TO THE PERI PLASMIC SIDE OF THE MEMBRANE FORMATE IS TAKEN UP FROM THE PERIPLASM AND THE PRODUCTS ARE RELEASED TO THE SAME SIDE OF THE MEMBRANE +RÚGER ET AL 4HE ELECTRONS REACTION K ARE THOUGHT TO BE PASSED TO K THE CYTOCHROME B SUBUNIT BY THE IRON SULFUR CENTERS OF &DH! AND &DH" )ZESPHFOBTF (YDROGENASE CONSISTS OF THE THREE SUBUNITS ENCODED
&IG (YPOTHETICAL ARRANGEMENT OF THE FOUR PREDICTED MEM BRANE SPANNING HELICES OF 7 SUCCINOGENES (YD# "IEL ET AL 4HE SCHEME IS BASED ON THE CRYSTAL STRUCTURE OF % COLI FORMATE DEHYDROGENASE . *ORMAKKA ET AL 4HE SHADED SQUARES REPRESENT THE HEME B GROUPS ! MOLECULE OF (1./ IS SHOWN BOUND TO THE SITE OF QUINONE REDUCTION WHICH IS CONlNED BY THE AXIAL LIGAND ( OF THE DISTAL HEME GROUP AND BY . AND 1 IN THE STRETCH CONNECTING THE HYDROPHOBIC PARTS OF HELICES )) AND )))
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PLACED 4HESE RESIDUES ARE PREDICTED TO BE LOCATED IN THE LOOP CONNECTING HELIX )) AND ))) &IG 2ESIDUES . AND 1 OF &DN) WHICH ARE IN CONTACT WITH (1./ CORRESPOND TO . AND 1 OF (YD# AND ARE ALSO CONSERVED IN 7 SUCCINOGENES &DH# -U TANTS .$ AND 1, DID NOT GROW BY ANAEROBIC RESPIRATION WITH FUMARATE OR POLYSULlDE AND ( AS ELECTRON DONOR NOT SHOWN 4HE MEMBRANE FRACTION OF MUTANTS .$ AND 1, GROWN WITH FORMATE AND FUMARATE CATALYZED BENZYL VIOLOGEN REDUCTION BY ( WITH WILD TYPE SPECIlC ACTIVITIES WHEREAS THE ACTIVITIES OF $-. OR POLYSULlDE REDUCTION BY ( WERE DRASTICALLY INHIBITED 4ABLE 2EPLACEMENT OF ( WHICH IS NOT CONSERVED DID NOT AFFECT THE ENZYMIC ACTIVITIES 4HE HEME GROUPS OF (YD# WERE PRESENT IN THE MUTANTS AND THEIR REDUCTION BY ( WAS NOT IMPAIRED BY THE MUTATIONS 4HIS IS SEEN FROM THE AMOUNT OF HEME B REDUCED BY ( AFTER THE ADDITION OF 4RITON 8 AND FUMARATE TO THE MEMBRANE FRACTION OF THE MUTANTS 2ESIDUES - AND ! AT THE CYTOPLASMIC END OF HELIX )6 OF (YD# ARE CONSERVED IN &DH# AND IN % COLI &DN) WHERE THEY MAKE CONTACT WITH (1./ *ORMAKKA ET AL 4HE CORRESPONDING 7 SUC CINOGENES MUTANTS -) AND !& WERE FOUND TO HAVE WILD TYPE PROPERTIES )N CONTRAST MUTANT 9& SHOWED NEGLIGIBLE ACTIVITIES OF $-. OR POLYSULlDE REDUCTION AND WILD TYPE ACTIVITY OF BENZYL VIOLOGEN REDUCTION BY ( (OWEVER HEME B WAS NOT REDUCED BY ( IN THIS MUTANT ALTHOUGH THE (YD# PROTEIN WAS DETECTED BY %,)3! 4HIS SUGGESTS THAT ELECTRON TRANSFER FROM (YD" TO (YD# WAS INTERRUPTED IN MU TANT 9& 4HIS VIEW IS SUPPORTED BY THE STRUCTURE OF % COLI FORMATE DEHYDROGENASE . 4HE TYROSINE RESIDUE CORRESPONDING TO 9 IS SEEN TO BE BOUND TO A CONSERVED HISTIDINE RESIDUE IN THE # TERMINAL MEMBRANE HELIX OF THE IRON SULFUR SUBUNIT ! MUTANT (- WITH THE CORRESPONDING HISTIDINE RESIDUE OF 7 SUCCINOGENES (YD! REPLACED WAS FOUND TO HAVE THE SAME PROPERTIES AS 9& 'ROSS ET AL B 4HIS RESULT INDICATES THAT 9 OF (YD# AND ( OF (YD! SERVE THE SAME FUNCTION AS THE CORRESPOND ING RESIDUES IN % COLI FORMATE DEHYDROGENASE . &URTHERMORE TIGHT BINDING OF 9 TO ( APPEARS TO BE REQUIRED FOR ELECTRON TRANSFER FROM (YD! TO (YD# WHICH IN TURN IS A PREREQUISITE FOR QUINONE AND POLYSULlDE REDUCTION 4HE RESULTS OF 4ABLE SUGGEST THAT THE SITE OF QUINONE REDUCTION ON (YD# IS LOCATED CLOSE TO THE CYTOPLASMIC MEMBRANE SURFACE AS ON % COLI &DH) &URTHERMORE (YD# AND ITS SITE OF QUINONE REDUC
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4ABLE %NZYMATIC ACTIVITIES AT # OF 7 SUCCINOGENES HYD# # MUTANTS GROWN WITH FORMATE AND FUMARATE 4HE MEASURE MENTS WERE PERFORMED AS DESCRIBED BY 'ROSS ET AL B 3TRAIN 7ILD TYPE (! .$ 1, 9& - ) !&
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TION APPEAR TO BE INVOLVED IN ELECTRON TRANSFER FROM HYDROGENASE TO POLYSULlDE REDUCTASE 4HE FUNCTION OF &DH# OF 7 SUCCINOGENES FORMATE DEHYDROGENASE IN QUINONE REDUCTION AND IN ELECTRON TRANSFER TO POLYSULlDE REDUCTASE IS THOUGHT TO BE EQUIVALENT TO THAT OF (YD# $&MFDUSPO5SBOTGFSGSPN)ZESPHFOBTFBOE 'PSNBUF%FIZESPHFOBTFUP1PMZTVMàEF3F EVDUBTF 0ROTEOLIPOSOMES CONTAINING POLYSULlDE REDUCTASE AND EITHER HYDROGENASE OR FORMATE DEHYDROGENASE ISOLATED FROM 7 SUCCINOGENES DO NOT CATALYZE POLYSULlDE RESPIRATION UNLESS METHYL MENAQUINONE IS PRESENT 4ABLE -ENAQUINONE WITH A SIDE CHAIN CONSISTING OF SIX OR FOUR ISOPRENE UNITS OR VITAMIN + SERVED IN RECONSTITUTING FUMARATE RESPIRATION BUT DID NOT RE PLACE METHYL MENAQUINONE IN POLYSULlDE RESPIRATION 4HE LOW ACTIVITIES OF POLYSULlDE RESPIRATION OBSERVED WITHOUT ADDED METHYL MENAQUINONE WERE PROBABLY DUE TO THE SMALL AMOUNTS OF THIS QUINONE ASSOCIATED WITH THE ENZYME PREPARATIONS USED -AXIMUM ACTIV ITY OF POLYSULlDE RESPIRATION REQUIRED ABOUT MOL METHYL MENAQUINONE PER GRAM OF PHOSPHOLIPID NOT SHOWN -OST OF THE METHYL MENAQUINONE OF 7 SUCCINO GENES IS PROBABLY DISSOLVED IN THE LIPID PHASE OF THE BACTERIAL MEMBRANE 4HE REDOX POTENTIAL OF METHYL MENAQUINONE DISSOLVED IN THE MEMBRANE WAS ESTIMATED TO BE M6 MORE POSITIVE THAN THAT OF POLYSULlDE 4ABLE $IETRICH AND +LIMMEK 4HEREFORE IT IS UNLIKELY THAT ELECTRON TRANSFER FROM THE DEHYDROGENASES TO POLYSULlDE REDUCTASE IS MEDIATED BY DIFFUSION OF METHYL MENAQUINONE IN THE MEMBRANE 4HE METHYL MENAQUINONE INVOLVED IN POLYSULlDE RESPIRATION IS THOUGHT TO BE BOUND TO 0SR# )N THE REDUCED STATE THE BOUND QUINONE --B IS THOUGHT TO FORM THE HYDROQUINONE ANION --B(
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(EME B REDUCED BY ( MOL q G Gn MEMBRANE PROTEIN P )
4HIS ASSUMPTION IS CONSISTENT WITH THE lNDING THAT AN ARGININE RESIDUE 2 IN ONE OF THE MEMBRANE HELI CES OF 0SR# IS ESSENTIAL FOR POLYSULlDE RESPIRATION SEE &IG -UTANTS WITH THE ARGININE RESIDUE REPLACED BY PHENYLALANINE OR LYSINE HAD NEGLIGIBLE ACTIVITIES OF POLYSULlDE RESPIRATION WITH ( OR FORMATE WHEREAS THE ACTIVITIES OF POLYSULlDE REDUCTASE REACTIONS H AND I WERE CLOSE TO THOSE OF THE WILD TYPE STRAIN AND 0SR! WAS BOUND TO THE MEMBRANE OF THE MUTANTS $IETRICH AND +LIMMEK 4HE HYDROQUINONE ANION --B( IS PROBABLY BOUND AND STABILIZED BY THE POSITIVE CHARGE OF ARGININE 4HE REDOX POTENTIAL OF THE --B--B( COUPLE IS EXPECTED TO BE CLOSE TO THAT OF POLYSULlDE OR MORE NEGATIVE 3INCE ELECTRON TRANSFER FROM THE DEHYDROGENASES TO POLYSULlDE REDUCTASE BY DIFFUSION OF METHYL MENA QUINONE IS UNLIKELY TWO ALTERNATIVE MECHANISMS HAVE TO BE CONSIDERED 4HE DEHYDROGENASES MAY EITHER FORM A STABLE ELECTRON TRANSPORT COMPLEX WITH POLYSULlDE REDUCTASE OR ELECTRON TRANSFER IS ACHIEVED BY DIFFUSION AND COLLISION OF THE ENZYMES WITHIN THE MEMBRANE 4HE LATTER MECHANISM IS SUPPORTED BY EXPERIMENTAL EVIDENCE &IG 7HEN EQUIMOLAR AMOUNTS OF A DEHY DROGENASE AND POLYSULlDE REDUCTASE ARE INCORPORATED INTO LIPOSOMES CONTAINING METHYL MENAQUINONE THE ACTIVITY OF ELECTRON TRANSPORT INCREASES IN PROPORTION TO THE PROTEIN PHOSPHOLIPID RATIO 4HE SAME LINEAR RELATIONSHIP WAS OBSERVED WHEN THE MEMBRANE FRAC TION OF 7 SUCCINOGENES WAS FUSED WITH INCREASING AMOUNTS OF LIPOSOMES CONTAINING METHYL MENAQUI NONE (EDDERICH ET AL 4HE LINEAR RELATIONSHIP ARGUES AGAINST THE EXISTENCE OF STABLE ELECTRON TRANS PORT COMPLEXES WITHIN THE MEMBRANE AND CANNOT BE EXPLAINED BY COMPLEX DISSOCIATION UPON DILUTION WITH PHOSPHOLIPID #OMPLEX DISSOCIATION WOULD RESULT IN A HYPERBOLIC RATHER THAN A LINEAR RELATIONSHIP OF ELECTRON TRANSPORT ACTIVITY TO THE PROTEINPHOSPHOLIPID RATIO ACCORDING TO /STWALDS LAW OF DILUTION 4HE LINEAR RELATIONSHIP SUGGESTS THAT THE ACTIVITY OF ELECTRON
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&IG !CTIVITY OF POLYSULlDE REDUCTION BY ( ( A ;3= OR FORMATE (#/ A ;3= IN PROTEOLIPOSOMES AT # 4HE PROTEOLIPOSOMES CONTAINED METHYL MENAQUINONE MOL q Gn PHOSPHOLIPID AND APPROXIMATELY THE SAME MOLAR AMOUNT OF HYDROGENASE OR FORMATE DEHYDROGENASE AS POLYSULlDE REDUCTASE 4HE ACTIVITIES WERE DETERMINED AS DESCRIBED BY $IETRICH AND +LIMMEK
TRANSPORT IS LIMITED BY THE DIFFUSION OF THE ENZYMES WITHIN THE MEMBRANE 4HE DIFFUSION COEFlCIENT $ EVALUATED FROM &IG ACCORDING TO %Q IS AP PROXIMATELY n CM q Sn IN AGREEMENT WITH DIRECT MEASUREMENTS OF THE DIFFUSION $ D T
COEFlCIENTS OF MEMBRANE PROTEINS OF SIMILAR SIZES #HAZOTTE AND (ACKENBROCK 4HE AVERAGE MEMBRANE SURFACE OCCUPIED BY AN ENZYME MOLECULE D WAS CALCULATED FROM THE PROTEIN PHOSPHOLIPID RATIO AND THE TURNOVER NUMBERS OF THE ENZYME IN ELEC TRON TRANSPORT WAS USED AS T 4HUS THE EXPERIMENT OF &IG SUGGESTS THAT THE ELECTRON TRANSFER FROM THE DEHYDROGENASES TO POLYSULlDE REDUCTASE IS ACHIEVED BY DIFFUSION AND COLLISION OF THE ENZYME MOLECULES WITHIN THE MEMBRANE 4HIS VIEW IS SUPPORTED BY THE lNDING THAT THE ACTIVITY OF FUMARATE RESPIRATION IS NOT DECREASED BY ENZYME DILUTION WITH PHOSPHOLIPID )N THIS CASE THE ELECTRON TRANSFER FROM THE DEHYDROGE NASES TO FUMARATE REDUCTASE IS MEDIATED BY DIFFUSION OF MENAQUINONE 4HE DIFFUSION OF MENAQUINONE IS TWO ORDERS OF MAGNITUDE FASTER THAN THAT OF THE ENZYMES AND THEREFORE DOES NOT LIMIT ELECTRON TRANSPORT 0SR# IS PREDICTED TO FORM EIGHT MEMBRANE HELICES 4HE NINETEEN RESIDUES OF 0SR# WHICH WERE REPLACED BY OTHERS ARE INDICTED IN &IG !CTIVE POLYSULlDE REDUCTASE WAS BOUND TO THE MEMBRANE IN ALL MUTANTS BUT SIX OF THEM HAD NEGLIGIBLE ELECTRON TRANSPORT ACTIVITY WITH ( OR FORMATE AND POLYSULlDE 4HE COR RESPONDING RESIDUES IN BOLD TYPE IN &IG ARE EITHER CHARGED AT NEUTRAL P( OR ARE TYROSINES THE PHENOLIC HYDROXYL GROUPS OF WHICH APPEAR TO BE ESSENTIAL FOR POLYSULlDE RESPIRATION 9 9 AND 2 MAY SERVE IN BINDING --B AND --B( TO 0SR# &IG $ AND % ARE WELL SUITED FOR RELEASE OF THE PRO TON FORMED BY --B( OXIDATION INTO THE PERIPLASMIC
&IG (YPOTHETICAL TOPOLOGY OF 0SR# $IETRICH AND +LIMMEK 2ESIDUES REPLACED IN 0SR# BY SITE DIRECTED MUTAGENESIS ARE INDICATED 2ESIDUES IN BOLD LETTERS CORRESPOND TO MUTANTS WITH OR LESS OF THE WILD TYPE SPECIlC ACTIVITIES OF POLYSULlDE RESPIRATION WITH ( OR FORMATE
0MJWFS,MJNNFL 8JFCLF%JFUSJDI 'FMJDJBO%BODFB :J+BO-JO 4UFGBOJB1GFJGGFS 'SBOL-ÕIS )FJO[3ÛUFSKBOT 3PMBOE(SPTT +ÕSH4JNPOBOE"DIJN,SÕHFS VIEW THAT ELECTRON TRANSPORT IS LIMITED BY --B( IN THE ABSENCE OF A 6P IS SUPPORTED BY THE lNDING THAT THE ACTIVITY OF POLYSULlDE REDUCTION BY ( IS STIMULATED TWOFOLD BY THE INCORPORATION OF ADDITIONAL METHYL MENAQUINONE INTO THE MEMBRANE FRACTION OF 7 SUCCINOGENES $IETRICH AND +LIMMEK #ONSIDERING ITS REDOX POTENTIAL 4ABLE THE METHYL MENAQUINONE DISSOLVED IN THE MEMBRANE SHOULD BE FULLY REDUCED IN THE STEADY STATE OF POLYSULlDE RESPIRA TION 4HEREFORE THE AMOUNT OF --B( IS PREDICTED TO INCREASE WITH THE QUINONE CONTENT OF THE MEMBRANE ACCORDING TO REACTION L %5IF$PVQMJOH.FDIBOJTN
&IG (YPOTHETICAL MECHANISM OF POLYSULlDE RESPIRATION WITH ( $IETRICH AND +LIMMEK 4HE REDUCTION OF METHYL MENAQUI NONE BOUND TO 0SR# --B IS COUPLED TO THE UPTAKE OF A PROTON FROM THE CYTOPLASMIC SIDE OF THE MEMBRANE 4HE OXIDATION OF THE HYDROQUINONE ANION OF --B --B(n BY POLYSULlDE IS COUPLED TO THE RELEASE OF A PROTON TO THE PERIPLASMIC SIDE 4HE DOTTED AND THE STRIPED AREAS DESIGNATE (YD# AND 0SR# RESPECTIVELY
AQUEOUS PHASE 4HE ACTIVITY OF POLYSULlDE RESPIRATION IN CELLS OF 7 SUCCINOGENES OR IN PROTEOLIPOSOMES IS DECREASED TO ABOUT UPON THE ADDITION OF A PROTONOPHORE $IETRICH AND +LIMMEK 4HIS EFFECT CAN BE EXPLAINED IF IT IS ASSUMED THAT THE ACTIVITY IS LIMITED BY THE AMOUNT OF --B( IN THE ABSENCE OF A 6P AND THAT --B( DISSOCIATES FROM 0SR# # ACCORDING TO REACTION L WHERE ( I DESIGNATES A PROTON --B( ( I @ A --( #
L
TAKEN UP FROM THE CYTOPLASMIC SIDE OF THE MEMBRANE AND --( REDUCED METHYL MENAQUINONE DISSOLVED IN THE LIPID PHASE OF THE MEMBRANE )N THE PRESENCE OF A 6P NEGATIVE INSIDE --B( FORMATION FROM --( IS FAVORED ACCORDING TO REACTION L )T IS FEASIBLE THAT $ WHICH IS LOCATED NEAR THE CYTOPLASMIC END OF A 0SR# MEMBRANE HELIX &IG IS INVOLVED IN THE UPTAKE AND RELEASE OF ( I 2EPLACEMENT OF $ BY ASPARGINE OR LEUCINE RESULTED IN MUTANTS WITH NEGLIGIBLE ACTIVI TIES OF POLYSULlDE RESPIRATION POSSIBLY BECAUSE THE FORMATION OF --B( FROM --( IS PREVENTED 4HE
4HE ELECTRON TRANSFER FROM (YD# OF HYDROGENASE TO METHYL MENAQUINONE BOUND TO 0SR# --B AT THE MOMENT OF COLLISION OF THE TWO ENZYMES IS IL LUSTRATED IN &IG !S DEPICTED THE 6P IS THOUGHT TO BE GENERATED BY PROTON TRANSLOCATION ACROSS THE MEMBRANE WHICH IS COUPLED TO THE REDOX REACTIONS OF --B 4HE SAME MECHANISM IS THOUGHT TO APPLY WHEN FORMATE DEHYDROGENASE REPLACES HYDROGENASE 4HE PROTON CONSUMED IN --B REDUCTION IS TAKEN UP FROM THE CYTOPLASMIC SIDE OF THE MEMBRANE AND THE PROTON LIBERATED BY --B( OXIDATION IS RELEASED ON THE PERIPLASMIC SIDE 0ROTON UPTAKE IS ENVISAGED TO BE ACCOMPLISHED BY (YD# AND PROTON RELEASE BY 0SR# $ AND % OF 0SR# &IG POSSIBLY SERVE IN THE RELEASE OF THE PROTON FORMED BY --B( OXIDA TION TO THE PERIPLASMIC SIDE -UTANTS $. AND %1 DID NOT CATALYZE POLYSULlDE RESPIRATION WITH ( OR FORMATE WHEREAS MUTANT %$ SHOWED OF THE WILD TYPE SPECIlC ACTIVITY 4HE ( En RATIO OF POLYSULlDE RESPIRATION WITH ( OR FORMATE PREDICTED BY THE MECHANISM IS CONSISTENT WITH THE VALUE GIVEN IN 4ABLE !CCORDING TO THIS MECHANISM 6P IS EXCLUSIVELY GENERATED BY --B REDUCTION WITH ( &IG )N THIS PROCESS TWO PROTONS ARE RELEASED ON THE PERIPLASMIC SIDE FROM ( OXIDATION AND ONE PROTON DISAPPEARS FROM THE CYTOPLASMIC SIDE FOR --B REDUCTION 4HIS IS CONSISTENT WITH THE LOCATION OF THE SITE OF QUINONE REDUCTION ON (YD# CLOSE TO THE CY TOPLASMIC MEMBRANE SURFACE &IG &URTHERMORE MENAQUINONE OR $-. REDUCTION BY ( CATALYZED BY HYDROGENASE IN THE MEMBRANE OF 7 SUCCINOGENES AND IN PROTEOLIPOSOMES WAS FOUND TO GENERATE A 6P +RÚGER ET AL "IEL ET AL AND THE SITE OF --B REDUCTION IS SUGGESTED TO BE CLOSE TO THAT OF MENAQUINONE OR $-. REDUCTION 4ABLE --B( OXIDATION BY POLYSULlDE DISSIPATES PART
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OF THE 6P GENERATED BY --B REDUCTION WITH ( AC CORDING TO THE MECHANISM &IG SINCE ONE PROTON PER TWO ELECTRONS DISAPPEARS FROM THE PERIPLASMIC POSITIVE SIDE IN THIS PROCESS (ENCE --B( OXIDA TION BY POLYSULlDE IS PREDICTED TO BE DRIVEN BY A 6P ACROSS THE MEMBRANE 4HE VIEW THAT 6P GENERATION IS COUPLED SOLELY TO --B REDUCTION IS CONSISTENT WITH THE ENERGETICS OF THE TWO REACTIONS $IETRICH AND +LIMMEK /NLY --B REDUCTION IS CONSIDERED TO BE SUFlCIENTLY EXERGONIC TO DRIVE THE TRANSLOCATION OF A PROTON AGAINST A 6P OF 6OLT )N CONTRAST THE OXIDATION OF --B( MIGHT BE SLIGHTLY ENDERGONIC UNDER STANDARD CONDITIONS &5IF'VODUJPOPG4VE1SPUFJO 'ROWTH AND SURVIVAL BY POLYSULlDE RESPIRATION MAY BE CRITICAL FOR BACTERIA LIVING AT LOW P( ANDOR LOW SULlDE CONCENTRATION SINCE THE CONCENTRATION OF POLYSULlDE MAY BECOME VERY LOW UNDER THESE CON DITIONS REACTION C 4HEREFORE IT IS NOT SURPRISING THAT SULFUR REDUCERS SYNTHESIZE A BINDING PROTEIN FOR POLYSULlDE SULFUR WHICH IS DESIGNED TO ALLOW RAPID POLYSULlDE RESPIRATION AT LOW POLYSULlDE CONCENTRA TION 4HE PERIPLASMIC 3UD PROTEIN OF 7 SUCCINOGENES APPEARS TO SERVE AS SUCH A BINDING PROTEIN +LIMMEK ET AL 3UD IS FORMED BY THE BACTERIUM WHEN GROWN WITH POLYSULlDE AND IS NEARLY ABSENT UPON GROWTH WITH FUMARATE 4HE APPARENT +- FOR POLYSULlDE SULFUR IS - IN POLYSULlDE REDUCTION BY ( REACTION E CATALYZED BY BACTERIA GROWN WITH FUMARATE AND IS - WITH POLYSULlDE GROWN BAC +- WAS OBSERVED UPON TERIA ! SIMILAR DECREASE OF + THE ADDITION OF 3UD PROTEIN TO THE BACTERIAL MEMBRANE FRACTION CATALYZING POLYSULlDE REDUCTION 3UD CONSISTS OF TWO IDENTICAL SUBUNITS K$A AND DOES NOT CONTAIN PROSTHETIC GROUPS OR HEAVY METAL IONS 3UD CATALYZES THE TRANSFER OF SULFUR FROM POLYSULlDE TO CYANIDE REACTION M AT A HIGH TURNOVER NUMBER Sn AT # 3Nnn #. A 3#. 3n Nn
M
3UD BINDS UP TO MOL SULFUR PER MOL SUBUNIT WHEN INCUBATED IN A POLYSULlDE SOLUTION )N SOLUTIONS CONTAINING LESS THAN M- POLYSULlDE THE DISSO CIATION CONSTANT OF SULFUR BOUND TO 3UD WAS - OR LESS 5NDER THESE CONDITIONS THE DECREASE OF + +WITH 3UD WAS OBSERVED IN POLYSULlDE REDUCTION !T HIGHER CONCENTRATIONS OF POLYSULlDE THE DISSOCIATION CONSTANT WAS M- AND NO EFFECT OF 3UD ON THE +-
FOR POLYSULlDE WAS OBSERVED 3UD CONTAINS A SINGLE CYSTEINE RESIDUE WHICH IS ESSENTIAL FOR SULFUR TRANSFERASE ACTIVITY REACTION M FOR SULFUR BINDING AND FOR THE DECREASE OF +- FOR POLYSULlDE IN POLYSULlDE REDUCTION +LIMMEK ET AL 3ULFUR APPEARS TO BE COVALENTLY BOUND TO THE CYSTEINE RESIDUE 7HEN SUBJECTED TO -!,$) MASS SPECTROMETRY 3UD INCUBATED WITH POLYSULlDE WAS FOUND TO CARRY ONE OR TWO SULFUR ATOMS PER MONOMER .O SULFUR WAS BOUND TO THE MONOMER AFTER TREATMENT OF 3UD WITH CYANIDE ! 3UD VARIANT CARRYING A SERINE INSTEAD OF THE CYSTEINE RESIDUE DID NOT CARRY SULFUR EVEN UPON INCUBATION WITH POLYSULlDE 4O EXPLAIN THE EFFECT OF 3UD ON THE APPARENT +- FOR POLYSULlDE IN POLYSULlDE REDUCTION IT IS ASSUMED THAT SULFUR IS TRANSFERRED FROM 3UD TO THE ACTIVE SITE OF POLYSULlDE REDUCTASE &ROM THE CONCENTRATION OF 3UD REQUIRED FOR +- DECREASE AND FROM ITS CONCENTRATION IN THE BACTERIAL PERIPLASM IT IS LIKELY THAT 3UD IS BOUND TO POLYSULlDE REDUCTASE DURING SULFUR TRANSFER 4HE TWO ENZYMES OCCUR IN ABOUT EQUIMOLAR AMOUNTS IN CELLS OF 7 SUCCINOGENES GROWN WITH POLYSULlDE 4HE STRUCTURE OF THE 3UD DIMER WAS DETERMINED BY .-2 SPECTROSCOPY ,IN ET AL ,ÚHR ET AL &IG ! 4HE TWO CYSTEINE RESIDUES # ONE ABOVE LEFT SIDE AND ONE BELOW THE PAPER PLANE RIGHT SIDE ARE HIGHLIGHTED ! VIEW OF THE LEFT MONO MER &IG ! FROM ITS LEFT SIDE AND ALONG THE PAPER PLANE IS PRESENTED IN &IG " 4HE MONOMER HAS AN _` TOPOLOGY WITH SIX _ HELICES PACKING AGAINST A CENTRAL CORE OF lVE PARALLEL ` STRANDS 4HE DIMER INTERFACE CONSISTS OF A FOUR HELIX BUNDLE AROUND THE ROTATIONAL SYMMETRY AXIS 4HERE IS NO INDICATION OF COOPERATIVE INTERACTION OF THE TWO CYSTEINE RESIDUES WHICH ARE ¯ APART 4HE INTERACTION OF 3UD WITH POLYSULlDE REDUCTASE WAS INVESTIGATED COMPARING THE ; ( . = n 42/39 SPECTRA OF 3UD AND OF AN EQUIMOLAR MIXTURE OF 3UD DIMER AND POLYSULlDE REDUCTASE IN THE PRESENCE OF POLYSULlDE AT LOW CON CENTRATION M- 4HE RESONANCES OF EIGHT RESIDUES $ $ - 3 2 ' $ AND + OF 3UD WERE FOUND TO BE SHIFTED BY THE PRESENCE OF POLYSULlDE REDUCTASE 4HESE RESIDUES WHICH FORM A RING AROUND THE CYSTEINE RESIDUE ARE THOUGHT TO MAKE CONTACT WITH POLYSULlDE REDUCTASE )N THE FUNCTIONAL COMPLEX CONSISTING OF 3UD AND POLYSULlDE REDUCTASE THE END OF THE SULFUR CHAIN BOUND TO THE CYSTEINE RESIDUE OF 3UD MAY COME CLOSE TO THE MOLYBDENUM WHERE A SULFUR ATOM IS REDUCTIVELY CLEAVED ACCORDING TO THE MECHANISM DEPICTED IN &IG 4HIS VIEW WAS SUPPORTED BY THE RESPONSE OF THE %02 SIGNAL OF THE
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&IG 3TRUCTURE OF 3UD 2IBBON REPRESENTATIONS OF 3UD DIMER DRAWN WITH PROGRAM -/,-/, +ORADI ET AL 4HE ! AND " VIEWS ARE RELATED BY A ROTATION AROUND THE SYMMETRY AXIS 4HE CYSTEINE RESIDUES WERE DEPICTED USING A #0+ MODEL SPHERES WITH 7D6 RADIUS 3EE #OLOR 0LATE FOR DETAILS
MOLYBDENUM ION IN POLYSULlDE REDUCTASE WHICH INDICATED A SLIGHT CHANGE IN THE GEOMETRY OF THE LIGAND SPHERE OF MOLYBDENUM WHEN 3UD WAS ADDED IN THE PRESENCE OF POLYSULlDE AT LOW CONCENTRATION 0RISNER ET AL 4HIS EFFECT OF 3UD WAS NOT OBSERVED IN THE ABSENCE OF POLYSULlDE OR WITH POLYSULlDE PRESENT AT A HIGH CONCENTRATION "DLOPXMFEHNFOUT 4HE WORK WAS SUPPORTED BY GRANTS FROM THE $EUTSCHE &ORSCHUNGSGEMEINSCHAFT 3&" AND THE &ONDS DER #HEMISCHEN )NDUSTRIE 4HE AUTHORS ARE INDEBTED TO -RS -ONICA 3ÊNGER FOR EDITING THE MANUSCRIPT
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!UTHOR FOR CORRESPONDENCE EMAIL PAULETTEVIGNAIS CEAFR $AVIDE :ANNONI ED 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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SUBGROUPS THE UPTAKE ;.I&E= (ASES THE CYTOPLASMIC ( SENSORS AND THE CYANOBACTERIAL UPTAKE ;.I&E= (ASES THE BIDIRECTIONAL CYTOPLASMIC ;.I&E= (ASES THE ( EVOLVING ENERGY CONVERTING ;.I&E= (ASES 5NLIKE THE ;.I&E= (ASES THE ;&E= (ASES FORM A HOMOGENEOUS GROUP AND ARE PRIMARILY INVOLVED IN ( EVOLUTION 4HE CLASSIlCATION OF (ASES ON THE BASIS OF PHYLOGENETIC ANALYSIS THE CORRELATION OF THE DIFFERENT PHYLOGENETIC GROUPINGS WITH (ASE FUNCTION THE POSSIBLE ROLE OF THE ;&E= (ASES IN THE GENESIS OF THE EUKARYOTIC CELL AND THE STRUCTURAL AND FUNCTIONAL RELATIONSHIPS OF (ASES SUBUNITS WITH THOSE OF #OMPLEX ) OF THE RESPIRATORY ELECTRON TRANSPORT CHAIN ARE ALL DISCUSSED 4HE DIVERSITY OF (ASES MAY SEEM SURPRISING IN VIEW OF THE SIMPLICITY OF THE REACTION CATALYZED ( C ( En BUT CAN BE UNDERSTOOD IN TERMS OF THE WIDE RANGE OF PHYSIOLOGICAL ROLES THAT THEY PLAY )N ADDITION TO THE DIRECT ROLES IN RESPIRATION DESCRIBED ABOVE WORK IN RECENT YEARS HAS REVEALED THAT (ASES MAY BE INVOLVED IN REGU LATING GENE EXPRESSION EG BY ACTING AS ( SENSORS OR MAY INTERACT DIRECTLY WITH MEMBRANE BOUND ELECTRON TRANSPORT SYSTEMS IN ORDER TO MAINTAIN REDOX POISE PARTICULARLY IN SOME PHOTOSYNTHETIC MICRO ORGANISMS SUCH AS CYANOBACTERIA AND GREEN ALGAE )N ADDITION SOME ( EVOLVING (ASES THOUGHT TO BE INVOLVED IN PURELY FERMENTATIVE PROCESSES HAVE BEEN SHOWN TO PLAY A ROLE IN MEMBRANE LINKED ENERGY CONSERVATION THROUGH THE GENERATION OF A PROTONMOTIVE FORCE 4HIS DIVERSITY OF METABOLIC ROLES IS REmECTED IN A DIVERSITY OF REGULATORY MECHANISMS AND OUR CURRENT KNOWLEDGE OF (ASES GENE REGULATION IS EXTENSIVELY DESCRIBED -OST STUDIES OF REGULATION HAVE BEEN CARRIED OUT ON MEMBERS OF THE 0ROTEOBACTERIA IN WHICH (ASES GENES ARE FREQUENTLY CLUSTERED BUT THESE STUDIES ARE NOW BEING EXTENDED TO MEMBERS OF THE !RCHAEA 4HE PURPOSE OF THIS CHAPTER IS TO EMPHASIZE RECENT ADVANCES THAT HAVE GREATLY INCREASED OUR KNOWLEDGE OF (ASES AND (ASES FUNCTION TO UNDERLINE THE IMPORTANCE OF ( METABOLISM IN BOTH PROKARYOTIC AND EUKARYOTIC ORGANISMS AND TO POINT OUT THAT NEVERTHELESS MUCH REMAINS TO BE DISCOVERED ABOUT THE STRUCTURE FUNCTION AND REGULATION OF THIS INTERESTING CLASS OF ENZYMES **OUSPEVDUJPO 4HE TITLE OF THIS CHAPTER ( RESPIRATION NEEDS SOME EXPLANATION 2ESPIRATION WAS INITIALLY UNDERSTOOD AS THE VITAL PROCESS THAT SUSTAINS LIFE IN THE PRESENCE OF / AS OPPOSED TO FERMENTATION WHICH SUSTAINS LIFE IN THE ABSENCE OF / )T IS NOW KNOWN THAT RES PIRATION WHICH INVOLVES ELECTRON TRANSFER BETWEEN REDOX COMPONENTS OF A RESPIRATORY CHAIN LOCATED IN A MEMBRANE AND IS COUPLED TO VECTORIAL PROTON TRANSFER ACROSS THE MEMBRANE CAN ALSO OCCUR IN THE ABSENCE OF / IN THE PRESENCE OF ALTERNATIVE ELECTRON ACCEPTORS SUCH AS NITRATE FUMARATE OR SULFATE 7E CAN THEREFORE DISTINGUISH AEROBIC RESPIRATION WHICH USES / AS TERMINAL ELECTRON ACCEPTOR AND ANAEROBIC RESPIRA TION IN WHICH OTHER COMPOUNDS ARE USED WITH THE TYPE OF RESPIRATION BEING REFERRED TO MORE SPECIlCALLY AS NITRATE RESPIRATION FUMARATE RESPIRATION OR SULFATE !BBREVIATIONS #YT n CYTOCHROME $-+ n DEMETHYLMENAQUINONE &!$ n mAVINE ADENINE NUCLEOTIDE &DH n FORMATE DEHYDROGENASE ;&E= (ASES n IRON ONLY CONTAINING HYDROGENASES ;&E3= n IRON SULFUR CLUSTER &(, n FORMATE HYDROGENASE &-. n mAVINE MONONUCLEOTIDE &.2 n FERREDOXIN .!$0 OXIDOREDUCTASE (ASE n UPTAKE HYDROGENASE ENZYME ;.I&E= (ASES n NICKEL IRON CONTAINING HYDROGENASES -+ n MENAQUINONE 03 n PHOTOSYN THETIC SYSTEM 3$( n SUCCINATE DEHYDROGENASE 4#! CYCLE n TRI CARBOXYLIC ACID CYCLE 51 n UBIQUINONE 51( n REDUCED UBIQUINONE OR UBIQUINOL 6P n PROTON MOTIVE FORCE
RESPIRATION DEPENDING ON THE ELECTRON ACCEPTOR USED )N ALL CASES RESPIRATION ALLOWS THE RECOVERY OF THE ENERGY RELEASED BY NUTRIENT OXIDATION IN THE FORM OF PROTONMOTIVE FORCE 4HE TERM ( RESPIRATION ON THE OTHER HAND MAY BE UNDERSTOOD IN TWO DIFFERENT WAYS DEPENDING ON WHETHER ( IS THE ELECTRON DONOR TO THE ELECTRON TRANSPORT CHAIN OR THE PRODUCT OF THE TERMINAL REDOX REACTION )N THE FORMER CASE ( IS RESPIRED BY MICROORGANISMS POSSESSING A SO CALLED UPTAKE HYDROGENASE (ASE WITH ELECTRONS FROM ( BEING TRANSFERRED VIA A RESPIRATORY CHAIN EITHER TO / OR TO AN ALTERNATIVE ELECTRON ACCEPTOR )N THE LATTER CASE PROTONS ARE USED AS THE lNAL ELECTRON AC CEPTORS IN THE OXIDATION OF LOW POTENTIAL SUBSTRATES SUCH AS FORMATE AND CARBON MONOXIDE UNDER STRICTLY ANAEROBIC CONDITIONS WITH ( EVOLUTION BY (ASE BEING COUPLED TO ENERGY CONSERVATION IN THE FORM OF AN ELECTROCHEMICAL PROTON GRADIENT 3TRICTLY THIS PROCESS BY ANALOGY TO OTHER FORMS OF ANAEROBIC RES PIRATION SHOULD BE REFERRED TO AS PROTON RESPIRATION AN AWKWARD EXPRESSION )N ADDITION TO THE RESPIRATORY (ASES SOME (ASES PARTICIPATE IN PURELY FERMENTATIVE PROCESSES AND ARE THEREFORE EXCLUDED FROM THE PRESENT ANALYSIS /N THE OTHER HAND SOME OF THESE @FERMENTATIVE ENZYMES HAVE RECENTLY BEEN SHOWN TO BE INVOLVED IN MEM BRANE LINKED ENERGY CONSERVATION AND THIS NOVEL
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5NLIKE ;.I&E= (ASES MANY ;&E= (ASES ARE MO NOMERIC FORMED BY THE CATALYTIC SUBUNIT CONTAINING THE SO CALLED ( CLUSTER OF CA RESIDUES AT THE ACTIVE SITE !DAMS BUT DIMERIC TRIMERIC AND TETRAMERIC ENZYMES ARE ALSO KNOWN !TTA AND -EYER AND REFERENCES THEREIN 6IGNAIS ET AL 4HE ( CLUSTER COMPRISES TWO &E ATOMS BOUND TO A ;&E 3= CLUSTER VIA A BRIDGING CYSTEINE 0ETERS ET AL .ICOLET ET AL 4HE CRYSTALLOGRAPHERS 0ETERS .ICOLET ET AL HAVE POINTED OUT STRIKING SIMILARITIES BETWEEN THE STRUCTURES OF THE .I CONTAIN ING ACTIVE SITE AND THAT CONTAINING &E ATOMS ONLY 4HE OTHER &E 3 CLUSTERS ARE LOCATED IN ADDITIONAL DOMAINS OF THE CATALYTIC SUBUNIT OR IN ADDITIONAL SUBUNITS !TTA AND -EYER 6IGNAIS ET AL 4HE EUKARYOTIC (ASES WHOSE GENES HAVE BEEN CLONED AND SEQUENCED APPEAR TO BE OF THE MONO MERIC ;&E= (ASE TYPE 4HEY ARE LOCALIZED EITHER IN HYDROGENOSOMES IN ANAEROBIC EUKARYOTES "UI AND *OHNSON (ACKSTEIN ET AL (ORNER ET AL 6ONCKEN ET AL OR IN CHLOROPLASTS OF GREEN ALGAE &LORIN ET AL (APPE AND +AMINSKI )NTERESTINGLY THE CHLOROPLASTIC ;&E= (ASES CONSIST OF AN ( CLUSTER DOMAIN ONLY AND LACK THE AD DITIONAL &E 3 CLUSTER IN THE . TERMINAL DOMAIN !LTHOUGH THE .I CONTAINING AND THE &E ONLY AC TIVE SITES DISPLAY STRIKING STRUCTURAL SIMILARITIES ALIGNMENTS OF FULL ;.I&E= AND ;&E= (ASE SEQUENCES FROM CA ONE HUNDRED ENZYMES IN TOTAL INDICATE THAT THE ;.I&E= AND THE ;&E= ENZYMES HAVE A DIFFERENT EVOLUTIONARY ORIGIN 6IGNAIS ET AL #5IF)ZESPHFOBTFT 4HE MOST NUMEROUS AND BEST STUDIED OF (ASES HAVE BEEN THE ;.I&E= (ASES OF THE DOMAIN "ACTERIA 6IGNAIS ET AL ;.I&E= (ASES HAVE ALSO BEEN ISOLATED FROM !RCHAEA ARCHAEAL GENES HAVE BEEN CLONED AND RECENTLY SEVERAL NEW ;.I&E= (ASES REVEALED BY ARCHAEAL GENOME SEQUENCING HAVE BEEN IDENTIlED AND ISOLATED +àNKEL ET AL -EUER ET AL 4ERSTEEGEN AND (EDDERICH 3APRA ET AL 3ILVA ET AL ;.I&E= (ASES ENABLE PROCARYOTES TO USE ( AS AN ELECTRON AND ENERGY SOURCE EITHER AEROBICALLY "OWIEN AND 3CHLEGEL 6IGNAIS ET AL OR ANAEROBICALLY AS IN METHANOGENS 4HAUER ET AL 4HAUER SULFATE REDUCERS &AUQUE ET AL PHOTOSYNTHETIC BACTERIA -EYER ET AL 6IGNAIS ET AL AND CYANOBACTERIA (OUCHINS -ANY ORGANISMS EG %SCHERICHIA COLI SULFATE
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REDUCERS AND METHANOGENS CONTAIN MORE THAN TWO DIFFERENT (ASES WHICH ARE DIFFERENTIALLY EXPRESSED UNDER VARIOUS GROWTH CONDITIONS 4HIS VARIETY IS IN KEEPING WITH THE IMPORTANCE OF ( IN THEIR METABO LISM AND WITH THE NEED TO ADAPT QUICKLY TO CHANGES IN THE ENVIRONMENT %VOLUTIONARY TREES HAVE BEEN DERIVED FROM THE FULL SEQUENCE ALIGNMENTS OF THE SMALL AND OF THE LARGE SUB UNITS OF ;.I&E= (ASES 4HE CLASSIlCATION OF ;.I&E= (ASES IN THE FOUR GROUPS DESCRIBED BELOW BASED ON SEQUENCE SIMILARITIES IS CONSISTENT WITH THE FUNCTIONS OF THE ENZYMES 6IGNAIS ET AL &IG 5IF6QUBLF)ZESPHFOBTFT (SPVQ
4HE UPTAKE (ASES ESSENTIALLY FOUND IN 0ROTEOBAC TERIA LINK THE OXIDATION OF ( TO THE REDUCTION OF ANAEROBIC ELECTRON ACCEPTORS SUCH AS ./n 3/n FUMARATE OR #/ OR IN THE PRESENCE OF OXYGEN TO AEROBIC RESPIRATION 4HE CORE (ASE DIMER IS ANCHORED TO THE MEMBRANE BY THE THIRD (ASE SUBUNIT A DI HEME CYTOCHROME B $ROSS ET AL "ERNHARD ET AL WHICH CONNECTS IT TO THE QUINONE POOL OF THE RESPIRATORY CHAIN IN THE MEMBRANE AND BY THE HYDROPHOBIC # TERMINUS OF THE SMALL SUBUNIT #AUVIN ET AL 'ROSS ET AL &IG ! MUTANT OF 2HODOBACTER CAPSULATUS LACKING THE THIRD SUBUNIT (UP# OF THE (UP3,# UPTAKE (ASE &IG WAS UN ABLE TO GROW AUTOTROPHICALLY OR TO TRANSFER ELECTRONS FROM ( TO THE RESPIRATORY CHAIN ALTHOUGH (ASE ACTIVITY WAS PRESENT AND MEASURABLE WITH ARTIlCIAL ELECTRON ACCEPTORS #AUVIN ET AL -AGNANI ET AL %LECTRONS FROM ( ARE DONATED TO THE QUINONE POOL (ENRY AND 6IGNAIS 6IGNAIS ET AL +OMEN ET AL AND THE ENERGY OF ( OXIDATION IS RECOVERED BY VECTORIAL PROTON TRANSFER AT THE LEVEL OF THE QUINOL OXIDASE +OMEN ET AL CYTOCHROME BC COMPLEX CYTOCHROME OXIDASE 0AUL ET AL 0ORTE AND 6IGNAIS AND FUMARATE REDUCTASE +RÚGER ET AL 4HE PERIPLASMIC (ASES OF SULFATE REDUCING BAC TERIA SUCH AS $ESULFOVIBRIO REPRESENT ANOTHER TYPE OF UPTAKE (ASE ABLE TO INTERACT WITH LOW POTENTIAL C TYPE CYTOCHROMES AND A TRANSMEMBRANE REDOX PROTEIN COMPLEX ENCODED BY THE HMC OPERON 2OSSI ET AL AND TO PARTICIPATE IN THE CREATION OF A PROTON GRADIENT ACROSS THE MEMBRANE FOR ENERGY CON SERVATION &AUQUE ET AL 6IGNAIS ET AL $ELETION OF THE HMC OPERON OF $ VULGARIS (ILDEN BOROUGH IMPAIRED GROWTH ON HYDROGEN BUT NOT THAT
&IG 3CHEMATIC REPRESENTATION OF THE PHYLOGENETIC TREE OF ;.I&E= (ASES BASED ON THE COMPLETE SEQUENCES OF THE SMALL AND THE LARGE SUBUNITS THE SAME TREE WAS OBTAINED WITH EACH TYPE OF SUBUNIT ORIGINALLY ESTABLISHED BY 6IGNAIS ET AL
ON LACTATE OR PYRUVATE CONlRMING THE IMPORTANCE OF THE (MC COMPLEX IN ELECTRON TRANSPORT FROM ( IN THE PERIPLASM TO SULFATE IN THE CYTOPLASM $OLLA ET AL (OWEVER (MC DOES NOT APPEAR TO BE THE ONLY COMPLEX CAPABLE OF COUPLING ELECTRON TRANSPORT TO PROTON PUMPING $OLLA ET AL AND SOME OF THE REDOX PARTNERS INVOLVED IN THE ESTABLISHMENT OF PROTON GRADIENT ACROSS THE MEMBRANE FOR ENERGY CONSERVATION DURING ( OXIDATION HAVE NOT YET BEEN IDENTIlED 4HE PRODUCTS OF HOMOLOGS OF ECH GENES SEE BELOW RECENTLY DISCOVERED IN THE GENOME OF $ VULGARIS CITED IN 0OHORELIC ET AL MIGHT PARTICIPATE IN THE CREATION OF A PROTONMOTIVE FORCE IN THE MEMBRANE /THER UPTAKE (ASES EG (ASE OF % COLI ENCODED BY THE HYB/!"#$%&' OPERON $U '
BINI ET AL &IG AND (ASE OF 4HIOCAPSA ROSEOPERSICINA ENCODED BY THE HYD3ISPISPHYD, OPERON 2ÈKHELY ET AL HAVE SUBUNITS THAT SHARE SIGNIlCANT DEGREES OF IDENTITY WITH TWO SUBUNITS OF THE (MC COMPLEX
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METHANE FROM METHYL 3 #O- 4HIS ELECTRON TRANSFER SYSTEM IS COUPLED TO VECTORIAL PROTON TRANSFER LEADING TO THE CREATION OF A PROTONMOTIVE FORCE 4HE SMALL SUBUNITS OF ALL THE ;.I&E= (ASES OF GROUP BEAR AT THEIR . TERMINUS A LONG SIGNAL PEPTIDE n AMINO ACID RESIDUES WHICH TARGETS THE FULLY FOLDED HETERODIMER TO THE MEMBRANE AND THE PERI PLASM "ERKS ET AL 6ORDOUW 7U ET AL 6IGNAIS ET AL 3ARGENT ET AL 4HE
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4HE ENZYMES OF THIS GROUP ARE MULTIMERIC SIX SUBUNITS OR MORE FUNCTION UNDER STRICTLY ANAEROBIC CONDITIONS AND REDUCE PROTONS IN ORDER TO DISPOSE OF EXCESS REDUCING EQUIVALENTS PRODUCED BY THE ANAEROBIC OXIDATION OF # ORGANIC COMPOUNDS 4HEY ARE ASSOCIATED WITH THE OXIDATION OF LOW POTENTIAL SUBSTRATES SUCH AS CARBON MONOXIDE AND FORMATE REVIEWED BY (EDDERICH 4HE PROTOTYPE OF THIS GROUP IS % COLI (ASE WHICH FORMS PART OF THE FORMATE HYDROGEN LYASE COMPLEX &,( ENCODED BY THE HYC OPERON "ÚHM ET AL 3AUTER ET AL CF &IG !NOTHER MEMBER OF THIS GROUP IS THE #/ INDUCED (ASE OF 2HODOSPIRILLUM RUBRUM &OX ET AL A B 4HIS ENZYME IS A COMPONENT OF THE #/ OXIDIZING SYSTEM THAT ALLOWS 2 RUBRUM TO GROW IN THE DARK WITH #/ AS SOLE ENERGY SOURCE #/ DEHYDROGENASE AND THE (ASE ENCODED BY THE COO OPERON OXIDIZE #/ TO #/ WITH CONCOMITANT PRODUCTION OF ( 3INCE THE #/ DEHYDROGENASE IS A PERIPHERAL MEMBRANE PROTEIN IT HAS BEEN PROPOSED THAT THE (ASE COMPONENT OF THE OXIDIZING SYSTEM CON STITUTES THE ENERGY COUPLING SITE &OX ET AL A B % COLI (ASE AND 2 RUBRUM #OO,( (ASE ARE LABILE ENZYMES AND THE EXACT NUMBER OF SUBUNITS IN EACH ENZYME REMAINS TO BE DETERMINED F )N % COLI THE RECENTLY DISCOVERED HYF OPERON IS THOUGHT TO ENCODE A PUTATIVE TEN SUBUNIT (ASE DESIGNATED (ASE WHICH COMBINES WITH FORMATE DEHYDROGENASE ( &DH ( TO FORM A SECOND FORMATE HYDROGENLYASE &(, (ASE WAS SUGGESTED TO BE ENERGY TRANSDUCING SINCE IN COMMON WITH OTHER GROUP (ASES SOME OF ITS SUBUNITS SHOW MARKED SEQUENCE SIMILARITIES WITH SUBUNITS OF #OMPLEX ) IN PARTICULAR TWO INTRINSIC MEMBRANE SUBUNITS WHICH PLAY A CRUCIAL ROLE IN PROTON TRANSLOCATION AND ENERGY COUPLING !NDREWS ET AL SEE BELOW ! RECENT REPORT "AGRAMYAN ET AL HAS SUGGESTED THAT AT SLIGHTLY ALKALINE P( ( PRODUCTION AND &DH ( ACTIVITY IN % COLI ARE DEPENDENT BOTH ON THE && !40ASE AND &(, 5NDER THESE CONDITIONS ( PRODUCTION WOULD BE DRIVEN BY A PROTON GRADIENT ESTABLISHED BY THE && !40ASE AS HAS BEEN REPORTED FOR FERMENTATIVE GAS ( AND (3 PRODUCTION IN 3ALMONELLA TYPHIMURIUM 3ASAHARA ET AL 4HE MAJORITY OF (ASES THAT HAVE BEEN ASSIGNED TO THIS GROUP HAVE BEEN FOUND IN !RCHAEA &IG IN
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CLUDING -ETHANOSARCINA BARKERI +àNKEL ET AL -ETHANOBACTERIUM THERMOAUTOTROPHICUM STRAIN -ARBURG NOW CALLED -ETHANOBACTER MARBURGENSIS 4ERSTEEGEN AND (EDDERICH AND 0YROCOCCUS FURIOSUS 3APRA ET AL 3ILVA ET AL 4HE GROUP (ASE FROM - BARKERI ENCODED BY THE ECH GENES HAS BEEN PURIlED FROM ACETATE GROWN CELLS IT HAS BEEN PROPOSED TO OXIDIZE THE CARBONYL GROUP OF ACETATE TO FORM #/ AND ( +àNKEL ET AL -EUER ET AL 4HE METHANOGENIC ARCHAEON - THERMOAUTOTROPHICUM WHICH IS ABLE TO GROW ON #/ ( AS CARBON AND ENERGY SOURCE CONTAINS IN ADDITION TO & REDUCING AND & NON REDUCING (ASES TWO GENE GROUPS DESIGNATED @ENERGY CONVERTING (ASE ! EHA AND @ENERGY CONVERTING (ASE " EHB WHICH ENCODE PUTATIVE MULTISUBUNIT MEMBRANE BOUND (ASES HOMOLOGOUS TO % COLI (ASE AND 2 RUBRUM #OO(, (ASE 4ERSTEEGEN AND (EDDERICH 4HE HYPERTHERMOPHILIC ARCHAEON 0 FURIOSUS CONTAINS TWO CYTOPLASMIC ( EVOLVING (ASES ) AND )) 0EDRONI ET AL -A ET AL MEMBERS OF GROUP AND A MEMBRANE BOUND (ASE MEMBER OF GROUP ENCODED BY A GENE OPERON 3CHUT ET AL TERMED MBH EITHER MBH 3APRA ET AL OR MBH! . . 3ILVA ET AL &OUR GENE PRODUCTS
OF THIS OPERON SHARE SIMILARITIES WITH SUBUNITS OF #OMPLEX ) 4HESE MULTIMERIC MEMBRANE BOUND (ASE COMPLEXES COMPRISE TRANSMEMBRANE SUBUNITS HOMOLOGOUS TO #OMPLEX ) SUBUNITS INVOLVED IN PROTON PUMPING AND ENERGY COUPLING 4ABLE AND APPEAR TO BE ABLE TO COUPLE THE OXIDATION OF A CARBONYL GROUP ORIGINATING FROM FORMATE ACETATE OR CARBON MONOXIDE AND THE REDUCTION OF PROTONS TO ( WITH ENERGY CONSERVATION $<'F>)ZESPHFOBTFT 7HILE ;.I&E= (ASES TEND TO BE INVOLVED IN ( CON SUMPTION ;&E= (ASES ARE USUALLY INVOLVED IN ( PRODUCTION (OWEVER THE PERIPLASMIC ;&E= (ASE OF $ VULGARIS HAS BEEN RECENTLY DEMONSTRATED TO FUNC TION AS AN UPTAKE (ASE 0OHORELIC ET AL 4HIS TYPE OF ENZYME IS FOUND IN ANAEROBIC PROKARYOTES SUCH AS CLOSTRIDIA AND SULFATE REDUCERS SEE REVIEWS BY !DAMS !TTA AND -EYER 6IGNAIS ET AL AND IN EUKARYOTES (ORNER ET AL &LORIN ET AL 7àNSCHIERS ET AL A (APPE AND +AMINSKY 6ONCKEN ET AL ;&E= (ASES ARE THE ONLY TYPE OF (ASE TO HAVE BEEN FOUND IN EUKARYOTES AND THEY ARE LOCATED EXCLUSIVELY
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IN MEMBRANE LIMITED ORGANELLES IE IN CHLOROPLASTS OR IN HYDROGENOSOMES REVIEWED IN 6IGNAIS ET AL AND (ORNER ET AL 4WO NOVEL HYPOTHESES FOR THE ORIGIN OF THE EU KARYOTIC CELL -ARTIN AND -àLLER -OREIRA AND ,ØPEZ 'ARCÓA ,ØPEZ 'ARCÓA AND -OREIRA POSIT THAT A METABOLIC SYMBIOSIS SYNTROPHY BETWEEN A METHANOGENIC ARCHEON AND A PROTEOBACTE RIUM ABLE TO RELEASE ( IN ANAEROBIOSIS WAS THE lRST STEP IN EUKARYOGENESIS 4HE HYDROGEN HYPOTHESIS -ARTIN AND -àLLER SUGGESTS THAT AN ANAEROBIC HETEROTROPHIC _ 0ROTEOBACTERIUM PRODUCING ( AND #/ AS WASTE PRODUCTS FORMED A SYMBIOTIC METABOLIC ASSOCIATION SYNTROPHY WITH A STRICTLY ANAEROBIC AU TOTROPHIC ARCHAEON POSSIBLY A METHANOGEN DEPENDENT ON ( 4HE INTIMATE RELATIONSHIP OVER LONG PERIODS OF TIME ALLOWED THE SYMBIONT AND THE HOST TO CO EVOLVE AND BECOME DEPENDENT ON EACH OTHER )N AN ANAEROBIC ENVIRONMENT THE SYMBIONT WAS EITHER LOST AS IN TYPE ) AMITOCHONDRIATE EUKARYOTES OR BECAME A HYDROGENO SOME IE A HYDROGEN GENERATING AND !40 SUPPLYING ORGANELLE AS IN TYPE )) AMITOCHONDRIATE EUKARYOTES -àLLER "Y FURTHER EVOLUTION THE HOST LOST ITS AUTOTROPHIC PATHWAY AND ITS DEPENDENCE ON ( AND THE ENDOSYMBIONT ADOPTED A MORE EFlCIENT AEROBIC RESPIRATION TO BECOME THE ANCESTRAL MITOCHONDRION 4HUS THE EUKARYOTIC CELL WOULD HAVE ARISEN THROUGH THE SYMBIOTIC ASSOCIATION OF TWO PROKARYOTES AN ARCHAEON THE HOST AND A EUBACTERIUM THE SYM BIONT THE COMMON ANCESTOR OF HYDROGENOSOMES AND MITOCHONDRIA 4HE SYNTROPHY HYPOTHESIS FOR THE ORIGIN OF EUKARYOTES PROPOSED AT THE SAME TIME AND INDEPENDENTLY BY -OREIRA AND ,ØPEZ 'ARCÓA WAS BASED ON SIMILAR METABOLIC CONSIDERATION IN TERSPECIES HYDROGEN TRANSFER BUT THE LATTER AUTHORS SPECULATED THAT THE ORGANISMS INVOLVED WERE b 0RO TEOBACTERIA ANCESTRAL SULFATE REDUCING MYXOBACTERIA AND METHANOGENIC !RCHAEA SEE ALSO ,ØPEZ 'ARCÓA AND -OREIRA 4HESE AUTHORS SUGGESTED ALSO THAT A SECOND ANAEROBIC SYMBIONT WAS INVOLVED IN THE ORIGIN OF MITOCHONDRIA (YDROGENOSOMES ARE EITHER CONSIDERED TO BE RELICS OF ANCESTRAL ENDOSYMBIONT AND TO SHARE A COMMON ORIGIN WITH MITOCHONDRIA -ARTIN AND -àLLER OR TO HAVE EVOLVED SEVERAL TIMES AS ADAPTATIONS OF MITOCHONDRIA TO ANAEROBIC ENVIRON MENTS (ACKSTEIN ET AL 7ITH RESPECT TO THE VARIOUS THEORIES ON THE ORIGIN OF MITOCHONDRIA AND HYDROGENOSOMES WHICH HAVE BEEN REVIEWED RECENTLY -ARTIN ET AL IT IS INTERESTING TO MENTION THAT EUKARYOTIC ORGANELLES CONTAIN ONLY ;&E= (ASES AND THAT ;&E= (ASES HAVE BEEN FOUND IN b 0ROTEOBACTERIA
-ALKI ET AL 0OHORELIC ET AL BUT NOT IN PRESENT DAY _ 0ROTEOBACTERIA WHICH CONTAIN ONLY ;.I&E= (ASES 6IGNAIS ET AL 0HYLOGENETIC ANALYSIS OF EUKARYOTIC ;&E= (ASES (ORNER ET AL SUGGESTS A POLYPHYLETIC ORIGIN OF THESE ENZYMES IMPLYING AN ACQUISITION BY LATERAL GENE TRANSFER FROM DIFFERENT PROKARYOTIC SOURCES /N THE OTHER HAND THE ;&E= (ASES FROM THE GREEN ALGAE EMERGE AS A MONOPHYLETIC GROUP WITH HYDROGENOSOMAL ;&E= (ASES FROM MICROAEROPHILIC PROTISTS (ORNER ET AL !LTHOUGH A VERTICAL INHERITANCE OF ORTHOLOGOUS ENZYMES FROM THE PUTATIVE ANCESTRAL ENDOSYMBIONT OF THE b 0ROTEOBACTERIUM TYPE COULD BE ENVISAGED FOR THE HYDROGENOSOMAL ENZYMES THE PLASTIDIAL ;&E= (ASES APPEAR TO HAVE A NON CYANOBACTERIAL ORIGIN SINCE CYANOBACTERIA THE PROGENITORS OF CHLOROPLASTS CONTAIN ONLY ;.I&E= (ASES AND NO ;&E= (ASE 6IGNAIS ET AL 4AMAGNINI ET AL %)ZESPHFOBTFTBOE $PNQMFY* 4HE PROTON PUMPING .!$( UBIQUINONE OXIDORE DUCTASE IS THE MAIN ENTRY SITE OF REDUCING EQUIVALENTS INTO THE MITOCHONDRIAL AND THE BACTERIAL RESPIRATORY CHAINS &EARNLY AND 7ALKER 7ALKER 4HE MITOCHONDRIAL ENZYME IS ALSO CALLED #OMPLEX ) WHEREAS THE BACTERIAL ENZYME IS MORE OFTEN REFERRED TO AS TYPE ) .!$( DEHYDROGENASE OR .$( 4HE BOVINE MITOCHONDRIAL #OMPLEX ) CONTAINS A TOTAL OF DIFFERENT SUBUNITS #ARROLL ET AL WHEREAS .$( FROM THE BACTERIA 0ARACOCCUS DENITRIlCANS 9AGI 9AGI ET AL AND 2B CAPSULATUS $UPUIS ET AL CONTAINS SUBUNITS AND THAT FROM % COLI 7EIDNER ET AL "LATTNER ET AL SUBUNITS ALL OF WHICH HAVE HOMOLOGS IN THE BOVINE ENZYME 4ABLE "OTH THE MITOCHON DRIAL AND BACTERIAL ENZYMES ARE , SHAPED WITH A MEMBRANE DOMAIN AND A PERIPHERAL ARM EXTENDING INTO THE CYTOSOL &IG &RIEDRICH 3AZANOV ET AL 3CHULTZ AND #HAN 4HE HYDROPHILIC .!$( OXIDIZING MODULE DISTAL FROM THE MEMBRANE COMPRISES THREE HYDROPHILIC SUBUNITS CONTAINING &-. AND lVE IRON SULFUR CLUSTERS A SECOND HYDROPHILIC MODULE CONSTITUTED OF FOUR SUBUNITS CONNECTS THE .!$( OXIDIZING PROTEINS TO THE MEMBRANE BOUND HYDROPHOBIC SUBUNITS 4HE TWO EXTRAMEMBRANOUS MODULES CONTAIN ALL THE REDOX CENTERS OF THE ENZYME 9AGI ET AL $UPUIS ET AL &RIEDRICH 3CHULTZ AND #HAN 3EQUENCE SIMILARITIES BETWEEN (ASES AND #OM
$IBQUFS )ZESPHFO3FTQJSBUJPO PLEX ) WERE lRST REPORTED BY "ÚHM ET AL AND BY 0ILKINGTON ET AL AND HAVE BEEN EMPHASIZED IN SEVERAL SUBSEQUENT REPORTS !LBRACHT AND DE *ONG &RIEDRICH AND 7EISS !LBRACHT AND (ED DERICH &RIEDRICH AND 3CHEIDE $UPUIS ET AL &RIEDRICH 6IGNAIS ET AL 9ANO AND /HNISHI 4O COMPARE THE SUBUNITS OF (ASES TO THOSE OF #OMPLEX ) WE HAVE ADOPTED THE NOMENCLATURE USED FOR % COLI AND 2B CAPSULATUS #OMPLEX ) 4ABLE &IG 3UBUNITS .UO% .UO& .UO) AND THE . TERMINAL &E 3 BINDING DOMAIN CA RESIDUES OF .UO' HAVE HOMOLOGOUS COUNTER PARTS IN ACCESSORY SUBUNITS AND DOMAINS OF SOLUBLE ;.I&E= AND ;&E= (ASES )N ADDITION THREE SUBUNITS LOCATED WITHIN THE CONNECTING MODULE OF #OMPLEX ) SHARE SIMILARITIES WITH SUBUNITS OF ;.I&E= (ASES THE .UO" SUBUNIT WITH THE SMALL (ASE SUBUNIT THE .UO# AND .UO$ SUBUNITS FUSED AS A SINGLE .UO#$ PROTEIN IN % COLI WITH THE LARGE (ASE SUBUNIT &URTHERMORE HYDROPHOBIC SUBUNITS OF MULTIMERIC MEMBRANE BOUND ;.I&E= (ASES BELONGING TO THE I GROUP NAMELY % COLI (YC "ÚHM ET AL 3AUTER ET AL 2 RUBRUM #OO &OX ET AL A B - BARKERI %CH +àNKEL ET AL -EUER ET AL - MARBURGENSIS %HA AND %HB 4ERSTEEGEN AND (EDDERICH AND 0 FURIOSUS -BH 3APRA ET AL 3ILVA ET AL ARE ALSO HOMOLOGOUS TO TRANSMEMBRANE SUBUNITS OF #OMPLEX ) .UO( .UO, .UO- .UO. )T SHOULD BE NOTED THAT THESE GROUP (ASES ARE ALSO PROTON PUMPS 4HUS THE PRESUMED EVOLUTIONARY LINKS BETWEEN (ASES AND #OMPLEX ) CONCERN NOT ONLY THE ELECTRON TRANSFERRING SUBUNITS BUT ALSO THE PROTON PUMPING UNITS IE THE COUPLING BETWEEN ELECTRON TRANSPORT AND ENERGY RECOVERY BY A CHEMIOSMOTIC MECHANISM /N THE BASIS OF THE SIMILARITIES BETWEEN ;.I&E= (ASES AND THE .UO" .UO$ DIMER OF THE CONNECTING MODULE $UPUIS ET AL HAVE RECENTLY PROPOSED THAT THE ;.I&E= ACTIVE SITE OF (ASES WAS REORGANIZED INTO A QUINONE REDUCTION SITE CARRIED BY THE .UO" .UO$ DIMER AND A HYDROPHOBIC SUBUNIT SUCH AS .UO( &IG AND THAT .UO$ MIGHT PROVIDE BOTH THE QUINONE GATE AND A POTENTIAL PROTON CHANNEL ENTRY FOR A MINIMAL @PROTON PUMPING MODULE COMPOSED OF SUBUNITS .UO" .UO$ .UO) .UO( AND .UO, &RIEDRICH AND 3CHEIDE $UPUIS ET AL SEE ALSO +ASHANI 0OOR ET AL 3UBUNIT .UO, OR .UO- OR .UO. WHICH APPARENTLY EVOLVED BY TRIPLICATION OF AN ANCESTRAL GENE RELATED TO BACTE RIAL ( + ANTIPORTERS &EARNLEY AND 7ALKER &RIEDRICH AND 7EISS WOULD HAVE PROVIDED
THE TRANSMEMBRANE CHANNEL REQUIRED TO COMPLETE THE PROTON PUMP $UPUIS ET AL ***1IZTJPMPHJDBM3PMFTPG3FTQJSBUPSZ)Z ESPHFOBTFT "&OFSHZ$POTFSWBUJPOWJB&MFDUSPO5SBOTGFS 1BUIXBZT %LECTRON TRANSFER PATHWAYS IN BACTERIA ARE INITIATED BY A SERIES OF SUBSTRATE SPECIlC DEHYDROGENASES FEEDING ELECTRONS INTO A COMMON QUINONE POOL FROM WHICH ELECTRONS ARE TRANSFERRED VIA SPECIlC QUINOL OXIDASES TO TERMINAL REDUCTASES EG IN THE ABSENCE OF OXYGEN 4-!/ OR NITRATE OR FUMARATE REDUCTASE AND IN THE PRESENCE OF OXYGEN CYTOCHROME OXIDASE 4HE CELLULAR CONTENT OF SPECIlC DEHYDROGENASES TERMINAL REDUC TASES AND ALSO OF THE TYPE OF QUINONE EG UBIQUINONE 51 MENAQUINONE -+ OR DEMETHYLMENAQUINONE $-+ IN % COLI 3BALLE AND 0OOLE DEPENDS ON THE PREVAILING ENVIRONMENTAL CONDITIONS 2ICH ARDSON AND IT IS NOT POSSIBLE TO GIVE A GENERAL PICTURE VALID IN ALL CASES 4HE OVERSIMPLIlED SCHEME OF &IG IS MEANT TO EMPHASIZE THE ROLE OF THE QUINONE POOL IN RESPIRATION 4HIS TYPE OF ELECTRON TRANSFER CHAIN IS ALSO FOUND IN PHOTOSYNTHETIC ORGANISMS WHICH HAVE THE CAPACITY TO CONVERT LIGHT ENERGY INTO CHEMICAL ENERGY .!$0 ( AND !40 )N PURPLE PHOTOSYNTHETIC BACTERIA SUCH AS 2B CAPSULATUS THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN COMPRISES TWO MULTISUBUNIT TRANS MEMBRANE COMPLEXES THE PHOTOCHEMICAL REACTION CENTER 03 AND THE CYTOCHROME BC COMPLEX CONNECTED BY THE WATER SOLUBLE CYTOCHROME C ANDOR CYTOCHROME CY AND IN THE MEMBRANE DOMAIN BY UBIQUINONE 51 0HOTOSYNTHETIC ELECTRON TRANSPORT IS CYCLIC IN THE LIGHT 03 ACCEPTS THE ELECTRONS FROM #YT C #YT CY AND DO NATES THEM TO THE 51 POOL UBIQUINOL 51( IN TURN BEING OXIDIZED BY THE CYTOCHROME BC COMPLEX WHICH REDUCES CYTOCHROME C &IG SEE ALSO #HAPTER BY #OOLEY ET AL 6OL OF THE SERIES AND #HAPTER BY 6ERMEGLIO ET AL THIS VOLUME #YCLIC ELECTRON TRANSPORT GENERATES A PROTON MOTIVE FORCE 6P ACROSS THE MEMBRANE WHICH IS CONSUMED BY !40 SYNTHASE TO MAKE !40 #YCLIC ELECTRON TRANSPORT GENERATES !40 BUT NOT .!$( )N THIS SYSTEM .!$( IS FORMED IN PART BY DIRECT OXIDATION OF LOW POTENTIAL DONORS AND IN PART BY REVERSE ELECTRON mOW FROM 51( TO .!$ A REACTION WHICH CONSUMES 6P REVIEWED BY 'EST AND +NAFF SEE ALSO $UPUIS ET AL
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&IG 3IMPLIlED AND GENERAL SCHEME OF ELECTRON PATHWAYS IN BACTERIA 4HE ENERGY COUPLING SITES ARE INDICATED BY ARROWS SHOWING VECTORIAL PROTON EJECTION 4HE PROTON PUMPING ACTIVITY OF 2B CAPSULATUS ALTERNATIVE OXIDASE CYTB :ANNONI IS INDICATED BY A DASHED ARROW SINCE IT IS NOT A COMMON CASE AMONG THE SO CALLED @CYANIDE INSENSITIVE OXIDASES
(ERTER ET AL )N THE LATTER CASE THE DONOR CAN BE OF HIGHER REDOX POTENTIAL THAN .!$( PROVIDED THAT IT CAN REDUCE QUINONES 4HE PHOTOSYNTHETIC APPARATUS OF OXYGENIC PHO TOSYNTHETIC PROKARYOTES #YANOBACTERIA COMPRISES TWO PHOTOSYSTEMS 03)) AND 03) LINKED BY A LINEAR ELECTRON TRANSPORT CHAIN &IG SIMILAR TO THOSE OF HIGHER PLANT AND ALGAL CHLOROPLASTS &IG )N CYA NOBACTERIA PHOTOSYNTHETIC ELECTRON TRANSPORT TAKES PLACE IN THE THYLAKOID MEMBRANE WHILE RESPIRATORY ELECTRON TRANSPORT OCCURS IN BOTH THE THYLAKOID AND THE CYTOPLASMIC MEMBRANES 3CHERER 3CHMETTERER #OOLEY AND 6ERMAAS SEE ALSO #HAPTER BY ' 3CHMETTERER THIS VOLUME 4HE TWO PHOTOSYS TEMS OPERATING IN SERIES ARE CAPABLE OF OXIDIZING WATER TO / AND GENERATING !40 AND LOW POTENTIAL REDUCTANT FOR USE IN BIOSYNTHETIC REACTIONS )N 3YNECHOCYSTIS THE PROTON PUMPING COMPLEX .$( LOCALIZED IN THE THYLAKOID MEMBRANE /HKAWA ET AL ACTS AS A PLASTOQUINONE OXIDOREDUCTASE WITH .!$( OR .!$0( AS THE REDUCTANT 3CHEMETTERER &IG )N THE THYLAKOID MEMBRANE THIS COMPLEX HAS BEEN SHOWN TO PARTICIPATE IN THE CYCLIC ELECTRON mOW AROUND 03) -I ET AL 4HE DISCOVERY OF A PLASTID ENCODED .!$0 ( DE
HYDROGENASE .DH COMPLEX HOMOLOGOUS TO #OMPLEX ) AND OF A PLASTID TERMINAL OXIDASE 04/8 #OURNAC ET AL HOMOLOGOUS TO THE PLANT MITOCHONDRIAL ALTERNATIVE OXIDASE HAS PROVIDED MOLECULAR SUPPORT FOR THE EXISTENCE OF A RESPIRATORY ELECTRON TRANSPORT CHAIN WHICH INTERACTS WITH THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN IN THE THYLAKOID MEMBRANES OF CHLORO PLASTS 4HIS RESPIRATORY ACTIVITY IN CHLOROPLASTS CALLED CHLORORESPIRATION PARTICIPATES IN THE NONPHOTOCHEMI CAL REDUCTION AND OXIDATION OF PLASTOQUINONES 01S 7HETHER THIS ACTIVITY HAS OR NOT A BIOENERGETIC ROLE IN ITSELF REMAINS CONTROVERSIAL )N ADDITION CHLORO RESPIRATION MAY BE INVOLVED IN THE INITIATION OF CYCLIC ELECTRON TRANSFER REACTIONS AROUND 03) SEE REVIEW BY 0ELTIER AND #OURNAC #%JTQPTBMPG&YDFTT3FEVDJOH&RVJWBMFOUT !LTHOUGH A PRIMARY FUNCTION OF THE RESPIRATORY SYSTEM IS TO GENERATE A PROTONMOTIVE FORCE UTILIZED FOR !40 SYNTHESIS AND A VARIETY OF OTHER ESSENTIAL FUNCTIONS THE RESPIRATORY SYSTEM IS ALSO NECESSARY TO REGENERATE .!$ FROM .!$( AND TO ELIMINATE EXCESS REDUCING EQUIVALENTS 'ROWTH OF BACTERIA DEPENDS ON DISSIMILATORY AND
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&IG %LECTRON TRANSFER PATHWAYS INVOLVED IN ( PRODUCTION IN CHLOROPLASTS OF #HLAMYDOMONAS REINHARDTII !BBREVIATIONS SEE LEGEND TO &IG .$( TYPE )) .!$0 ( DEHYDROGENASE 04/8 PLASTID TERMINAL OXIDASE 4HE 4#! CYCLE TAKES PLACE IN THE STROMA 4HE FERREDOXIN FUNCTIONS AS ELECTRON DONOR LINKING THE (ASE TO THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN
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ASSIMILATORY PROCESSES /XIDATION OF INORGANIC OR ORGANIC SUBSTRATES RESULTS IN THE FORMATION OF REDUC ING POWER .!$( AND !40 WHICH IS USED TO DRIVE ASSIMILATORY PROCESSES LEADING TO THE SYNTHESIS OF CELL MATERIALS 'ROWTH RATES DEPEND ON !40 CONTENT AND THE PHOTO PHOSPHORYLATION RATE IS REGULATED BY REDOX BALANCE @/VER REDUCTION OR @OVER OXIDATION OF THE REDOX COMPONENTS OF THE ELECTRON TRANSPORT CHAIN INCLUDING THE QUINONE POOL LEADS TO INHIBI TION OF PHOSPHORYLATION "OSE AND 'EST 4HE REQUIREMENT FOR A MEMBRANE REDOX POISE CLOSE TO THE OXIDATION REDUCTION POTENTIAL OF THE UBIQUINONE POOL #ANDELA ET AL CAN BE EXPLAINED BY THE INVOLVEMENT OF A SEMIQUINONE INTERMEDIATE IN THE 1 CYCLE .ICHOLLS AND &ERGUSON $UTTON ET AL "RANDT 4O DISSIPATE EXCESS REDUCING EQUIVALENTS FROM THE PHOTOSYNTHETIC MEMBRANE BACTERIA SUCH AS 2B CAPSULATUS CAN USE AN ALTERNATIVE QUINOL OXIDASE WHICH ALLOWS THE CELL TO CONTROL THE REDOX STATE OF THE 1 POOL AND THE RATE OF PHOTOPHOSPHORYLATION ACTIVITY :ANNONI AND -ARRS 4HIS PATHWAY DRIVES A LIGHT INDUCED OXYGEN UPTAKE WHEN THE 1 POOL REDUCTION LEVEL REACHES :ANNONI 5NDER RESPIRATORY STEADY STATE CONDITIONS CONTINUOUS LIGHT DOES NOT PERTURB THE REDOX STATE OF THE 1 POOL WHEN THE QUINOL OXIDASE PATHWAY IS FUNCTIONING :ANNONI AND -OORE SEE ALSO #HAPTER BY 6ERMEGLIO ET AL THIS VOLUME $URING ANAEROBIOSIS AUXILIARY OXIDANTS SERVE TO SUSTAIN THE REDOX POISE OF THE 1 POOL FOR PHOTOSYN THETIC ELECTRON TRANSPORT -C%WAN ET AL &ERGU SON ET AL )N 2B CAPSULATUS UNDER ANAEROBIC CONDITIONS IN THE LIGHT EXCESS REDUCING EQUIVALENTS ARE TRANSFERRED TO .!$ BY REVERSE ELECTRON mOW THROUGH #OMPLEX ) +LEMME $UPUIS ET AL 2EDUCING EQUIVALENTS STORED IN .!$( CAN BE DISSIPATED BY METABOLIC SYSTEMS SUCH AS #/ lXATION #ALVIN CYCLE NITROGEN lXATION OR OXIDA TION OF AUXILIARY OXIDANTS (ILLMER AND 'EST 4ICHI ET AL )N THE CASE OF NITROGEN lXATION WHICH IS CATALYZED BY NITROGENASE ( IS PRODUCED AS AN INTRINSIC PART OF THE ENZYMATIC REACTION AND IN THE ABSENCE OF . NITROGENASE FUNCTIONS AS A (ASE REDUCING PROTONS TO ( 6IGNAIS ET AL 7ILLI SON 3INCE NITROGENASE IS AN !40 DEPENDENT ENZYME THIS REACTION DISSIPATES ENERGY AS WELL AS REDUCING EQUIVALENTS ( PRODUCTION AS A MECHANISM TO DISSIPATE REDUCING EQUIVALENTS IS ALSO OBSERVED IN SOME ALGAE )T IS THE
CASE FOR EXAMPLE WHEN DARK ADAPTED #HLAMYDO MONAS REINHARDTII CELLS ARE ILLUMINATED #OURNAC ET AL )N 3CENEDESMUS OBLIQUUS &LORIN ET AL 7àNSCHIERS ET AL B OR # REINHARDTII (APPE AND +AMINSKY THE ELECTRONS PROVIDED BY FERMENTATIVE METABOLISM ARE TRANSFERRED TO 03) VIA THE PLASTOQUINONE POOL PROBABLY THROUGH A TYPE .!$0 ( DEHYDROGENASE SEE 0ELTIER AND #OURNAC )N TURN 03) REDUCES A ;&E 3= FERREDOXIN THE PHYSIOLOGICAL ELECTRON DONOR TO ;&E= (ASE &IG URE SHOWS HOW ELECTRONS ORIGINATING AT 03)) UPON PHOTOOXIDATION OF WATER OR AT THE PLASTOQUINONE POOL UPON OXIDATION OF CELLULAR ENDOGENOUS SUBSTRATES ARE TRANSPORTED VIA 03) TO FERREDOXIN AND THEN SERVE EITHER TO REDUCE .!$0 TO .!$0( BY &.2 OR ( TO ( BY THE ;&E= (ASE -ELIS AND (APPE #OURNAC ET AL 4HE CYANOBACTERIUM 3YNECHOCYSTIS 0## CONTAINS AN .!$0 DEPENDENT BIDIRECTIONAL ;.I&E= (ASE LOCATED IN THE CYTOPLASM 3IGNIlCANT ( PRODUC TION WAS OBSERVED WHEN CELLS ACHIEVED ANAEROBIOSIS THE RATE OF ( PRODUCTION BEING HIGHER IN THE PRESENCE OF FERMENTATIVE SUBSTRATES SUCH AS GLUCOSE #OURNAC ET AL 4HE TRANSIENT ( BURST OBSERVED UPON ILLUMINATION !PPEL ET AL #OURNAC ET AL PROBABLY REmECTS THE INCREASE IN .!$0 ( CONCENTRATION IN RESPONSE TO 03) ACTIVITY !PPEL ET AL HAVE PROPOSED THAT THE BIDIRECTIONAL (ASE FUNCTIONS AS AN ELECTRON VALVE FOR THE DISPOSAL OF LOW POTENTIAL ELECTRONS GENERATED AT THE ONSET OF ILLUMINATION &IG SHOWS THAT THE PRODUCTION OF ( DEPENDS ON THE SUPPLY OF .!$0 ( .!$0 ( CAN ALSO DONATE ELECTRONS TO THE .$( COMPLEX BUT THE MECHANISM OF THIS TRANSFER IS NOT CLEAR 4HE ;.I&E= (ASE IS THEREFORE IN COMPETITION WITH .$( FOR THE .!$0 ( PRODUCED EITHER BY &.2 OR BY GLYCOLYSIS AND THE 4#! CYCLE &IG (OWEVER IT IS NOT YET CLEAR HOW .!$0 ( CAN TRANSFER ELECTRONS TO .$( SINCE THE 3YNECHOCYSTIS .$( COMPLEX LACKS OF THE USUAL SUBUNITS INCLUDING THE .!$0 ( BINDING SUBUNIT 3INCE HOMOLOGS OF THE MISSING SUBUNITS ARE PRESENT IN THE (ASE IT HAS BEEN SUGGESTED THAT THE BIDIRECTIONAL (ASE MAY INTERACT DIRECTLY WITH .$( THUS COMPLEMENTING ITS FUNCTION AS ILLUSTRATED IN &IG 3CHMITZ AND "OTHE !PPEL AND 3CHULZ ALTHOUGH SEE "OISON ET AL
$IBQUFS )ZESPHFO3FTQJSBUJPO *73FHVMBUJPOPG)ZESPHFOBTF(FOF&Y QSFTTJPO "$MVTUFSJOHPG)ZESPHFOBTF(FOFTJO1SP UFPCBDUFSJB 4HE 0ROTEOBACTERIA FORMERLY KNOWN AS @PURPLE BAC TERIA AND RELATIVES COMPRISE lVE SUBDIVISIONS AND FORM A LARGE GROUP OF PHENOTYPICALLY DIVERSE 'RAM NEGATIVE BACTERIA MANY OF WHICH ARE ENDOWED WITH ;.I&E= (ASES 4HE ORGANIZATION OF GENES ENCODING ;.I&E= (ASES HAS BEEN MOST EXTENSIVELY STUDIED IN THIS GROUP 4HE GENES ARE OFTEN FOUND TO BE CLUSTERED A FEATURE THAT HAS FACILITATED THEIR IDENTIlCATION AND THE STUDY OF THEIR PRODUCTS 4HE BEST STUDIED OF THE (ASE GENE CLUSTERS IE THOSE FROM THE _ 0ROTEOBACTERIA 2B CAPSULATUS 2 LEGUMINOSARUM AND " JAPONICUM THE ` 0ROTEOBACTERIUM 2 EUTROPHA AND THE a 0RO TEOBACTERIUM % COLI ARE ILLUSTRATED IN &IG 4HE DIFFERENT OPERONS HAVE BEEN TERMED VARI H UPPTAKE HOX FOR HYDROGEN H OUSLY HUP FOR HYDROGEN OXIDATION OR IN THE CASE OF % COLI HYA HYB HYC AND HYF FOR (ASES AND RESPECTIVELY )N EACH STRUCTURAL OPERON CODING FOR UPTAKE (ASE THE GENE ENCODING THE SMALL SUBUNIT PRECEDES THAT OF THE LARGE SUBUNIT FOLLOWED BY A THIRD GENE NAMED HUP# HOX: : HYA# # OR HYB" ENCODING A CYTOCHROME B THE % COLI (ASES AND ARE PART OF FORMATE HYDROGEN LYASE COMPLEXES AND EVOLVE ( 4HE STRUCTURAL GENES ARE FOLLOWED BY GENES NECESSARY FOR THE PROCESSING AND MATURATION OF THE ENZYME PROTEIN AND BY THE HYP GENES NECESSARY FOR THE INSERTION AT THE ACTIVE SITE OF THE .I AND THE &E ATOM WITH ITS LIGANDS #/ AND #. !LTOGETHER THERE ARE GENES INVOLVED IN THE BIOSYNTHESIS AND THE MATURATION OF THESE (ASES 4HE INVOLVEMENT OF HYP GENES IN THE ASSEMBLY OF THE .I&E ACTIVE CENTER IN % COLI (ASE HAS BEEN INTEN SIVELY STUDIED BY THE GROUP OF ! "ÚCK IN -UNICH (OMOLOGOUS HYP GENES ARE PRESENT IN ALL (ASE GENE CLUSTERS AND HAVE BEEN FOUND TO FUNCTION SIMILARLY IN % COLI AND IN OTHER PROTEOBACTERIAL ORGANISMS SEE RELEVANT REFERENCES CONCERNING THE FUNCTION OF VARIOUS ACCESSORY (ASE GENES IN THE REVIEWS OF &RIEDRICH AND 3CHWARTZ 6IGNAIS ET AL #ASALOT AND 2OUSSET "LOKESCH ET AL 6IGNAIS AND #OLBEAU #4JHOBMJOHBOE5SBOTDSJQUJPO$POUSPM 2EGULATION OF ;.I&E= (ASE BIOSYNTHESIS HAS MAINLY
BEEN STUDIED IN THE ORGANISMS LISTED IN &IG 3OME OF THESE ORGANISMS ARE ENDOWED WITH PHYSIOLOGICAL ATTRIBUTES ALLOWING THEIR GROWTH UNDER VERY DIVERSE ENVIRONMENTAL CONDITIONS AUTOTROPHICALLY OR HETERO TROPHICALLY IN THE LIGHT OR IN DARKNESS AEROBICALLY OR ANAEROBICALLY 4HE CONTROL OF (ASE SYNTHESIS REPRESENTS A MEANS TO QUICKLY AND EFlCIENTLY RESPOND TO CHANGES IN THE ENVIRONMENT AND IN PARTICULAR TO NEW ENERGY DEMANDS 4HIS CONTROL IS EXERTED AT THE TRANSCRIPTION LEVEL AS DEMONSTRATED IN PARTICULAR BY THE USE OF TRANSCRIPTIONAL REPORTER SYSTEMS MEASURING LEVELS OF PROMOTER ACTIVITY OF STRUCTURAL OPERONS #OL BEAU AND 6IGNAIS (ASE SYNTHESIS RESPONDS TO THREE MAIN SIGNALS A -OLECULAR HYDROGEN WHICH IS ALSO THE SUBSTRATE ACTIVATES (ASE GENE EXPRESSION IN AEROBIC BACTERIA EG 2 EUTROPHA IN PHOTOSYNTHETIC BACTERIA EG 2B CAPSULATUS AND IN FREE LIVING 2HIZOBIA EG " JAPONICUM )N ALL THESE BACTERIA THE REGULATORY CAS CADE RESPONDING TO ( USES THE SAME ELEMENTS B -OLECULAR OXYGEN IS AN INHIBITORY SIGNAL FOR MOST OF THE (ASES )N SYMBIOTIC 2HIZOBIA THE REGULATION OF (ASE GENE EXPRESSION IS LINKED TO THE EXPRESSION OF NITROGEN lXATION GENES C -ETABOLITES FUNCTIONING AS ELECTRON DONORS OR AC CEPTORS SUCH AS FORMATE NITRATE OR SULFUR REGULATE THE EXPRESSION OF (ASES 3FTQPOTFUP) 4HE PRODUCTS OF FOUR GENES FUNCTION TOGETHER AS A ( SPECIlC REGULATORY CASCADE WHICH CONTROLS THE TRANSCRIPTION OF THE STRUCTURAL OPERONS HUP3,# IN 2B CAPSULATUS AND " JAPONICUM AND HOX+': IN 2 EUTROPHA 4HE ( SPECIlC REGULATORY SYSTEM COMPRISES I AN ( SENSING ( (ASE 6IGNAIS ET AL ,ENZ AND &RIEDRICH ENCODED BY THE HUP56 GENES IN " JAPONICUM "LACK ET AL ANDD 2B CAPSULATUS %LSEN ET AL AND BY THE HOMOLOGOUS HOX"# # GENES IN 2 EUTROPHA ,ENZ ET AL &IG 4HIS ( SENSING (ASE ALSO CALLED REGULATORY (ASE +LEIHUES ET AL IS THE ( SENSOR FOR A TWO COMPONENT REGULATORY SYSTEM WHICH CONTROLS THE TRANSCRIPTION OF THE STRUCTURAL (ASE OP ERONS II A TWO COMPONENT REGULATORY SYSTEM WHICH CONSISTS OF A PROTEIN HISTIDINE KINASE TERMED (UP4 IN 2B CAPSULATUS %LSEN ET AL AND IN
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&IG %XAMPLES OF ;.I&E= (ASE GENE CLUSTERS AND TRANSCRIPTIONAL FACTORS REGULATING GENE EXPRESSION IN SOME 0ROTEOBACTERIA )N 2B CAPSULATUS 2 CAP (ASE EXPRESSION IS ACTIVATED BY AN ( RESPONDING SPECIlC SYSTEM (UP56(UP4(UP2 AND REPRESSED BY THE GLOBAL REDOX SENSING TWO COMPONENT REGULATORY SYSTEM 2EG"! )N 2 EUTROPHA 2 EUT THE (OX"#(OX*(OX! IS HOMOLOGOUS TO (UP56 (UP4(UP2 FROM 2B CAPSULATUS 4HE SAME REGULATORY SYSTEM IS INVOLVED IN FREE LIVING " JAPONICUM CELLS NOT SHOWN )N BACTEROID CELLS EXPRESSION OF (ASE IS LINKED TO THAT OF NITROGENASE BY THE INTERMEDIARY OF THE TWO COMPONENT SYSTEM &IX,* IN " JAPONICUM " JAP OR THE .IF! REGULATOR IN 2 LEGUMINOSARUM 2 LEG 4HE SCHEME FOR % COLI CONCERNS THE CONTROL OF THE EXPRESSION OF (ASE
$IBQUFS )ZESPHFO3FTQJSBUJPO " JAPONICUM 6AN 3OOM ET AL AND (OX* IN 2 EUTROPHA ,ENZ ET AL ,ENZ AND &RIEDRICH AND A RESPONSE REGULATOR AN .TR# LIKE TRAN SCRIPTION FACTOR TERMED (UP2 2ICHAUD ET AL 4OUSSAINT ET AL $ISCHERT ET AL OR (OX! :IMMER ET AL $URMOWICZ AND -AIER 6AN 3OOM ET AL 4HE .TR# SUPERFAMILY OF RESPONSE REGULATORS PRES ENTS THREE DOMAINS THE . TERMINAL DOMAIN WITH THE CONSERVED PHOSPHORYLABLE ASPARTATE RESIDUE A CENTRAL DOMAIN WITH 7ALKER MOTIFS FOR !40 BINDING AND A # TERMINAL DOMAIN WITH A HELIX TURN HELIX MOTIF )N HUP2 MUTANTS 2ICHAUD ET AL 4OUSSAINT ET AL EXPRESSION OF (ASE NO LONGER RESPONDED TO THE PRESENCE OF ( AND WAS EVEN ABOLISHED %XPRES SION OF THE RESPONSE REGULATOR (OX! GENE IS ITSELF REGULATED IN 2 EUTROPHA 3CHWARZ ET AL WHILE (UP2 IN 2B CAPSULATUS IS CONSTITUTIVELY EXPRESSED $ISCHERT ET AL 4HE GENE CODING FOR THE HISTIDINE KINASE HUP4 IS 4 LOCALIZED UPSTREAM FROM THE (ASE STRUCTURAL GENES IN 2B CAPSULATUS &IG AND IS COTRANSCRIBED WITH THE HUP5 5 AND HUP6 GENES %LSEN ET AL 6 (UP4 MUTANTS EXHIBIT HIGH (ASE ACTIVITIES EVEN IN ABSENCE OF ( INDICATING THAT (UP4 NEGATIVELY CONTROLS (ASE GENE EXPRESSION %LSEN ET AL 4HIS IS THE OP POSITE PHENOTYPE TO THAT SHOWN BY HUP2 MUTANTS (OWEVER THE TWO TYPES OF MUTANT ARE SIMILAR IN THAT EXPRESSION OF THEIR UPTAKE (ASE DOES NOT RESPOND TO ( SUGGESTING THAT BOTH PROTEINS ARE INVOLVED IN THE TRANSDUCTION OF THE ( SIGNAL )NDEED IT WAS LATER CLEARLY DEMONSTRATED THAT THE (UP4 AND (UP2 PROTEINS ACT AS PARTNERS OF A TWO COMPONENT REGULA TORY SYSTEM WHICH REGULATES THE EXPRESSION OF THE HUP3,# # (ASE GENES IN 2B CAPSULATUS $ISCHERT ET AL 4HE (UP4 HISTIDINE KINASE IS CAPABLE OF AUTOPHOS PHORYLATION IN THE PRESENCE OF ;0= a !40 %LSEN ET AL AND IN THE PRESENCE OF (UP4 0 THE RESPONSE REGULATOR (UP2 BECOMES PHOSPHORYLATED $ISCHERT ET AL &OOTPRINTING EXPERIMENTS AND IN VIVO : EXPERIMENTS WITH PLASMID BORNE HUP3LAC: FUSIONS HAVE DEMONSTRATED THAT THE TRANSCRIPTION FACTOR (UP2 BINDS TO THE HUP33 PROMOTER AND PROTECTS A PALINDROMIC SEQUENCE 44' . #!! LOCALIZED AT NT FROM THE TRANSCRIPTION START SITE 4OUSSAINT ET AL $ISCHERT ET AL !LTHOUGH (UP2 BINDS TO AN ENHANCER SITE OF THE HUP3 PROMOTER AND REQUIRES )(& 3 TO ACTIVATE TRANSCRIPTION 4OUSSAINT ET AL THE 2B CAPSULATUS HUP3, GENES ARE TRANSCRIBED BY A m LINKED 2.! POLYMERASE $ISCHERT ET AL 4HIS
IS A RATHER EXCEPTIONAL CASE SINCE ENHANCER BINDING TRANSCRIPTION FACTORS USUALLY ACTIVATE m DEPENDENT 2.! POLYMERASE AS IS THE CASE FOR THE ACTIVATION OF HOX+' ' GENE TRANSCRIPTION IN 2 EUTROPHA 2ÚMERMANN ET AL OR HUP3, TRANSCRIPTION IN " JAPONICUM "LACK AND -AIER ! SECOND PECULIARITY CONCERNING THE FUNCTIONING OF THE TWO COMPONENT (UP4(UP2 AND (OX*(OX! REG ULATORY SYSTEMS IS THAT THE RESPONSE REGULATOR (UP2 OR (OX! ACTIVATES THE TRANSCRIPTION OF (ASE GENES IN THE NONPHOSPHORYLATED FORM ,ENZ AND &RIEDRICH $ISCHERT ET AL IN CONTRAST TO .TR# IN ENTERIC BACTERIA WHICH ACTIVATES TRANSCRIPTION UNDER THE PHOSPHORYLATED FORM !USTIN AND $IXON 4HIS WAS DEMONSTRATED IN VIVO BY COMPLEMENTING A HUP2 NULL MUTANT OF 2B CAPSULATUS WITH MUTATED FORMS OF THE (UP2 PROTEIN IN WHICH THE CONSERVED PHOSPHORYLABLE !SP RESIDUE HAD BEEN REPLACED BY OTHER AMINOACIDS OR HAD BEEN DELETED )N ALL CASES THE MUTANT (UP2 PROTEINS FULLY ACTIVATED (UP3, SYNTHESIS $ISCHERT ET AL 3IMILAR TYPES OF EXPERIMENT HAVE DEMONSTRATED THAT (OX! IN 2 EUTROPHA IS ACTIVE IN THE NONPHOSPHORYLATED FORM ,ENZ AND &RIEDRICH AND A SIMILAR CONCLUSION WAS REACHED FOR " JAPONICUM (OX! 6AN 3OOM ET AL 4HE RELATIONSHIPS BETWEEN THE ( SENSOR (UP56 THE (UP4(UP2 REGULATORY SYSTEM AND THE PROMOTER OF THE HUP3, (ASE ARE SCHEMATIZED IN &IG !LTHOUGH IN 2B CAPSULATUS 2 EUTROPHA AND " JAPONICUM THE REGULATORY CASCADE IS SIMILAR 2B CAPSULATUS (UP56 n MUTANTS HAVE HIGH LEVEL OF (ASE ACTIVITY WHILE IN 2 EUTROPHA AND " JAPONI CUM MUTANTS DEFECTIVE IN THE ( SENSOR ARE DEVOID OF (ASE ACTIVITY 3FTQPOTFUP0 BOE3FEPY3FHVMBUJPO 'LOBAL REGULATORY PROTEINS WITH COFACTORS THAT ARE ABLE EITHER TO BIND / HEME AND mAVINS OR TO TRANSFER ELECTRONS HEME mAVINS AND &E 3 CLUSTERS $IXON REVIEWED BY "AUER ET AL "EINERT AND +ILEY 3AWERS FUNCTION IN BIOLOGICAL / SENSING PATHWAYS 4HE % COLI ANAEROBIC GLOBAL REGULATOR &NRR FOR F FUMARATE NITRATE REDUCTION 3PIRO AND 'UEST +ILEY AND "EINERT IS A CYTOPLASMIC / RE SPONSIVE REGULATOR WITH A SENSORY AND A REGULATORY $.! BINDING DOMAIN &NR REGULATES TRANSCRIPTION INITIATION IN RESPONSE TO OXYGEN STARVATION )T ACTIVATES THE TRANSCRIPTION OF GENES INVOLVED IN ANAEROBIC RE SPIRATORY PATHWAYS WHILE REPRESSING THE EXPRESSION
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&IG 2EGULATORY CASCADE IN RESPONSE TO ( IN 2B CAPSULATUS )N THE ABSENCE OF ( THE HETERODIMER (UP56 INTERACTS WITH THE HIS TIDINE KINASE (UP4 %LSEN ET AL WHICH IS ABLE TO AUTOPHOSPHORYLATE ON THE (IS RESIDUE 4HE TRANSFER OF THE PHOSPHATE GROUP INACTIVATES THE RESPONSE REGULATOR (UP2 )N THE PRESENCE OF ( SENSED BY (UP56 THE INTERACTION OF (UP56 WITH (UP4 IS BLOCKED OR MODIlED !S A RESULT THE REGULATOR (UP2 REMAINS IN AN UNPHOSPHORYLATED ACTIVE FORM WHICH IS ABLE TO ACTIVATE THE TRANSCRIPTION OF THE # HUP4 4 AND HUP56 6 MUTANTS ON THE ONE HUP3,# GENES $ISCHERT ET AL 4HIS SCHEME EXPLAINS THE OPPOSING PHENOTYPES OBSERVED IN HAND AND IN HUP2 MUTANTS ON THE OTHER
OF GENES INVOLVED IN AEROBIC ENERGY GENERATION )UCHI AND ,IN 5PSTREAM REGIONS OF &NR REGULATED GENES ARE CHARACTERIZED BY &NR CONSENSUS SEQUENCES OF DYAD SYMMETRY 44'!4 . !4#!! TO WHICH THE PROTEIN BINDS AS A DIMER &NR ACTIVITY DEPENDS ON THE PRESENCE OF A ;&E 3= CLUSTER WHICH IS LABILE IN THE PRESENCE OF / BEING CONVERTED RAP IDLY TO A MORE / STABLE ;&E 3= FORM +ILEY AND "EINERT 4HAT THE / SENSING ABILITY OF &NR IS RELATED TO THE / LABILITY OF THE ;&E 3= CLUSTER WAS DEMONSTRATED USING A MUTANT &NR PROTEIN BEARING A SUBSTITUTION OF ,EU WITH (IS &NR ,( WHICH STABILIZES THE ;&E 3= CLUSTER IN THE PRESENCE OF / 4HE MUTANT PROTEIN WAS FUNCTIONAL UNDER AEROBIC CONDITIONS "ATES ET AL &NR HOMOLOGS ARE PRESENT IN OTHER 0ROTEOBACTERIA AND HAVE BEEN SHOWN TO BE REQUIRED FOR GENE EXPRESSION UNDER ANAEROBIC OR MICROAEROBIC CONDITIONS 4HEY ARE CLASSIlED INTO TWO TYPES &NR LIKE SUCH AS &IX+ IN " JAPONICUM OR &NR. IN 2 LEGUMINOSARUM AND &IX+ LIKE SUCH AS &IX+ IN " JAPONICUM &IX+ LIKE PROTEINS LACK THE REDOX SENSITIVE CYSTEINES AND ARE ACTIVATED BY AN ASSOCIATED / SENSITIVE TWO COMPONENT SYSTEM &IX,* 'UTTIÏREZ ET AL &IG 2HIZOBIA CAN GROW IN SYMBIOSIS WITH LEGUMINOUS PLANTS FORMING BACTEROIDS THAT ARE LOCATED WITHIN SPECIlC ROOT NODULES 5NDER THESE CONDITIONS EX PRESSION OF (ASE IS INCLUDED IN A COMPLEX NETWORK COORDINATING THE PROCESS OF . lXATION )N " JA PONICUM THE SENSING OF LOW / CONCENTRATIONS AND SIGNAL TRANSDUCTION ARE ORGANIZED IN TWO REGULATORY CASCADES INVOLVING THE ACTIVATORS &IX+ AND .IF! FOR HUP AND NIF GENES RESPECTIVELY 4HE TRANSCRIPTION OF F (ASE STRUCTURAL GENES IS ACTIVATED BY $.! BINDING OF &IX+ WHOSE EXPRESSION DEPENDS ON THE &IX,&IX*
TWO COMPONENT REGULATORY SYSTEM $URMOWICZ AND -AIER 4HE TRANSMEMBRANE SENSOR &IX, HAS A HEME BINDING DOMAIN WHICH BELONGS TO THE 0!3 SUPERFAMILY 4AYLOR AND :HULIN 4HE LOSS OF / FROM &IX, INDUCES A CONFORMATIONAL CHANGE WHICH ACTIVATES ITS KINASE ACTIVITY 'ILLES 'ONZALES ET AL 'ONG ET AL TRIGGERS THE PHOSPHORYLATION OF &IX* AND INITIATES THE REGULATORY CASCADE AT REDUCED LEVELS OF / " JAPONICUM CONTAINS TWO HOMOLOGS OF &IX+ BUT ONLY &IX+ AND &IX* MUTANTS ARE DEVOID OF (ASE ACTIVITY (UPn AND ARE ALSO .IFFn 4HE (ASE PROMOTER PRESENTS TWO POTENTIAL BINDING SITES FOR &IX+ 44'! # '!4#!! ' )N " JAPONICUM BESIDES THE (ASE GENES &IX+ REGULATES THE EXPRES SION OF THE lX./10CCO./10 0 OPERON ENCODING A TERMINAL OXIDASE WITH A HIGH AFlNITY FOR / RPO. lX+ AND SEVERAL GENES INVOLVED IN NITRATE METABOLISM IN RESPONSE TO LOW OXYGEN CONCENTRATIONS !S IN " JAPONICUM (ASE GENE TRANSCRIPTION IN 2 LEGUMINOSARUM IS CO REGULATED WITH THAT OF NITROGE NASE GENES )T IS CONTROLLED BY TWO GLOBAL REGULATORS .IF! AND &NR. IN RESPONSE TO LOW OXYGEN CONCENTRA TIONS IN THE NODULES (OWEVER IN THIS BACTERIUM .IF! DIRECTLY ACTIVATES (ASE GENE EXPRESSION BY BINDING TO AN 5!3 OF THE PROMOTER REGION OF THE STRUCTURAL HUP3, GENES 4HIS PROMOTER ALSO BINDS )(& AND IS TRANSCRIBED BY A m 2.! POLYMERASE )N ADDITION &NR. INDIRECTLY ACTIVATES (ASE GENE EXPRESSION BY BINDING TO THE m DEPENDENT PROMOTER OF THE HYP . GENE 'UT GENES 4HERE ARE TWO COPIES OF THE FNR. TIÏREZ ET AL AND ONLY DOUBLE MUTANTS FORM INEF F FECTIVE NODULES LACKING BOTH (ASE AND NITROGENASE #OLOMBO ET AL !NOTHER REDOX DEPENDENT SENSORREGULATOR SYSTEM IS THE !RC"!RC! REGULATORY SYSTEM OF % COLI )UCHI
$IBQUFS )ZESPHFO3FTQJSBUJPO AND ,IN !RC" IS A TRANSMEMBRANE SENSOR PROTEIN THE HISTIDINE KINASE ACTIVITY OF WHICH IS STIMULATED IN ABSENCE OF / !RC" CAN THEN AUTOPHOS PHORYLATE AND THE PHOSPHORYL GROUP IS TRANSFERRED TO A CONSERVED ASPARTATE OF F !RC! 5PON PHOSPHORYLATION ANOXIC CONDITIONS !RC! IS CONVERTED TO !RC PHOS PHATE !RC 0 THE ACTIVE FORM WHICH REPRESSES TARGET GENES OF AEROBIC METABOLISM AND ACTIVATES GENES OF ANAEROBIC METABOLISM 1UINONES ARE REDOX SIGNALS FOR THE !RC SYSTEM /XIDIZED FORMS OF QUINONE ELECTRON CARRIERS INHIBIT AUTOPHOSPHORYLATION OF !RC" DURING AEROBIOSIS THUS PROVIDING A LINK BETWEEN THE ELECTRON TRANSPORT CHAIN AND GENE EXPRESSION 'EORGELLIS ET AL 4HIS TWO COMPONENT SYSTEM IS INVOLVED IN THE SYNTHESIS OF (ASE AND (ASE OF % COLI ENCODED BY HYA AND HYB OPERONS RESPECTIVELY (OW EVER WHILE !RC! ACTIVATES THE EXPRESSION OF THE HYA GENES IT REPRESSES THAT OF THE HYB GENES 2ICHARD ET AL % COLI (ASE IS ENCODED BY THE HYA!"#$%& OPERON )TS PHYSIOLOGICAL ROLE IS UNCLEAR BUT IT IS GENERALLY ASSUMED THAT IT RECYCLES THE HYDROGEN PRODUCED BY (ASE )T IS ACTIVE UNDER FERMENTA TIVE CONDITIONS GROWTH ON GLUCOSE AND FORMATE OR DURING ANAEROBIC RESPIRATION GROWTH ON GLYCEROL AND FUMARATE !NAEROBIC EXPRESSION OF THE HYA OP ERON IS INDEPENDENT OF &NR AND IS INDUCED BY TWO GLOBAL REGULATORS !RC! AND !PP9 $EPENDING ON THE GROWTH CONDITIONS THE !RC! AND !PP9 PROTEINS ACT EITHER COOPERATIVELY OR INDEPENDENTLY !PP9 IS ITSELF REGULATED BY A TWO COMPONENT REDOX SENSI TIVE SYSTEM $PI!" WHICH FUNCTIONS IN AEROBIOSIS AND REPRESSES !RC! SYNTHESIS IN AEROBIOSIS )NGMER ET AL 4HUS THE HYA OPERON IS CONTROLLED BY TWO TWO COMPONENT REDOX SENSITIVE SYSTEMS ONE ACTIVE UNDER ANAEROBIC CONDITIONS !RC"!RC! AND THE OTHER !PP9$PI!" UNDER AEROBIC CONDITIONS 2ICHARD ET AL 4HE TRANSCRIPTIONAL REGULATION OF THE HYB/!"#$%&' ' OPERON OF % COLI CODING FOR (ASE IS LESS WELL UNDERSTOOD THAN THAT OF THE OTHER % COLI (ASES !NAEROBIC INDUCTION IS ONLY SLIGHTLY AFFECTED BY FNRR MUTATIONS AND SEEMS TO BE NEGATIVELY REGULATED BY !RC! BUT UNAFFECTED BY !PP9 /XIDATION OF ( BY UPTAKE (ASE PRODUCES ELECTRONS THAT CAN SUPPLY VARIOUS ENERGY CONSUMING PROCESSES SUCH AS #/ REDUCTION NITROGEN lXATION AND !40 SYNTHESIS &IG )N 2B CAPSULATUS A GLOBAL TWO COMPONENT SIGNAL TRANSDUCTION SYSTEM 2EG"2EG! ALSO CALLED 0RR"0RR! IN 2B SPHAEROIDES WHICH WAS ORIGINALLY FOUND TO REGULATE KEY OPERONS INVOLVED IN PHOTOSYNTHESIS REVIEWED BY "AUER ET AL AND
MORE RECENTLY TO CONTROL RESPIRATORY ELECTRON TRANSFER COMPONENTS 3WEM ET AL IS IMPLICATED ALSO IN THE CONTROL OF #/ lXATION 1IAN AND 4ABITA 6ICHIVANIVES ET AL AS WELL AS NITROGEN lXATION AND ( OXIDATION *OSHI AND 4ABITA 1IAN AND 4ABITA %LSEN ET AL )N 2B CAPSULATUS THE 2EG! PROTEIN REGULATES NEGATIVELY THE EXPRESSION OF (ASE BY BINDING TO PHUP3 THE PROMOTER OF THE STRUCTURAL (ASE GENES 2EG" AND 2EG! DEFECTIVE MUTANT STRAINS HAVE THREE TO lVE TIMES MORE ACTIV ITY THAN THE WILD TYPE )NTERESTINGLY THIS EFFECT WAS OBSERVED UNDER BOTH AEROBIC AND ANAEROBIC GROWTH CONDITIONS %LSEN ET AL "Y FOOT PRINTING EX PERIMENTS TWO BINDING SITES FOR 2EG! WERE FOUND A HIGH AFlNITY SITE LOCATED BETWEEN THE BINDING SITES FOR THE (UP2 REGULATOR AND FOR 2.! POLYMERASE AND A LOW AFlNITY SITE OVERLAPPING THE BINDING SITE FOR THE GLOBAL REGULATOR )(& %LSEN ET AL 4HE EFFECT OF THE 2EG0RR SYSTEM IS MEDIATED BY A REGULATORY CASCADE ORIGINATING FROM THE RESPIRATORY CHAIN 4HIS CASCADE HAS BEEN ELUCIDATED IN 2B SPHAER R OIDES BY THE GROUP OF +APLAN %RASO AND +APLAN 4HE SIGNAL COMES FROM THE CBB CYTOCHROME C OXIDASE VIA ITS INTERACTION WITH / IN CONJUNCTION WITH THE REDOX PROTEIN 2DX 4HE CBB CYTOCHROME C OXIDASE PRESENT IN CELLS GROWN EITHER IN AEROBIOSIS OR ANAEROBIOSIS SHOWS PROPERTIES OF AN / SENSOR )T HAS A HIGH AFlNITY FOR / BUT IT IS THE RATE OR THE VOLUME OF THE ELECTRON mUX THROUGH THE CBB #YT C OXIDASE WHICH IS THE SIGNAL RATHER THAN THE BINDING OF / TO THE OXIDASE PER SE /H AND +APLAN 4HE CONFORMATION OF THE CBB OXIDASE CHANGES WITH THE VOLUME OF THE ELECTRON mUX THIS LONG RANGE CON FORMATIONAL CHANGE IN THE CBB OXIDASE IS SENSED AND ULTIMATELY TRANSPONDED BY #CO1 SUBUNIT TO THE TWO COMPONENT 0RR"0RR! SYSTEM BY THE INTERMEDIARY OF THE MEMBRANE LOCALIZED 0RR# POLYPEPTIDE REVIEWED BY /H AND +APLAN SEE ALSO #HAPTER BY +A PLAN AND /H 6OL OF THIS SERIES AND #HAPTER BY 6ERMEGLIO ET AL PRESENT VOLUME )N 2B CAPSULATUS SIGNAL TRANSDUCTION BY 2EG" IS MEDIATED BY A REDOX ACTIVE CYSTEINE 3WEM ET AL 5NDER OXIDIZING CONDITIONS FORMATION OF AN INTERMOLECULAR DISULlDE BOND CONVERTS THE 2EG" KINASE FROM AN ACTIVE DIMER INTO AN INACTIVE TETRAMER STATE )N " JAPONICUM THE TWO COMPONENT SYSTEM 2EG32EG2 IS HOMOLOGOUS TO 0RR"0RR! OR 2EG"2EG! (OWEVER IT IS NOT KNOWN IF THIS SYSTEM REGULATES (ASE SYNTHESIS 5NDER SYM BIOTIC CONDITIONS (ASE SYNTHESIS IS REGULATED BY THE &IX,* &IX+ SYSTEM SEE ABOVE WHICH IS INDEPENDENT OF 2EG32EG2 %MMERICH ET AL
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3FTQPOTFUP.FUBCPMJUFT &MFDUSPOEPOPST PS"DDFQUPST B'PSNBUF3FHVMBUJPO % COLI (ASE ENCODED BY THE HYC OPERON IS THE (ASE COMPONENT OF FORMATE HYDROGEN LYASE &(, WHICH EVOLVES ( UNDER FERMENTING GROWTH CONDITIONS AND IS REGULATED BY THE INTRACELLULAR CONCENTRATION OF FORMATE 4HE GENES CODING FOR % COLI (ASE ALSO BELONG TO THE FORMATE REGULON 2OSSMANN ET AL WHICH IS REGULATED BY THE TRANSCRIPTIONAL REGULATOR &HL! &HL! SHARES HOMOLOGY WITH m DEPENDENT REGULATORS OF THE .TR# FAMILY IN ITS CENTRAL AND # TER MINAL DOMAINS BUT DIFFERS IN POSSESSING AN EXTENDED . TERMINAL DOMAIN LACKING THE ASPARTATE RESIDUE WHICH IS THE SITE OF PHOSPHORYLATION OF RESPONSE REGULATORS 4HUS &HL! IS NOT ACTIVATED BY PHOSPHORYLATION BUT BY BINDING AN EFFECTOR MOLECULE FORMATE &HL! IS AN HOMOTETRAMER WHICH BINDS TO AND ACTI VATES THE HYC HYP FHL& AND & HYD.HYP& & PROMOTERS4HE HYC AND HYP OPERONS ARE DIVERGENTLY TRANSCRIBED &HL! BINDS IN THE INTERGENIC REGION AND ACTIVATES THE HYC OPERON &IG !N ADDITIONAL BINDING SITE BETWEEN THE HYC! CODING FOR A REPRESSOR AND THE HYC" GENES IS RESPONSIBLE OF THE ACTIVATION OF THE HYP OPERON 4HUS THE REGULATOR &HL! CONTROLS THE EXPRESSION OF THE STRUCTURAL AND ACCESSORY GENES OF (ASE 4HESE PROMOTERS ARE OF THE m TYPE AND ARE ACTIVATED BY THE GLOBAL REGULATOR )(& 2OSSMAN ET AL 4HE (ASE OPERON HYF OF F % COLI IS REGULATED BY THE m DEPENDENT ACTIVATORS &HL! THUS HYF EXPRESSION F RESEMBLES THAT OF THE HYC OPERON AND (YF2 RELATED TO &HL! (YF2 DEPENDENT INDUCTION IS DEPENDENT ON LOW P( ANAEROBIOSIS AND POSTEXPONENTIAL GROWTH AND IS WEAKLY ENHANCED BY FORMATE (OWEVER NO HYF RE F LATED HYDROGENASE OR FORMATE DEHYDROGENASE ACTIVITY HAS SO FAR BEEN DETECTED 3KIBINSKI ET AL 4HE PARTICULAR CASE DESCRIBED ABOVE CONCERNS FERMENTATION RATHER THAN RESPIRATION .O EVIDENCE HAS YET BEEN OBTAINED FOR THE REGULATION OF UPTAKE (ASES BY SPECIlC METABOLIC INTERMEDIATES 'LOBAL REGULATORY NETWORKS HAVE BEEN DISCOVERED IN SOME BACTERIA THAT CONTROL BOTH CARBON AND HYDROGEN ME TABOLISM AS WELL AS NITROGEN METABOLISM EG 1IAN AND 4ABITA C$BSCPO.POPYJEF3FHVMBUJPO )N 2 RUBRUM #/ INDUCES THE SYNTHESIS OF #OO,(
(ASE 4HIS SYNTHESIS IS ACTIVATED BY #OO! #/ OXIDATION ACTIVATOR A HEME CONTAINING TRANSCRIPTION FACTOR MEMBER OF THE #!0#20 &.2 SUPERFAMILY OF REGULATORY PROTEINS ,ANZILOTTA ET AL #OO! IS UNABLE TO BIND #/ WHEN THE &E HEME IS OXIDIZED UPON REDUCTION THERE IS AN UNUSUAL SWITCH OF PROTEIN LIGANDS TO THE SIX COORDINATE HEME AND THE REDUCED HEME IS ABLE TO BIND #/ #/ BINDING STABILIZES A CONFORMATION OF THE DIMERIC PROTEIN THAT ALLOWS SEQUENCE SPECIlC $.! BINDING AND TRANSCRIPTION IS ACTIVATED THROUGH CONTACTS BETWEEN #OO! AND 2.! POLYMERASE 4HUS THE #OO! DIMER FUNCTIONS BOTH AS A REDOX SENSOR AND AS A SPECIlC #/ SENSOR !ONO ET AL REVIEWED BY 2OBERTS ET AL !ONO D/JUSBUF3FHVMBUJPO %XPRESSION OF HYA AND HYB OPERONS ARE STRONGLY REPRESSED IN PRESENCE OF NITRATE REPRESSION BEING MEDIATED BY THE TWO COMPONENT SYSTEMS .AR1.AR0 AND ESPECIALLY .AR8.AR, 3EVERAL POTENTIAL BINDING SITES FOR THESE REGULATORS ARE PRESENT ON PHYA AND PHYB 2ICHARD ET AL (OWEVER IT HAS BEEN SUGGESTED FOR (ASE THAT THIS REPRESSION COULD BE INDIRECTLY LINKED TO !RC! )NDEED PHOSPHORYLATION OF !RC! BY !RC" DEPENDS ON THE RESPIRATORY STATE OF THE CELL WHEN NITRATE IS PRESENT !RC! MAY BE MORE ACTIVATED THAN IN ITS ABSENCE "RNSTED AND !TLUNG -ANY ( OXIDIZING BACTERIA SUCH AS 2 EUTROPHA ! EUTROPHUS ARE ALSO CAPABLE OF NITRATE RESPIRATION AND DENITRIlCATION AND ARE ABLE TO GROW ANAEROBICALLY WITH ( AS ELECTRON DONOR AND NITRATE AS ELECTRON ACCEPTOR (OWEVER NO REGULATORY LINK HAS YET BEEN ESTABLISHED BETWEEN ( UPTAKE AND THE PRESENCE OF NITRATE OR ANY OF THE DENITRIlCATION PATHWAY INTERMEDIATES )N 2 EUTROPHA ( ( OXIDATION AND NITRATE REDUCTION ARE BOTH UNDER THE CONTROL OF THE m FACTOR OF 2.! POLYMERASE AND PLEIOTROPIC MUTANTS DElCIENT IN BOTH ACTIVITIES HAVE BEEN ISOLATED 2ÚMERMANN ET AL 4HE GENES FOR UPTAKE (ASE ARE LOCATED ON A KB MEGAPLASMID IN THIS STRAIN AND THE ELIMINATION OF THIS PLASMID RESULTED IN THE SIMULTANEOUS LOSS OF ( OXIDATION AND NITRATE REDUCTION SUGGESTING THAT DENITRIlCATION GENES MIGHT ALSO BE PLASMID LOCATED (OWEVER THE LOSS OF THE ABILITY TO GROW WITH NITRATE WAS SUBSEQUENTLY FOUND TO BE DUE TO THE PRESENCE ON THE MEGA PLASMID OF A GENE FOR ANAEROBIC RIBO NUCLEOTIDE REDUCTASE WHICH IS ABSOLUTELY REQUIRED FOR ANAEROBIC GROWTH 3IEDOW ET AL
$IBQUFS )ZESPHFO3FTQJSBUJPO ! REGULATORY INTERACTION BETWEEN (ASE ACTIVITY AND THE AEROBIC ELECTRON TRANSPORT CHAIN IN ! EUTROPHUS ( WAS SHOWN BY THE ANALYSIS OF (ASE DElCIENT MUTANTS +ÚMEN ET AL -UTANTS LACKING THE MEMBRANE BOUND (ASE CONTAINED INCREASED AMOUNTS OF THE CYTOCHROME AA TYPE CYTOCHROME C OXIDASE AND USED THIS TERMINAL OXIDASE PREFERENTIALLY FOR RES PIRATION WHEREAS MUTANTS DElCIENT IN SOLUBLE (ASE USED PREDOMINANTLY THE QUINOL OXIDASES FOR ELECTRON TRANSFER )T WOULD CLEARLY BE OF INTEREST TO DETERMINE THE EFFECTS OF THESE MUTATIONS ON THE ACTIVITIES OF NITRATE REDUCTASE AND OTHER ANAEROBIC RESPIRATORY ENZYMES E4VMGVS3FHVMBUJPO 4HE HYPERTHERMOPHILIC ARCHAEON 0 FURIOSUS CAN GROW ON MALTOSE EITHER IN THE ABSENCE OF ELEMENTAL SULFUR 3 IT PRODUCES ( AS AN END PRODUCT RATHER THAN (3 OR IN THE PRESENCE OF 3 2ECENTLY THE EFFECT OF 3 ON THE LEVELS OF GENE EXPRESSION IN 0 FURIOSUS CELLS GROWN AT # WITH MALTOSE AS THE CARBON SOURCE WAS INVESTIGATED WITH THE USE OF $.! MICROARRAYS 3CHUT ET AL %IGHTEEN /2&S THAT ENCODE SUBUNITS ASSOCIATED WITH THE THREE (ASES CHARACTERIZED IN 0 FURIOSUS TWO CYTOPLASMIC (ASES ) AND )) AND ONE MEMBRANE BOUND WERE FOUND TO BE STRONGLY DOWN REGULATED BY 3 AN INDICATION THAT THESE (ASES ARE PROBABLY NOT DIRECTLY INVOLVED IN 3 REDUCTION 4HE NATURE OF THE ENZYME SYSTEM THAT REDUCES 3 REMAINS UNKNOWN AS IS UNKNOWN THE MECHANISM BY WHICH 3 AFFECTS (ASE GENE EXPRESSION IN 0 FURIOSUS 7$PODMVTJPOTBOE1FSTQFDUJWFT (ASE ENZYMES PLAY MANY DIFFERENT ROLES IN RESPIRA TION #HIEF AMONG THESE IS CLEARLY THE OXIDATION OF ( OR THE REDUCTION OF PROTONS COUPLED TO ENERGY CONSERVING ELECTRON TRANSFER CHAIN REACTIONS WHICH ALLOW ENERGY TO BE OBTAINED EITHER FROM ( OR FROM THE OXIDATION OF LOWER POTENTIAL SUBSTRATES 4HESE ENERGY CONSERVING REACTIONS ARE GENERALLY RESTRICTED TO THE PROKARYOTES BUT ARE WIDELY DISTRIBUTED AMONG THE BACTERIAL AND ARCHAEAL DOMAINS OF LIFE )N THE LAST DECADE ADDITIONAL ROLES HAVE BEEN REVEALED 4HUS THE SO CALLED ( SENSOR (ASES ARE INVOLVED IN REGULATING THE BIOSYNTHESIS OF UPTAKE ;.I&E= (ASES IN RESPONSE TO THEIR SUBSTRATE ( 4HESE (ASES FORM A SEPARATE CLASS WITH SOME CYANOBACTERIAL (ASES WHICH ARE APPARENTLY NON REGULATORY AND FUNCTION AS UPTAKE
(ASES /THER BIDIRECTIONAL (ASES MAY INTERACT WITH RESPIRATORY ELECTRON TRANSPORT CHAINS AND ACT AS ELECTRON @VALVES TO CONTROL THE REDOX POISE OF THE RESPIRATORY CHAIN AT THE LEVEL OF THE QUINONE POOL 4HIS IS ESSENTIAL TO ENSURING THE CORRECT FUNCTIONING OF THE RESPIRATORY CHAIN IN THE PRESENCE OF EXCESS REDUCING EQUIVALENTS PARTICULARLY IN PHOTOSYNTHETIC MICROORGANISMS )T IS INTERESTING TO NOTE THAT A SIMILAR ROLE HAS LONG BEEN PROPOSED FOR THE NITROGENASE OF PHOTOSYNTHETIC BACTERIA WHICH FUNCTIONS AS A ( EVOLVING (ASE IN THE ABSENCE OF . !N ADDITIONAL lNDING CONCERNS SOME (ASES WHICH WERE ORIGINALLY THOUGHT TO PLAY A PURELY FERMENTATIVE ROLE BUT WHICH ARE NOW KNOWN TO BE INVOLVED IN MEMBRANE LINKED ENERGY CONSERVATION THROUGH THE GENERATION OF A TRANSMEMBRANE PROTONMOTIVE FORCE (ASES ARE A STRUCTURALLY DIVERSE AS WELL AS A FUNC TIONALLY DIVERSE GROUP OF ENZYMES AND PHYLOGENETIC ANALYSIS HAS LED TO THE IDENTIlCATION OF SEVERAL PHY LOGENETICALLY DISTINCT GROUPS AND SUBGROUPS WHICH FORM THE BASIS OF A COHERENT SYSTEM OF CLASSIlCATION 4HE LARGE NUMBER OF (ASE GENE SEQUENCES HAS BEEN AUGMENTED BY WHOLE GENOME SEQUENCING WHICH HAS REVEALED THE PRESENCE OF MULTIPLE (ASES IN SEVERAL "ACTERIA AND !RCHAEA 0OST GENOMIC ANALYSIS TRAN SCRIPTOME PROTEOME METABOLOME HAS AND WILL BE ESSENTIAL TO ELUCIDATING THE METABOLIC ROLES OF THESE ENZYMES AND THE REGULATION OF THEIR BIOSYNTHESIS AND ACTIVITY 3TUDIES OF REGULATION HAVE ESSENTIALLY BEEN RESTRICTED TO THE UPTAKE ;.I&E= (ASES OF 0ROTEO BACTERIA BUT HAVE RECENTLY BEEN EXTENDED TO OTHER TYPES OF (ASE AND OTHER MICROORGANISMS SUCH AS THE HYPERTHERMOPHILIC ARCHAEON 0 FURIOSUS 4HE PHYLOGENETIC DIVERSITY OF (ASES ATTESTS TO THE IMPORTANCE OF ( METABOLISM IN A WIDE RANGE OF ENVIRONMENTS AND SUGGESTS THAT (ASES MAY HAVE APPEARED VERY EARLY IN EVOLUTION )T HAS BEEN PROPOSED THAT ( METABOLISM MAY HAVE BEEN THE DRIVING FORCE THAT LED TO THE CELLULAR SYMBIOSIS AND FUSION EVENTS THAT ARE THOUGHT TO HAVE BEEN INVOLVED IN THE FORMATION OF THE lRST EUKARYOTIC CELLS 4HE PRESENT DAY (ASES THAT ARE FOUND IN THE ORGANELLES HYDROGENOSOMES OR CHLOROPLASTS OF SOME UNICELLULAR EUKARYOTES MAY BE RELICS OF THESE EVOLUTIONARY EVENTS OR THE RESULTS OF MORE RECENT LATERAL GENE TRANSFER EVENTS 4HE CON TINUED STUDY OF THESE ENZYMES WILL ASSUREDLY LEAD TO FURTHER PROGRESS IN THIS lELD 4HE CURRENT INTEREST IN ( AS AN ALTERNATIVE ENERGY SOURCE TO FOSSIL FUELS HAS LED TO A RESURGENCE OF INTER EST IN THE BIOLOGICAL PRODUCTION OF ( AND RESEARCH INTO (ASES WILL CLEARLY PLAY A MAJOR ROLE IN THIS
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AREA 3TRUCTURAL STUDIES OF (ASES WILL BE IMPORTANT IN DIRECTING PROTEIN ENGINEERING EG IN DECREASING THE / SENSITIVITY OF THESE ENZYMES 3TUDIES OF ( METABOLISM AND REGULATION WILL ALSO BE IMPORTANT IN ENGINEERING MICROORGANISM AT THE CELLULAR LEVEL IN ORDER TO MAXIMIZE ( PRODUCTION 4HE ISOLATION OF NOVEL ( PRODUCING ORGANISMS WILL ALSO BE A PRIOR ITY 2ECENT ADVANCES IN MOLECULAR TAXONOMY EG THE ANALYSIS OF ENVIRONMENTAL 3 R$.! SEQUENCES HAVE REVEALED THAT THE NUMBER OF EXISTING BACTERIAL SPECIES IS AT LEAST TWO ORDERS OF MAGNITUDE HIGHER THAN THE NUMBER OF ISOLATED ONES 0ROKARYOTIC BIODIVERSITY IS THEREFORE MUCH GREATER THAN PREVIOUSLY THOUGHT AND WHOLE PHYLOGENETIC GROUPINGS EXIST EG THE MESOPHILIC #RENARCHAEOTA WHICH HAVE NEVER BEEN CULTIVATED 'IVEN THE IMPORTANCE OF ( METABOLISM AMONG MICROORGANISMS GENERALLY IT CAN BE ANTICI PATED THAT MANY OF THESE SO FAR UNCULTIVATED SPECIES WILL CONTAIN (ASES AND THAT NOVEL TYPES OF (ASE AND OF ( METABOLISM REMAIN TO BE DISCOVERED "DLOPXMFEHNFOUT 7E ARE GRATEFUL TO OUR COLLEAGUES AND FRIENDS ,AU RENT #OURNAC !LAIN $UPUIS 3YLVIE %LSEN AND !NDRÏ 6ERMEGLIO FOR CAREFUL READING OF THE MANUSCRIPT AND FOR MANY HELPFUL SUGGESTIONS AND COMMENTS AND )SABELLE 0RIEUR FOR THE SCHEME OF 2B CAPSULATUS #OMPLEX ) USED IN &IG 4HE EXPERT ASSISTANCE OF 'ÏRARD +LEIN WITH THE PREPARATION OF THE lGURES IS GRATEFULLY ACKNOWLEDGED 3FGFSFODFT !DAMS -77 4HE STRUCTURE AND MECHANISM OF IRON HY DROGENASES "IOCHIM "IOPHYS !CTA n !LBRACHT 30* AND DE *ONG !-0H "OVINE HEART .!$( UBIQUINONE OXIDOREDUCTASE IS A MONOMER WITH &E 3 CLUSTERS AND &-. GROUPS "IOCHIM "IOPHYS !CTA n !LBRACHT 30* AND (EDDERICH 2 ,EARNING FROM HYDROG ENASES ,OCATION OF A PROTON PUMP AND OF A SECOND &-. IN BOVINE .!$( UBIQUINONE OXIDOREDUCTASE #OMPLEX ) &%"3 ,ETT n !NDREWS 3# "ERKS "# -CCLAY * !MBLER ! 1UAIL -! 'OLBY 0 F AND 'UEST *2 ! CISTRON %SCHERICHIA COLI OPERON HYF ENCODING A PUTATIVE PROTON TRANSLOCATING FORMATE HYDROGENLYASE SYSTEM -ICROBIOLOGY n !ONO 3 (ONMA 9 /HKUBO + 4AWARA 4 +AMIYA 4 AND .AKA JIMA ( #/ SENSING AND REGULATION OF GENE EXPRESSION BY THE TRANSCRIPTIONAL ACTIVATOR #OO! * )NORG "IOCHEM n !ONO 3 "IOCHEMICAL AND BIOPHYSICAL PROPERTIES OF THE
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4VNNBSZ #YANOBACTERIAL RESPIRATION HAS SOME UNIQUE FEATURES #YANOBACTERIA CONTAIN TWO INDEPENDENT RESPIRATORY CHAINS ONE IN THE CYTOPLASMIC MEMBRANE AND ONE IN THE INTRACYTOPLASMIC MEMBRANES ALSO CALLED THYLAKOIDS 4HE LATTER IS INTIMATELY LINKED WITH THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN AND SOME COMPONENTS EG THE QUINONE POOL THE CYTOCHROME B F COMPLEX AND CYTOCHROME C ARE SHARED BY BOTH PHOTOSYNTHESIS AND RESPIRATION (OWEVER ALL COMPONENTS OF THE RESPIRATORY ELECTRON TRANSPORT CHAIN MIGHT HAVE SOMEPOSSIBLY REGULATORYFUNCTION IN PHOTOSYNTHESIS )N RECENT YEARS THE TOTAL GENOMIC SEQUENCES OF SEVERAL CYANOBACTERIA HAVE BEEN INVALUABLE TOOLS FOR UNDERSTANDING CYANOBACTERIAL RESPIRATION %SPECIALLY IN 3YNECHOCYSTIS SP STRAIN 0## THE MAJORITY OF THE COMPONENTS OF THE RESPIRATORY CHAINS WERE lNALLY IDENTIlED 2ESPIRATORY ELEC TRON TRANSPORT IS HIGHLY BRANCHED WITH SEVERAL ROUTES OF ELECTRON INPUT AND SEVERAL ROUTES TO THE lNAL ELECTRON ACCEPTOR WHICH IS ALWAYS DIOXYGEN 4HE SINGLE COMPONENT COMMON TO ALL RESPIRATORY ELECTRON PATHWAYS IS THE QUINONE POOL &URTHERMORE TOTAL GENOMIC SEQUENCING HAS SHOWN THAT DIFFERENT CYANOBACTERIA MAY CON TAIN DIFFERENT ELECTRON TRANSPORT COMPONENTS 3EVERAL STRAINS ESPECIALLY BUT NOT EXCLUSIVELY THOSE THAT ARE CAPABLE OF CELL DIFFERENTIATION CONTAIN MORE THAN ONE COPY OF GENES FOR SOME RESPIRATORY ELECTRON TRANSPORT CHAIN COMPONENTS 4HE DIFFERENT FUNCTIONS OF THESE GENES AND THEIR GENE PRODUCTS HAVE ONLY PARTIALLY BEEN ELUCIDATED 3EVERAL COMPONENTS OF THE RESPIRATORY CHAINS ARE SUBJECT TO COMPLEX GENE REGULATIONS ABOUT WHICH INFORMATION IS ONLY STARTING TO APPEAR 3OME PROTEINS EG CYTOCHROME C- AND THE DEHYDROGENASE $RG! ARE LIKELY TO BE INVOLVED IN RESPIRATORY ELECTRON TRANSPORT BUT THEIR POSITIONS IN THE ELECTRON TRANSPORT CHAIN IE THEIR IN VIVO ELECTRON DONORS AND ACCEPTORS HAVE NOT YET BEEN FULLY CLARIlED 4HE ELECTRON TRANSPORT CHAIN OF THE CYTOPLASMIC MEMBRANE IS NOT YET WELL CHARACTERIZED IN ANY STRAIN
**OUSPEVDUJPO )N RECENT YEARS CONSIDERABLE PROGRESS HAS BEEN MADE IN THE UNDERSTANDING OF CYANOBACTERIAL RESPIRATION AND A NUMBER OF REVIEWS HAVE APPEARED ON THE TOPIC EG 3CHMETTERER 6ERMAAS 4HIS REVIEW STRESSES THE FACT THAT DESPITE MUCH WORK MANY QUES TIONS ARE STILL OPEN NOT THE LEAST BEING THAT ALMOST CERTAINLY THE COMPONENTS OF THE CYANOBACTERIAL RE SPIRATORY CHAINS ARE NOT YET KNOWN IN THEIR ENTIRETY 4HE AVAILABILITY OF A STEADILY GROWING NUMBER OF GENOMIC SEQUENCES OF DIFFERENT CYANOBACTERIA HAS HAD A PROFOUND IMPACT ON THE STUDY OF CYANOBACTE RIA AND SPECIlCALLY THEIR RESPIRATORY CHAINS /NE IMPORTANT LESSON LEARNED WAS THAT A DETAILED PICTURE OF RESPIRATION IN CYANOBACTERIA AS A WHOLE CANNOT BE GIVEN BECAUSE NOT ALL RESPIRATORY COMPONENTS ARE PRESENT IN ALL STRAINS AND FOR MANY PERTINENT GENES THE NUMBER OF HOMOLOGUES PER CHROMOSOME DIFFER FROM STRAIN TO STRAIN 4HEREFORE THIS REVIEW ATTEMPTS TO LIST ALL COMPONENTS THAT ARE CURRENTLY KNOWN TO BE PART OF CYANOBACTERIAL RESPIRATORY CHAINS .OT ALL !BBREVIATIONS !24/ n ALTERNATE RESPIRATORY TERMINAL OXIDASES #- n CYTOPLASMIC MEMBRANE #OX n CYTOCHROME C ME OXIDASE &.2 n FERREDOXIN .!$0 OXIDOREDUCTASE (1./ n HEPTYL HY DROXY QUINOLINE . OXIDE )#- n INTRACYTOPLASMIC MEMBRANES OR THYLAKOIDS 0#0 n PENTACHLOROPHENOL 1OX n QUINOL OXIDASE TSP n TRANSCRIPTIONAL STARTING POINT
COMPONENTS ARE PRESENT IN ALL CYANOBACTERIA AND ONLY FOR 3YNECHOCYSTIS SP 0## THE lRST TOTALLY SEQUENCED CYANOBACTERIUM +ANEKO ET AL AN ATTEMPT WILL BE MADE TO DElNE THE FUNCTION OF EACH COMPONENT IDENTIlED SO FAR )N ORDER TO KEEP THE LIST OF REFERENCES TO A MANAGE ABLE SIZE THE MAJORITY OF REFERENCES PRESENTED IN THIS REVIEW ARE FROM OR YOUNGER &OR OLDER REFERENCES THE READER IS REFERRED TO AN EARLIER REVIEW ON THE SAME TOPIC 3CHMETTERER "$ZBOPCBDUFSJB #YANOBACTERIA ARE PROKARYOTES CAPABLE OF OXYGENIC PHOTOSYNTHESIS 4HEY ARE VERY WIDELY DISTRIBUTED IN MANY DIFFERENT HABITATS AND PLAY A HUGE ROLE IN TOTAL PRIMARY PRODUCTION OF ORGANIC MATERIAL 0ARTENSKY ET AL !LL CYANOBACTERIA ARE ALSO CAPABLE OF AERO BIC RESPIRATION 4HIS IS AN IMPORTANT PROPERTY SINCE CYANOBACTERIA ARE THUS THE ONLY CELLS IN WHICH THE TWO MOST IMPORTANT BIOENERGETIC PROCESSES AEROBIC RESPIRATION AND OXYGENIC PHOTOSYNTHESIS OCCUR IN THE SAME COMPARTMENT -UCH ATTENTION HAS BEEN PLACED ON THE QUESTION OF THE INTERACTION OF PHOTOSYNTHESIS AND RESPIRATION IN CYANOBACTERIA 3INCE THE OVERALL REACTIONS OF THESE TWO PROCESSES FORMALLY ARE THE REVERSE OF EACH OTHER THEIR SIMULTANEOUS OCCURRENCE IN THE SAME COMPARTMENT SHOULD LEAD TO FUTILE CYCLES
$IBQUFS $ZBOPCBDUFSJBM3FTQJSBUJPO $ESPITE GREAT PROGRESS IN THE UNDERSTANDING OF THE INTERACTION BETWEEN RESPIRATION AND PHOTOSYNTHESIS IN CYANOBACTERIA IT SEEMS LIKELY THAT NOT ALL THE COMPO NENTS OF THE RESPIRATORY CHAINS HAVE BEEN IDENTIlED AT LEAST NOT WITH A CLEAR IDEA OF THEIR FUNCTION 7ITH ONE SINGLE EXCEPTION 'LOEOBACTER SP THAT R HAS NO )#- ALL CYANOBACTERIA HAVE THREE DISTINCT LIPID BILAYER MEMBRANES THE OUTER MEMBRANE THAT IS PART OF THE GRAM NEGATIVE CELL WALL AND LOCATED OUTSIDE OF THE PEPTIDOGLYCANE LAYER IS NOT BIOENERGET ICALLY ACTIVE AND THUS NOT OF IMMEDIATE RELEVANCE FOR RESPIRATORY PROCESSES THE CYTOPLASMIC MEMBRANE #- THAT ENCLOSES THE CYTOPLASM AND THE NET WORK OF INTRACELLULAR MEMBRANES )#- ALSO KNOWN AS CYANOBACTERIAL THYLAKOIDS THAT ENCLOSE A FURTHER COMPARTMENT THE INTRATHYLAKOID LUMEN WHICH IS DISTINCT FROM THE CYTOPLASM AND MAY HAVE A PHYSICAL CONNECTION TO THE PERIPLASM 0URIlCATION TECHNIQUES HAVE SHOWN THAT #- AND )#- ARE CLEARLY DISTINCT IN THEIR CHEMICAL COMPOSITION /MATA AND -URATA BUT THE QUESTION WHETHER THERE IS PHYSICAL CONTACT BETWEEN THE TWO MEMBRANES HAS NOT BEEN SOLVED YET )T SEEMS NOW CERTAIN THAT BOTH THE #- AND THE )#- CARRY RESPIRATORY ELECTRON TRANSPORT CHAINS THAT MAY HOWEVER CONSIST OF DIFFERENT COMPONENTS 0HOTOSYNTHETIC ELECTRON TRANSPORT IS CONlNED TO THE )#- AND THE #- CONTAINS NO OR AT MOST VERY LITTLE CHLOROPHYLL ! GENERAL SCHEME OF CYANOBACTERIAL MEMBRANES THE CELLULAR COMPARTMENTS FORMED BY THEM AND THEIR BIOENERGTIC SIGNIlCANCE IS PRESENTD IN &IG #4QFDJàD1SPQFSUJFTPG$ZBOPCBDUFSJBM 3FTQJSBUJPO 3EVERAL PROPERTIES OF CYANOBACTERIAL RESPIRATION ARE UNIQUE !S MENTIONED ABOVE THERE ARE TWO BIOEN ERGETICALLY ACTIVE MEMBRANES AND EACH MEMBRANE APPARENTLY CONTAINS A DIFFERENT RESPIRATORY CHAIN )N THE )#- RESPIRATORY AND PHOTOSYNTHETIC ELECTRON TRANSPORT CHAINS SHARE SEVERAL COMPONENTS SO THAT BOTH PROCESSES MUST BE CONSIDERED TO BE PART OF A SINGLE LARGE MULTI ENZYME BIOENERGETIC COMPLEX #YANOBACTERIAL RESPIRATION IS ALSO CHARACTERIZED BY SOME COMPONENTS THAT ARE NOT USUALLY PART OF RESPIRATORY ELECTRON TRANSPORT CHAINS AMONG THEM .!$0( THE CYTOCHROME B F COMPLEX AND F PLASTO CYANIN .!$( NOT .!$0( IS GENERALLY A DONOR TO THE ELECTRON TRANSPORT CHAIN #YANOBACTERIA ARE THE F ONLY ORGANISMS TO USE THE CYTOCHROME B F COMPLEX AND THE PERIPHERAL ELECTRON CARRIER PLASTOCYANIN THAT
&IG ! SCHEMATIC MODEL OF THE BIOENERGETICS OF A @TYPICAL CYA NOBACTERIAL CELL /- OUTER MEMBRANE /03 OUTER PERIPLASMIC SPACE 0' PEPTIDOGLYCAN CELL WALL )03 INNER PERIPLASMIC SPACE #- CYTOPLASMIC MEMBRANE # CYTOPLASM )#- INTRACELLULAR MEMBRANES OR THYLAKOIDS )42 INTRATHYLAKOID SPACE 03 PHOTO SYNTHETIC 30 ACTIVE TRANSPORT BY SYMPORTERS !0 ACTIVE TRANSPORT BY ANTIPORTERS
ARE COMPONENTS OF PHOTOSYNTHETIC ELECTRON TRANSPORT IN CHLOROPLASTS AND CYANOBACTERIA IN RESPIRATORY ELECTRON TRANSPORT 3OME lLAMENTOUS CYANOBACTERIA ARE CAPABLE OF CELL DIFFERENTIATION SUCH AS HETEROCYSTS SPECIALIZED FOR NITROGEN lXATION AKINETES SPORES OR HORMOGONIA HIGHLY MOTILE IMPORTANT FOR THE INFECTION PROCESS ESTABLISHING SYMBIOSES WITH OTHER ORGANISMS %S PECIALLY IN HETEROCYSTS WHERE THE EXTREMELY OXYGEN SENSITIVE NITROGENASE IS LOCALIZED RESPIRATION AP PARENTLY PLAYS AN IMPORTANT ROLE IN MAINTAINING THE NECESSARY MICROAEROBIC ENVIRONMENT #YANOBACTERIA WERE THE lRST ORGANISMS ON %ARTH THAT PRODUCED / IN MASSIVE AMOUNTS SEE ALSO #AS TRESANA #HAPTER 6OL AND THUS MAY HAVE BEEN THE lRST ORGANISMS THAT USED THE AVAILABLE / TO GENER ATE !40 BY AEROBIC RESPIRATION IN THE DARK )NDEED ALL COMPONENTS OF CYANOBACTERIAL RESPIRATION APPARENTLY HAVE AT LEAST SOME FUNCTION IN PHOTOSYNTHESIS 4HIS REASONING LED "RODA TO THE FORMULATION OF HIS @CONVERSION HYPOTHESIS WHICH STATES ESSENTIALLY THAT ALL AEROBIC RESPIRERS EVOLVED FROM PHOTOSYNTHETIC
ORGANISMS SPECIlCALLY CYANOBACTERIA BY LOSS OF THE PHOTOSYSTEMS 5NTIL NOW NO EVIDENCE HAS TURNED UP THAT MAKES THIS HYPOTHESIS OBSOLETE **$PNQPOFOUTPG$ZBOPCBDUFSJBM3FTQJSB UPSZ$IBJOT "&YUFSOBM4PVSDFTPG&MFDUSPOTGPS3FTQJSB UJPO )T HAS BEEN KNOWN FOR A LONG TIME THAT THE NUMBER OF SUBSTANCES THAT ARE USED AS EXTERNAL ELECTRON SOURCES IN CYANOBACTERIA IS SMALL 4HE MOST IMPORTANT ONE IS OF COURSE (/ THAT IS PROCESSED BY 0HOTOSYSTEM )) TO YIELD / AND ELECTRONS THAT ARE TRANSFERRED TO THE QUINONE POOL $URING PHOTOSYNTHESIS CARBOHYDRATE IN THE FORM OF GLYCOGEN IS ACCUMULATED THAT IS USED DURING DARK PERIODS AS A PRIMARY SOURCE OF ELECTRONS FOR RESPIRATION !LTHOUGH IN NATURE GLYCOGEN IS MOST LIKELY THE MAJOR ELECTRON SOURCE FOR RESPIRATORY ELECTRON TRANSPORT IT SEEMS THAT THERE IS NO STUDY THAT DESCRIBES QUANTITATIVELY RESPIRATORY ACTIVITY AT THE EXPENSE OF GLYCOGEN DESPITE THE EXISTENCE OF A USEFUL METHOD TO QUANTIFY GLYCOGEN %RNST ET AL 3EVERAL CYANOBACTERIA CAN USE EXOGENOUS SUGARS AND A FEW RELATED SUBSTANCES SUCH AS GLYCEROL IN 3YNECHOCOCCUS SP 0## THAT ARE TAKEN UP AND THEN OXIDIZED TO YIELD .!$( ANDOR .!$0( A LIST IS AVAILABLE IN 2IPPKA ET AL %XOGENOUS ( CAN BE USED AS A PRIMARY ELECTRON DONOR THROUGH THE ACTION OF ONE OF THE HYDROGENASES SEE BELOW #*OUSBDFMMVMBS1SJNBSZ&MFDUSPO%POPST /"%1)
(FPSH4DINFUUFSFSBOE%JFUNBS1JMT AEROBIOSIS TO ANAEROBIOSIS THE AMOUNT OF .!$0( INCREASED BUT THE AMOUNT OF .!$( REMAINED CON STANT AND CONCLUDED THAT .!$0( NOT .!$( WAS THE PRIMARY DONOR !LTHOUGH SEVERAL PRIMARY DEHY DROGENASES HAVE BEEN IDENTIlED THAT OXIDIZE .!$( IN VITRO THE IN VIVO FUNCTION OF .!$( IN RESPIRATION REMAINS LARGELY UNKNOWN %VEN THE MAJOR ROUTES OF FORMATION OF .!$( FROM .!$ ARE NOT KNOWN WITH CERTAINTY .OTABLY IN THE SEQUENCED GENOMES OF CYANOBACTERIA PYRIDIN NUCLEOTIDE TRANSHYDROGENASES THAT USE .!$0( AS AN ELECTRON DONORR TO GENERATE .!$( HAVE BEEN IDENTIlED 4VDDJOBUF !N IMPORTANT RESULT FROM THE lRST AVAILABLE COM PLETE SEQUENCE OF A CYANOBACTERIUM ;3YNECHOCYS TIS SP 0## +ANEKO ET AL = WAS THE IDENTIlCATION OF SUCCINATE DEHYDROGENASES THAT HAVE BEEN SHOWN TO BE IMPORTANT FOR RESPIRATION #OOLEY ET AL 4HE SOURCE OF SUCCINATE IS NOT KNOWN WITH CERTAINTY BUT #OOLEY ET AL HAVE OBTAINED EVIDENCE THAT DESPITE THE ABSENCE OF A RECOGNIZABLE OXOGLUTARATE DEHYDROGENASE IN CYANOBACTERIAL GENOMES AND THUS THE EXISTENCE OF AN INCOMPLETE CITRIC ACID CYCLE OXOGLUTARATE CAN BE CONVERTED INTO SUCCINATE #OOLEY AND 6ERMAAS HAVE PRESENTED EVIDENCE THAT THE LEVELS OF .!$0 ( AND SUCCINATE ARE INTERRELATED ESPECIALLY A MUTANT LACKING TYPE ) .!$0 ( DEHYDROGENASE HAVING ONLY ABOUT OF THE SUCCINATE LEVELS OF THE WILD TYPE 4HEY THEREFORE CONSIDERED THAT A CONSIDERABLE PART OF THE OXIDIZED .!$0 FORMED BY .!$0 ( DEHYDROGENASES MAY BE USED FOR THE CYCLE PRODUCING SUCCINATE SPECIlCALLY FOR CONVERSION OF ISOCITRATE TO OXOGLUTARATE AND MALATE TO OXALOACETATE
!N UNUSUAL PROPERTY OF CYANOBACTERIAL RESPIRATORY CHAINS IS THAT .!$0( IS AN IMPORTANT PRIMARY RE DUCTANT )N THE DARK .!$0( IS PROBABLY GENERATED PRIMARILY BY THE OXIDATION OF GLUCOSE PHOSPHATE IN THE OXIDATIVE PENTOSE PHOSPHATE CYCLE )N THE LIGHT .!$0( IS PRODUCED BY FERREDOXIN .!$0 OXIDOREDUCTASE
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- OF 3YNECHOCYSTIS SP 0## THAT LACKS THE NDH" GENE IS THE MOST INTERESTING /GAWA 2ESPIRATION IN THIS STRAIN WAS REDUCED TO ABOUT OF WILD TYPE ACTIVITY THE BEST INDICATION AVAILABLE THAT TYPE ) .!$0 ( DEHYDROGENASE IS IMPORTANT FOR RESPIRATION IN CYANOBACTERIA )NTERESTINGLY MUTANT - ALSO SHOWED A SEVERE REDUCTION IN UPTAKE OF INORGANIC CARBON WHICH MADE THIS STRAIN HIGH #/ REQUIRING 4HE REASON FOR THIS LINK BETWEEN INORGANIC CARBON UPTAKE AND RESPIRATION HAS NOT YET BEEN EXPLAINED 3IMILARLY AN NDH" MUTANT IN 3YN ECHOCOCCUS SP 0## WAS HIGH #/ REQUIRING BUT ITS RESPIRATORY CAPACITY WAS ONLY REDUCED BY n -ARCO ET AL )N 3YNECHOCOCCUS SP 0## AN ATTEMPTED NDH" MUTANT DID NOT SEGREGATE TO HOMOZYGOCITY .OMURA 4HE NDH, GENE IS ONLY KNOWN FROM CYANOBACTERIA )TS MUTATION IN 3YNECHOCYSTIS SP 0## ALSO LED TO A DEFECT IN INORGANIC CARBON UPTAKE /GAWA !NOTHER INTERESTING .!$0 ( DEHYDROGENASE MUTANT WAS CONSTRUCTED BY 3CHLUCHTER ET AL $ESPITE THE OCCURRENCE OF ABOUT FOUR DIFFERENT GENES FOR NDH& & SEE ABOVE IN THIS STRAIN MUTATION IN ONE SUCH GENE THE NDH& GENE DISPLAYED SEVERAL STRIKING PHENOTYPES RESPIRATORY ACTIVITY WAS LOWERED TO ABOUT n OF THE WILD TYPE THE CAPACITY FOR PHOTOHETEROTROPHIC GROWTH WITH GLYCEROL AS THE SUBSTRATE WAS LOST AND CYCLIC ELECTRON TRANSPORT AROUND 0HOTOSYSTEM ) WAS PARTIALLY INHIBITED 9U ET AL 5ZQF**/"% 1 )%FIZESPHFOBTF -ANY BACTERIA CONTAIN A TYPE )) .!$0 ( DEHY DROGENASE THAT CONSISTS IN CONTRAST TO THE MANY SUBUNITS OF TYPE ) ONLY OF A SINGLE SUBUNIT AND HAS NO IRON SULFUR CLUSTERS 3EVERAL CYANOBACTERIA HAVE BEEN FOUND TO CONTAIN A NUMBER OF HOMOLOGUES TO THIS ENZYME 3YNECHOCYSTIS SP 0## CONTAINS THREE SUCH GENES DESIGNATED NDB! SLR NDB" SLR AND NDB# SLL +ANEKO ET AL # !NABAENA SP 0## CONTAINS AT LEAST SIX ALL MOST SIMILAR TO NDB! ALR MOST SIMILAR TO NDB" ALR MOST SIMILAR TO NDB# ALL ALL AND # ALL +ANEKO ET AL 4HERMOSYNECHOCOC CUS ELONGATUS "0 CONTAINS ONLY ONE TLR MOST SIMILAR TO NDH" AND 3YNECHOCOCCUS SP 0## CONTAINS TWO THAT DISPLAY HIGH SIMILARITIES WITH NDH! AND NDH" RESPECTIVELY .OMURA /NLY THE 3YNECHOCYSTIS ENZYMES ARE CHARACTERIZED IN SOME DETAIL (OWITT ET AL NDB! AND NDB" HAVE NO DISCERNIBLE TRANSMEMBRANE STRETCHES AND MAY BE
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4WO OTHER ENZYMES WITH .!$0 ( DEHYDROGENASE ACTIVITIES HAVE BEEN IDENTIlED IN CYANOBACTERIA THE $RG! PROTEIN AND &ERREDOXIN .!$0 /XIDOREDUCTASE &.2 4HE DRG! GENE WAS ORIGINALLY DISCOVERED AS A GENE WHOSE INACTIVATION CONFERRED RESISTANCE TO HERBICIDES SUCH AS DINOSEB OR METRONIDAZOLE IN 3YNECHOCYSTIS SP 0## %LANSKAYA ET AL -ATSUO ET AL SHOWED THAT THE $RG! PROTEIN WAS AN .!$0 ( QUINONE OXIDOREDUCTASE )T IS PROB ABLY A SOLUBLE PROTEIN AND ITS FUNCTION IN THE ELECTRON TRANSPORT CHAIN IS STILL UNCERTAIN &ERREDOXIN .!$0 OXIDOREDUCTASE &.2 WHOSE MAIN FUNCTION IS UN DOUBTEDLY THE SYNTHESIS OF .!$0( IN NON CYCLIC PHOTOSYNTHETIC ELECTRON TRANSPORT HAS BEEN SUG GESTED TO PLAY A ROLE AS AN .!$0 ( DEHYDROGENASE WITH QUINONES AS ELECTRON AND PROTON ACCEPTORS SEE 3CHMETTERER )T IS STILL NOT KNOWN WHETHER THIS ACTIVITY OF &.2 CONTRIBUTES IN A SIGNIlCANT WAY TO THE TOTAL IN VIVO .!$0 ( DEHYDROGENASE ACTIVITY (OWITT ET AL HAVE PERFORMED NATIVE GEL ELECTROPHORESIS OF .!$0 ( DEHYDROGENASES OF 3YNECHOCYSTIS SP 0## AND FOUND EVIDENCE FOR AT LEAST TWO MORE SUCH ENZYMES ABOUT WHICH NO DETAILS ARE AVAILABLE
4VDDJOBUF%FIZESPHFOBTFT 3YNECHOCYSTIS SP 0## CONTAINS TWO SUCCINATE QUINONE OXIDOREDUCTASES THAT CONTRIBUTE SIGNIlCANTLY TO THE TOTAL RESPIRATORY ELECTRON TRANSPORT ACTIVITY )N % COLI SUCCINATE DEHYDROGENASE CONSISTS OF FOUR SUBUNITS ENCODED BY THE SDH#$!" OPERON #YANO BACTERIA CONTAIN HOMOLOGUES TO SDH! BINDING &!$ AND SDH" BINDING THE &E3 CLUSTER BUT NOT TO SDH# # OR SDH$ WHICH ENCODE THE SUBUNITS BINDING TO HEME B (OWEVER IN NONE OF THE CYANOBACTERIA INVESTIGATED SO FAR THE SDH! AND SDH" GENES ARE LOCATED CLOSE TO EACH OTHER ON THE CHROMOSOME 3YNECHOCYSTIS SP 0## SDH! SLR AND SDH" SLL AND SDH" SLL !NABAENA SP 0## SDH! ALL AND SDH" ALL 4HERMOSYNECHOCOCCUS ELONGATUS "0n SDH! TLR AND SDH" TLR 'LOEOBACTER VIOLACEUS 0## SDH! GLR AND SDH" GLR #OOLEY ET AL CONSTRUCTED MU TANTS IN EACH OF THE TWO SDH" GENES OF 3YNECHOCYSTIS SP 0## AND ALSO A DOUBLE MUTANT LACKING BOTH GENES 4HEY CLEARLY SHOWED THAT THESE PROTEINS FORM PART OF A FUNCTIONAL SUCCINATE DEHYDROGENASE THAT IS LACKING IN THE DOUBLE MUTANT .O INFORMATION IS AVAIL ABLE AS TO OTHER SUBUNITS THIS ENZYMES MIGHT CONTAIN #OOLEY AND 6ERMAAS HAVE SHOWN THAT SUCCINATE DEHYDROGENASE MAY BE A MAJOR ELECTRON DONOR TO THE RESPIRATORY CHAIN IN 3YNECHOCYSTIS SP 0## )N WILD TYPE CELLS RESPIRATION WAS APPROXIMATELY INHIBITED BY MALONATE AN INHIBITOR OF SUCCINATE DEHYDROGENASE AND IN A MUTANT LACKING SUCCINATE DE HYDROGENASE THE RESIDUAL RESPIRATORY OXYGEN UPTAKE WAS ONLY ABOUT OF THE WILD TYPE )ZESPHFOBTFT !T LEAST THREE ENZYMES EXIST IN CYANOBACTERIA THAT HAVE HYDROGENASE ACTIVITY .ITROGENASE WHICH PRODUCES HYDROGEN 5PTAKE HYDROGENASE WHICH OXIDIZES HYDROGEN AND TRANSFERS ELECTRON INTO THE QUINONE POOL 4HIS ENZYME CAN BE CONSIDERED PART OF A RESPIRATORY ELECTRON TRANSPORT BRANCH "IDIRECTIONAL HYDROGENASE WHOSE BIOLOGICAL FUNCTION IS NOT CLEAR YET ;! RECENT REVIEW ON CYANOBACTERIAL HYDROGENASES HAS APPEARED 4AMAGNINI ET AL AND THEREFORE
THESE ENZYMES WILL NOT BE TREATED FURTHER HERE= %2VJOPOF1PPM 3TOICHIOMETRICALLY THE QUINONES REPRESENT BY FAR THE MOST ABUNDANT COMPONENTS OF THE CYANOBACTERIAL RESPIRATORY ELECTRON TRANSPORT CHAINS #YANOBACTERIAL MEMBRANES CONTAIN LARGE AMOUNTS OF PLASTOQUINONE AND LESS PHYLLOQUINONE WITH THEIR RELATIVE AMOUNTS VARYING IN THE )#- AND #- 4HE QUINONE POOL OF THE )#- THYLAKOIDS IS ALSO USED BY THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN AND IT IS OF CENTRAL IMPORTANCE IN DElNING THE REDOX STATE OF THE CELL 'IVEN THEIR IMPORTANCE IN BIOENERGETIC PROCESSES COMPARATIVELY LITTLE IS KNOWN ABOUT THE BIOSYNTHESIS OF THE QUINONES 5SING THE COMPLETE GENOMIC SEQUENCE OF 3YNECHOCYS TIS SP 0## *OHNSON ET AL HAVE PROPOSED A HYPOTHETICAL PATHWAY FOR THE BIOSYNTHESIS OF PHYL LOQUINONE FROM CHORISMATE BASED ON THE ASSUMPTION THAT THIS PATHWAY CONSISTS OF HOMOLOGUES OF EIGHT GENES THAT HAVE BEEN IDENTIlED IN % COLI AND "ACILLUS SUBTILIS 4HEY SHOWED THAT INACTIVATION OF THE MEN! OR MEN" GENES DIHYDROXY NAPHTHOIC ACID PHYTYL TRANSFERASE AND DIHYDROXY NAPHTHOATE SYNTHASE RESPECTIVELY LEADS TO CELLS THAT LACK PHYLLOQUINONE BUT HAVE ALMOST NORMAL PHOTOSYNTHETIC ELECTRON TRANSPORT RATES 5NFORTUNATELY NO DATA ON RESPIRATORY ELECTRON TRANSPORT RATES ARE PRESENTED BUT THE ABILITY OF THE MUTANTS TO GROW PHOTOHETEROTROPHICALLY AT RATES SIMILAR TO WILD TYPE CELLS COULD INDICATE THAT ELECTRON TRANSPORT FROM .!$0 ( TO CYTOCHROME BB F MAY BE LARGELY UNALTERED IN THE MUTANTS 4HUS PHYLLOQUINONE MAY NOT BE ESSENTIAL FOR RESPIRATION &$ZUPDISPNFC G "IOENERGETICALLY THE CYTOCHROME BB F COMPLEX IS THE F MOST IMPORTANT COMPONENT OF CYANOBACTERIAL ELECTRON TRANSPORT SINCE ITS REDOX REACTION FROM PLASTOQUINOL TO EITHER CYTOCHROME C OR PLASTOCYANIN IS COUPLED TO THE CHEMIOSMOTIC TRANSLOCATION OF PROTONS )N THE )#- IT IS USED IN BOTH RESPIRATORY AND PHOTOSYNTHETIC ELECTRON TRANSPORT PROCESSES $ESPITE EARLIER REPORTS INDICATING THAT CYANOBACTERIA CONTAIN A MITOCHONDRIAL TYPE CYTOCHROME BC COMPLEX THE COMPLETE GENOMIC SEQUENCES HAVE SHOWN THAT THIS IS NOT THE CASE 4HE USE OF CYTOCHROME BB F IN RESPIRATORY ELECTRON TRANSPORT F IS UNIQUE TO CYANOBACTERIA #YANOBACTERIAL CYTOCHROME B FF CONSISTS OF FOUR SUBUNITS CYTOCHROME B CYTOCHROME FF A C TYPE CY TOCHROME WHICH IS NOT RELATED TO THE CYTOCHROME C OF
(FPSH4DINFUUFSFSBOE%JFUNBS1JMT THE CYTOCHROME BC COMPLEX THE SO CALLED SUBUNIT )6 AND THE 2IESKE PROTEIN 4HE ACTIVE PROTEIN MAY BE A DIMER 0OGGESE ET AL )N THE HOMOLO GOUS CYTOCHROME BC COMPLEX CYTOCHROME B AND SUBUNIT )6 ARE FUSED TO ONE PROTEIN 4HE THREE lRST NAMED SUBUNITS ARE PRESENT AS SINGLE COPY GENES WHILE THE NUMBER OF GENES FOR THE 2IESKE PROTEIN VARIES 4HUS 3YNECHOCYSTIS SP 0## CONTAINS THREE SLL SLL SLL !NABAENA SP 0## FOUR ALL ALL ALL ALL AND 4HERMOSYNECHOCOCCUS ELONGATUS "0 ONE GENE TLR CODING FOR PUTATIVE 2IESKE &E 3 PROTEINS 4HE SIGNIlCANCE OF THE DIFFERENT 2IESKE PROTEINS HAS NOT BEEN ELUCIDATED YET 3EVERAL REPORTS HAVE PROVIDED EVIDENCE FOR THE EXISTENCE OF A CHLOROPHYLL A MOLECULE AS A COMPONENT OF CYTOCHROME BB F IN CYANOBACTERIA SEE 0OGGESE ET AL AND THE LITERATURE CITED THERE 4HESE AUTHORS ALSO PROVIDE EVIDENCE THAT THE CYTOCHROME B SUBUNIT IS THE ACTUAL BINDING SITE FOR THE CHLOROPHYLL A MOL ECULE 7HILE IT IS UNCLEAR WHAT THE POSSIBLE FUNCTION OF THIS CHLOROPHYLL COMPONENT MIGHT BE IT APPEARS NEVERTHELESS REMARKABLE THAT A CHLOROPHYLL CONTAIN ING PROTEIN COMPLEX IS AN IMPORTANT COMPONENT OF A RESPIRATORY CHAIN !TTEMPTS TO CONSTRUCT MUTANTS LACKING THE CYTO CHROME B F COMPLEX HAVE NOT BEEN SUCCESSFUL 4HIS F PROBABLY IMPLIES THAT CYTOCHROME B F IS AN ESSENTIAL COMPONENT IN ALL BIOENERGETIC REGIMES THAT EFFECT GROWTH (OWEVER ,EE ET AL SUCCEEDED IN CONSTRUCTING SITE SPECIlC MUTATIONS IN THE CYTO CHROME B F COMPLEX OF F 3YNECHOCOCCUS SP 0## %SPECIALLY STRIKING WAS A $' SUBSTITUTION IN 3YN ECHOCOCCUS SP 0## CYTOCHROME B )N CONTRAST TO CYTOCHROME BC THE CYTOCHROME BB F COMPLEX IS F INSENSITIVE TO THE INHIBITOR MYXOTHIAZOL 4HE ABOVE MENTIONED SUBSTITUTION GENERATED A FUNCTIONAL BUT MYXOTHIAZOL SENSITIVE CYTOCHROME B F COMPLEX /N THE OTHER HAND IN 2HODOBACTER CAPSULATUS WHICH CONTAINS A CYTOCHROME BC THE MUTATION '$ OF THE CORRESPONDING AMINO ACID YIELDED A STRAIN THAT WAS INSENSITIVE TO MYXOTHIAZOL SEE ALSO #OOLEY ET AL #HAPTER 6OL ! CYTOCHROME B F COMPLEX WITH REDUCED ACTIVITY F WAS GENERATED BY 4ICHY AND 6ERMAAS 4HE CCS" GENE OF 3YNECHOCYSTIS SP 0## WHICH IS INVOLVED IN THE MATURATION OF CYTOCHROME F COULD NOT F BE DELETED COMPLETELY FROM THE GENOME (OWEVER A CCS" GENE LACKING THE lRST AMINO ACIDS COULD BE CONSTRUCTED 4HIS STRAIN WAS PARTIALLY DElCIENT IN MATURATION OF CYTOCHROME F AND ACCUMULATED THE F
$IBQUFS $ZBOPCBDUFSJBM3FTQJSBUJPO CYTOCHROME F APOPROTEIN -ATURE CYTOCHROME F WAS PRODUCED TO A LEVEL OF ABOUT OF THE WILD TYPE AND THE MUTANT STRAIN GREW ONLY UNDER ANAEROBIC CONDITIONS '1FSJQIFSBM*OUFSNFEJBUF&MFDUSPO$BSSJFST #YANOBACTERIA CONTAIN TWO TYPES OF PERIPHERAL INTER MEDIATE ELECTRON CARRIERS CYTOCHROME C AND PLASTO CYANIN THE FORMER CONTAINING A HEME IRON COFACTOR THE LATTER CONTAINING COPPER 4HE MAIN FUNCTION OF THESE SOLUBLE PROTEINS IN CYANOBACTERIA IS THE TRANS FER OF ELECTRONS FROM THE CYTOCHROME BB F COMPLEX F TO 0HOTOSYSTEM ) (OWEVER THEY ALSO ARE IMPORTANT PARTS OF THE RESPIRATORY CHAINS 7ITH RESPECT TO THE EXPRESSION OF THE PERIPHERAL INTERMEDIATE ELECTRON CARRIERS IN RESPONSE TO THE COPPER CONCENTRATION OF THE MEDIUM 3ANDMANN FOUND THAT ALL KNOWN CYANOBACTERIA BELONG TO ONE OF THREE GROUPS 3TRAINS CONTAINING ONLY CYTOCHROME C GENE FOR PLASTOCYANIN ABSENT 3TRAINS FORMING CYTOCHROME C IN LOW AND PLAS TOCYANIN IN HIGH ABOUT - #U #ELLS GROWN IN INTERMEDIATE #U CONCENTRATIONS ABOUT N- CONTAIN BOTH PROTEINS 3TRAINS FORMING CYTOCHROME C CONSTITUTIVELY AND PLASTOCYANIN ONLY IN HIGH #U CONCENTRATION /F THE SEVERAL STRAINS THAT WERE THOUGHT TO BELONG TO GROUP ONLY A SINGLE STRAIN HAS REMAINED THAT DOES NOT SEEM TO CONTAIN A PLASTOCYANIN GENE 3YN ECHOCOCCUS SP STRAIN 0## $ZUPDISPNFTD 4HERE ARE THREE TYPES OF SOLUBLE CYTOCHROME CS IN CYANOBACTERIA NAMELY CYTOCHROME C ALSO CALLED CYTOCHROME C CYTOCHROME C- AND CYTOCHROME C 4HE LATTER CYTOCHROME HAS AN UNUSUALLY LOW MIDPOINT REDOX POTENTIAL ABOUT n M6 IS A COMPONENT OF 0HOTOSYSTEM )) AND THUS IT IS NOT INVOLVED IN RESPIRA TION #YTOCHROME C IS HIGHLY RELATED TO MITOCHONDRIAL CYTOCHROME C AND SEVERAL CYTOCHROME CS FROM MANY BACTERIAL SPECIES WHILE CYTOCHROME C- SEEMS TO OCCUR ONLY IN CYANOBACTERIA
B$ZUPDISPNFD !LL CYANOBACTERIA CONTAIN AT LEAST ONE COPY OF A GENE FOR CYTOCHROME C )T CONSTITUTES A BRANCHING POINT IN THE ELECTRON TRANSPORT CHAINS OF THE CYANOBACTERIAL )#- )T ACCEPTS ELECTRONS FROM THE CYTOCHROME B F COMPLEX AND TRANSFERS THEM EITHER TO 0HOTOSYSTEM ) OR TO CYTOCHROME C OXIDASE )N STRAINS WHERE THE EXPRESSION OF CYTOCHROME C DEPENDS ON THE COPPER CONCENTRATION SUCH AS 3YNECHOCYSTIS SP 0## ITS FUNCTION IN THE )#- CAN APPARENTLY BE TAKEN OVER COMPLETELY BY PLASTOCYANIN SINCE MUTANTS SHOW VERY LITTLE PHENOTYPE )N CONTRAST THE RESPIRATORY ELECTRON TRANSPORT CHAIN IN THE #- IS FULLY DEPENDENT ON THE PRESENCE OF CYTOCHROME C 4HIS HAS BEEN DEMONSTRATED BY 0ILS AND 3CHMETTERER USING THE UPTAKE OF / METHYL GLUCOSE BY 3YNECHOCYSTIS SP 0## CELLS AS A PROBE 4HEY SHOWED THAT THE ENERGIZATION OF THE #- NECESSARY FOR THE ACTIVE UPTAKE OF THE NON METABOLIZABLE GLUCOSE ANALOG IS LOST WHEN A MUTANT CONTAINING !24/ STANDS FOR !LTERNATE 2ESPIRATORY 4ERMINAL /XIDASE AS THE ONLY RESPIRATORY TERMINAL OXIDASE SEE BELOW ALSO LACKS THE PET** GENE ENCODING CYTOCHROME C AND THEREFORE CONCLUDED THAT CYTOCHROME C IS AN ESSENTIAL PART OF THE ELECTRON TRANSPORT CHAIN ENDING IN !24/ WHICH IS LIKELY A COMPONENT OF THE #- 6ERY LITTLE IS KNOWN ABOUT THE MECHANISMS THAT TARGET PROTEINS INTO EITHER THE #- OR THE )#- IN CYANOBACTERIA 4HIS IS ALSO TRUE FOR SOLUBLE PROTEINS THAT ARE FULLY TRANSPORTED THROUGH THE MEMBRANES INTO THE INTRATHYLAKOID LUMEN TRANSPORT THROUGH THE )#- OR INTO THE PERIPLASMIC SPACE TRANSPORT THROUGH THE #- 4HEREFORE WHILE AT LEAST IN 3YNECHOCYSTIS SP 0## IT IS QUITE LIKELY THAT CYTOCHROME C IS PRESENT IN BOTH CELLULAR SPACES * WHAT PROPERTY OF THE PET* GENE IS RESPONSIBLE FOR THIS DUAL TARGETING REMAINS UNKNOWN 4HE TOTAL GENOMIC SEQUENCE OF !NABAENA SP 0## +ANEKO ET AL HAS REVEALED THE PRESENCE OF THREE COPIES OF GENES THAT MAY CODE FOR CYTOCHROME CS .O DATA ARE AVAILABLE WHAT THE FUNCTION OF THESE GENES AND THE PROTEINS THEY MAY ENCODE IS BUT ONE MIGHT SPECULATE THAT THE MULTITUDE OF GENES IS RELATED TO THE FACT THAT THIS STRAIN DEVELOPS HETEROCYSTS UPON REMOVAL OF COMBINED NITROGEN !N UNUSUAL PROPERTY HAS BEEN OBSERVED FOR CYTO CHROME C OF 3YNECHOCYSTIS SP 0## 4HIS STRAIN CONTAINS A SINGLE GENE PET* CODING FOR * CYTOCHROME C )N CONTRAST TO ALL OTHER CYANOBACTERIA THIS GENE CANNOT BE REMOVED FROM THE GENOME AND IS THEREFORE ESSEN TIAL UNDER BOTH AUTOTROPHIC AND HETEROTROPHIC GROWTH
CONDITIONS .OMURA 4HIS IS CLEARLY DUE TO THE ABSENCE OF A PLASTOCYANIN GENE IN THIS STRAIN WHICH IN OTHER CYANOBACTERIA CAN TAKE OVER THE FUNCTIONS OF CYTOCHROME C SEE BELOW #URIOUSLY EVEN THE AT TEMPTED SUBSTITUTION OF THE 3YNECHOCOCCUS SP 0## * PET** GENE OR THE PET% GENE % PET* GENE WITH THE ENCODING PLASTOCYANIN FROM 3YNECHOCYSTIS SP 0## WAS NOT POSSIBLE .OMURA )N VIEW OF THE HIGH SIMILARITY BETWEEN THE CYTOCHROME CS FROM THE TWO ORGANISMS IDENTITY SIMILARITY IT IS UNCLEAR WHAT SPECIlC PROPERTY OF THE CYTOCHROME C FROM STRAIN 0## MAKES IT UNIQUE C $ZUPDISPNFD. #YTOCHROME C- WAS DISCOVERED BY SEQUENCING A REGION OF 3YNECHOCYSTIS SP 0## -ALAKHOV ET AL $ESPITE EARLIER REPORTS THAT CYTOCHROME CMIGHT OCCUR ONLY IN A FEW STRAINS A HOMOLOG HAS BEEN IDENTIlED IN EACH CYANOBACTERIUM WHOSE TOTAL SE QUENCE WAS DETERMINED 3YNECHOCYSTIS SP 0## !NABAENA SP 0## 4HERMOSYNECHOCOCCUS ELONGATUS "0 'LOEOBACTER VIOLACEUS 0## 0ROCHLOROCOCCUS MARINUS 33 0ROCHLOROCOCCUS MARINUS -%$ 0ROCHLOROCOCCUS MARINUS -)4 3YNECHOCOCCUS SP 7( 3YNECHOCOCCUS SP 0## )T IS PRESENT IN VERY SMALL AMOUNTS UNDER NORMAL GROWTH CONDITIONS BUT INDUCED UNDER STRESS SUCH AS LOW TEMPERATURE # AND HIGH LIGHT % Mn Sn UNDER WHICH CONDITIONS THE EXPRESSION OF THE OTHER TWO PERIPHERAL INTERMEDIATE ELECTRON CARRIERS CYTOCHROME C AND PLASTOCYANIN IS DOWN REGULATED -ALAKHOV ET AL 4HIS HAS LED TO THE IDEA THAT CYTOCHROME C- MIGHT SUBSTITUTE FOR THE OTHER TWO PROTEINS UNDER STRESS )NDEED 3HUVALOV ET AL HAVE SHOWN THAT IT IS REDOX ACTIVE IN VIVO AND LIGHT ABSORBED BY 0HOTOSYSTEM ) LED TO THE OXIDATION OF REDUCED CYTOCHROME C- SUGGESTING THAT CYTOCHROME C- MIGHT DONATE ELECTRONS TO 0HOTOSYSTEM ) (OW EVER -OLINA (EREDIA ET AL FOUND VERY LOW ELECTRON TRANSPORT RATES IN VITRO FROM PURIlED REDUCED CYTOCHROME C- TO ISOLATED 03 ) PARTICLES SO THAT THIS REACTION MAY NOT BE OF IMPORTANCE IN THE WHOLE CELL &URTHERMORE THE REDOX MIDPOINT POTENTIAL OF CYTO CHROME C- FROM 3YNECHOCYSTIS 0## HAS BEEN DETERMINED INDEPENDENTLY BY TWO GROUPS #HO ET AL -OLINA (EREDIA ET AL TO BE M6 AT P( WHICH IS VERY DIFFERENT FROM THE REDOX MIDPOINT POTENTIALS OF CYTOCHROME C AND PLASTOCYANIN FROM THE SAME STRAIN ABOUT M6 SO THAT A SIMILAR REDOX FUNCTION ALSO APPEARS UNLIKELY -UTANTS LACKING
(FPSH4DINFUUFSFSBOE%JFUNBS1JMT THE CYT- GENE ENCODING CYTOCHROME C- HAVE LITTLE PHENOTYPE (OWEVER 0ILS ET AL FOUND THAT A 3YNECHOCYSTIS 0## DOUBLE MUTANT LACKING BOTH THE PET% % GENE ENCODING PLASTOCYANIN AND THE CYTGENE WILL NOT SEGREGATE TO HOMOZYGOCITY SO THAT IN - MUTANT PLASTOCYANIN APPEARS TO BE ESSENTIAL A CYT-ANNA AND 6ERMAAS FOUND THAT IN A 0HOTO SYSTEM ) MINUS STRAIN CYTOCHROME C- COULD NOT BE REMOVED FROM THE GENOME AND THUS IS ESSENTIAL IN THIS MUTANT 3INCE 0HOTOSYSTEM ) MINUS STRAINS NEED CY TOCHROME C OXIDASE AS THE TERMINAL ELECTRON TRANSPORT COMPONENT THEY SUGGESTED THAT CYTOCHROME C- MIGHT BE A PART OF THE ELECTRON TRANSPORT BRANCH ENDING IN CYTOCHROME C OXIDASE 7HILE THIS IDEA IS COMPATIBLE WITH THE RESULTS OF 0ILS ET AL THE POSITION OF CYTOCHROME C- IN THIS BRANCH TAKING INTO ACCOUNT ITS RATHER LOW MIDPOINT REDOX POTENTIAL REMAINS UNCLEAR )N SUMMARY ALTHOUGH CYTOCHROME C- MAY BE UBIQUITOUS AMONG CYANOBACTERIA FURTHER WORK IS WARRANTED FOR UNDERSTANDING ITS REAL FUNCTION 1MBTUPDZBOJO 0LASTOCYANIN IS A SOLUBLE PROTEIN CONTAINING ONE ATOM OF COPPER PER POLYPEPTIDE )T IS ONLY EXPRESSED IN MEDIA CONTAINING HIGH AMOUNTS OF COPPER ABOUT - OR HIGHER )TS PRIMARY FUNCTION IN CYANOBACTERIA IS THE ELECTRON TRANSFER FROM THE CYTOCHROME B F COM PLEX TO 0HOTOSYSTEM ) AS IN CHLOROPLASTS (OWEVER 0ILS ET AL HAVE ALSO OBTAINED EVIDENCE FOR THE INVOLVEMENT OF PLASTOCYANIN IN THE RESPIRATORY CHAIN IN 3YNECHOCYSTIS SP 0## 5SING A NUMBER OF MUTANTS LACKING DIFFERENT RESPIRATORY TERMINAL OXI DASES AND PERIPHERAL SOLUBLE INTERMEDIATE ELECTRON CARRIERS THEY FOUND THAT THE ABILITY OF THIS STRAIN TO GROW CHEMOHETEROTROPHICALLY IS STRICTLY COUPLED TO THE FUNCTIONAL PRESENCE OF CYTOCHROME C OXIDASE )NTEREST INGLY A MUTANT CONTAINING CYTOCHROME C OXIDASE BUT LACKING THE PET* GENE ENCODING * CYTOCHROME C THE NORMAL ELECTRON DONOR TO CYTOCHROME C OXIDASE IS STILL ABLE TO GROW CHEMOHETEROTROPHICALLY 4HUS THERE MUST BE AN ALTERNATIVE PATHWAY FOR ELECTRONS TO CYTO CHROME C OXIDASE AND PLASTOCYANIN MAY THUS BE NOT ONLY AN ALTERNATIVE ELECTRON DONOR TO 0HOTOSYSTEM ) BUT ALSO TO CYTOCHROME C OXIDASE -UTANTS LACKING THE PET% GENE ENCODING % PLASTOCYA NIN ARE EASY TO OBTAIN AND HAVE LITTLE PHENOTYPE #LARKE AND #AMPBELL REPORTED AN ENHANCED SENSITIVITY TO CHILLING IN A PET% MUTANT OF % 3YNECHOCOCCUS SP 0## $ESPITE ATTEMPTS IN SEVERAL LABORATORIES IT HAS NOT BEEN POSSIBLE TO CONSTRUCT A DOUBLE MUTANT LACKING
$IBQUFS $ZBOPCBDUFSJBM3FTQJSBUJPO BOTH THE GENES FOR CYTOCHROME C AND PLASTOCYANIN 4HUS ONE MUST CONCLUDE THAT ONE OF THESE PERIPHERAL INTERMEDIATE ELECTRON CARRIERS MUST BE PRESENT TO ACHIEVE TRANSFER OF ELECTRONS TO 0HOTOSYSTEM ) FOR PHOTOTROPHIC GROWTH OR TO CYTOCHROME C OXIDASE FOR CHEMOHETEROTROPHIC GROWTH 4WO REPORTS HAVE CLAIMED THAT BOTH PROTEINS MIGHT BE DISPENSIBLE IN 3YNECHOCOCCUS SP STRAIN 0## ,AUDENBACH ET AL OR IN 3YNECHOCYSTIS SP 0## :HANG ET AL )N THE FORMER CASE A MUTANT LACKING THE PET** GENE WAS CONSTRUCTED AND NO GENE FOR PLASTOCYANIN WAS FOUND AT THE TIME AND STILL NORMAL PHOTOSYNTHETIC AND RESPIRATORY ELECTRON TRANSPORT CAPACITIES WERE FOUND IN THE MUTANT (OWEVER IT IS NOW KNOWN THAT THIS STRAIN DOES CONTAIN A PET% % GENE SO THAT THE ALTERNATIVE PERIPHERAL INTERMEDIATE ELECTRON CARRIER MOST LIKELY IS PLASTOCYANIN IN THIS STRAIN AS IN OTHER CYANOBACTERIA )N THE CASE OF 3YNECHOCYSTIS SP STRAIN 0## A STRAIN BELONGING TO 3ANDMANN GROUP SEE ABOVE A MUTANT LACKING THE PET* GENE AND THUS CYTOCHROME C WAS CONSTRUCTED AND GROWN UNDER VERY LOW COPPER CONCENTRATIONS ABOUT N- SO THAT NO PLASTOCYANIN WAS DETECTABLE 4HIS RESULT WAS THOUGHT TO BE EVIDENCE FOR THE EXISTENCE OF A THIRD ELECTRON CARRIER BETWEEN CYTOCHROME B F AND 0HOTOSYSTEM ) (OWEVER 0ILS ET AL UNPUBLISHED RESULTS HAVE FOUND THAT THE REPRESSION OF THE PET* GENE IN HIGH COPPER OR PET% GENE IN LOW COPPER % IS VERY EFFECTIVE BUT NOT FULLY COMPLETE SO THAT WILD TYPE CELLS GROWN IN HIGH COPPER STILL CONTAIN TINY AMOUNTS OF CYTOCHROME C THAT MAY BE SUFlCIENT FOR FUNCTION 4HEREFORE CURRENTLY THERE IS NO COMPELLING EVIDENCE FOR THE EXISTENCE OF ANY OTHER PERIPHERAL INTERMEDIATE ELECTRON CARRIER BESIDES CYTOCHROME C AND PLASTOCYANIN EXCEPT IN ONE CASE SEE BELOW ! POSSIBLE FUNCTION OF PLASTOCYANIN AS A DONOR TO CYTOCHROME C OXIDASE HAS BEEN DESCRIBED ABOVE SEE #YTOCHROME C #MVF$PQQFS1SPUFJO#DQ" ! NEW POTENTIAL PERIPHERAL INTERMEDIATE ELECTRON CAR R RIER WAS DISCOVERED IN 3YNECHOCOCCUS SP 0## .OMURA )T IS A COPPER PROTEIN AND RELATED TO PLASTOCYANIN THE MAIN SIMILARITIES BEING AROUND THE COPPER BINDING SITE BCP! GENES ARE ALSO FOUND IN THE GENOMES OF !NABAENA SP 0## ALL +ANEKO ET AL AND .OSTOC PUNCTIFORME .#)" ACCESSION NUMBER :0 BUT ABSENT IN THE OTHER COMPLETELY SEQUENCED CYANOBACTERIAL GENOMES CURRENTLY AVAILABLE )T IS EXPRESSED AND TRANSLATED IN
3YNECHOCYSTIS SP 0## ! MUTANT LACKING THE BCP! GENE CAN BE CONSTRUCTED BUT HAS NO KNOWN PHENOTYPE .OMURA 4HUS IT MUST REMAIN UNCERTAIN WHAT THE FUNCTION OF "CP! MIGHT BE AND WHETHER IT IS ASSOCIATED WITH PHOTOSYNTHETIC ANDOR RESPIRATORY ELECTRON TRANSPORT (3FTQJSBUPSZ5FSNJOBM0YJEBTFT #YANOBACTERIAL RESPIRATORY TERMINAL OXIDASES ARE THE KEY ENZYMES OF RESPIRATION SINCE THEY MAY BE THE ONLY COMPONENTS OF THE RESPIRATORY CHAIN IN THE )#- THAT ARE NOT DIRECTLY INVOLVED IN PHOTOSYNTHESIS !LL CYANOBACTERIAL RESPIRATORY TERMINAL OXIDASES BELONG TO ONLY TWO PROTEIN FAMILIES THE RELATIVES OF THE HEME COPPER OXIDASES #OX AND !24/ AND THE QUINOL OXIDASES OF THE CYTOCHROME BD TYPE 1OX 4HE BEST D CHARACTERIZED MEMBERS OF THE lRST GROUP ARE MITO CHONDRIAL CYTOCHROME C OXIDASE FROM MAMMALS FROM THE SECOND GROUP THE CYTOCHROME BDD FROM %SCHERICHIA COLI 4HE HEME COPPER OXIDASES OF CYANOBACTERIA ARE OF TWO TYPES THE GENUINE CYTOCHROME C OXIDASES #OX AND A GROUP OF RELATED ENZYMES WHOSE FUNCTION IS NOT WELL UNDERSTOOD AND WHICH WE THEREFORE FOR THE TIME BEING HAVE CALLED !24/ !LL KNOWN CYANOBACTERIAL RESPIRATORY TERMINAL OXIDASES ARE SENSITIVE TO +#. 4HIS AND THE COMPLETE GENOMIC SEQUENCES SHOW DElNITELY DESPITE EARLIER REPORTS TO THE CONTRARY THE ABSENCE OF CYANIDE INSENSITIVE TERMINAL RESPIRATORY OXIDASES IN CYANOBACTERIA AND SPECIlCALLY OF ALTERNATIVE TERMINAL OXIDASE AS FOUND IN MITOCHONDRIA OF PLANTS AND SOME OTHER EUKARYOTES 6ANLERBERGHE AND -C)NTOSH !N IMPORTANT RESULT OF THE SEQUENCING OF CYA NOBACTERIAL GENOMES IS THAT EVEN CLOSELY RELATED STRAINS MAY HAVE DIFFERENT SETS OF RESPIRATORY TERMI NAL OXIDASES 4ABLE 4HIS MAKES IT VERY DIFlCULT TO ASSIGN A SPECIlC FUNCTION TO ANY OF THE TERMINAL RESPIRATORY OXIDASES IN CYANOBACTERIA SINCE RESULTS OBTAINED IN ONE STRAIN CANNOT NECESSARILY BE APPLIED TO ANOTHER ONE 5NFORTUNATELY THERE IS CURRENTLY NO METHOD TO ASSAY THE IN VIVO CONTRIBUTION OF THE DIFFERENT RESPIRATORY TERMINAL OXIDASES TO TOTAL RESPIRATION IN ANY STRAIN ! NUMBER OF MUTANTS EXIST IN 3YNECHOCYSTIS SP 0## 3CHMETTERER ET AL (OWITT AND 6ER MAAS 0ILS AND 3CHMETTERER !NABAENA SP 0## 6ALLADARES ET AL !NABAENA VARIABILIS!4## 3CHMETTERER ET AL AND 3YNECHOCOCCUS SP 0## .OMURA THAT LACK ONE OR MORE RESPIRATORY TERMINAL OXIDASES )N
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'LOEOBACTER VIOLACEUS 0##
3YNECHOCOCCUS SP 7(
0ROCHLOROCOCCUS MARINUS 33
0ROCHLOROCOCCUS MARINUS -%$
0ROCHLOROCOCCUS MARINUS -)4
3YNECHOCOCCUS SP 0##
.OSTOC PUNCTIFORME
!NABAENA VARIABILIS !4##
4HE NUMBERS GIVEN REPRESENT THE NUMBER OF GENE SETS ENCODING THE CORRESPONDING RESPIRATORY TERMINAL OXIDASES 4HE LAST THREE NAMED STRAINS ARE NOT YET SEQUENCED COMPLETELY SO THAT THE ACTUAL NUMBER OF RESPIRATORY TERMINAL OXIDASES IN THESE STRAINS MAY BE HIGHER #ONCERNING THE QUESTION MARK FOR THE !24/ OF 'LOEOBACTER VIOLACEUS 0## SEE TEXT
3YNECHOCYSTIS SP 0## AND ONLY IN THIS STRAIN ALL POSSIBLE COMBINATIONS OF MUTANTS ARE AVAILABLE AND A STRIKING RESULT IS THAT REMOVAL OF ONE OR MORE RESPIRATORY TERMINAL OXIDASES INmUENCES THE TOTAL RESPIRATORY ACTIVITY AS DETERMINED BY THE UPTAKE OF / IN A CURRENTLY UNPREDICTABLE WAY %SPECIALLY THE RESPIRATORY RATES OF ALL THOSE MUTANTS THAT CONTAIN ONLY ONE RESPIRATORY TERMINAL OXIDASE DO BY FAR NOT ADD UP TO THE RESPIRATORY RATE OF THE WILD TYPE 4HIS IMPLIES THAT REMOVAL OF ANY ONE OF THE TERMINAL RESPIRATORY OXIDASES CHANGES THE CONTRIBUTION THROUGH DIFFERENT EXPRESSION OR ACTIVITY OR DEGREE OF SATURATION WITH SUBSTRATES OF ALL THE OTHER RESPIRATORY TERMINAL OXI DASES TO TOTAL RESPIRATION -UTANTS LACKING ALL KNOWN RESPIRATORY TERMINAL OXIDASES ARE AVAILABLE CURRENTLY ONLY FROM 3YNECHOCYSTIS SP 0## (OWITT AND 6ERMAAS AND 3YNECHOCOCCUS SP 0## .OMURA 4HE FORMER SHOWS NO RESPIRATORY ACTIVITY DEMONSTRATING THAT ALL RESPIRATORY TERMINAL OXIDASES ARE KNOCKED OUT AND THUS KNOWN THE LATTER DISPLAYS SOME RESIDUAL UPTAKE OF / AND MAY THEREFORE CONTAIN ANOTHER RESPIRATORY TERMINAL OXIDASE )FNF$PQQFS'BNJMZ B $ZUPDISPNFD0YJEBTFT !LL CYANOBACTERIA CONTAIN AT LEAST ONE SET OF THREE
GENES CALLED COX"!# ORR CTA#$% CODING FOR A CYTOCHROME C OXIDASE #OX 4HE THREE SUBUNITS CORRESPOND TO THE THREE MITOCHONDRIALLY ENCODED SUBUNITS ) )) AND ))) OF CYTOCHROME C OXIDASE FROM EUKARYOTES 4HE TOTAL GENOMIC SEQUENCES OF CYANO BACTERIA HAVE PRODUCED NO EVIDENCE FOR GENES WITH SEQUENCE SIMILARITY TO THOSE ENCODED IN THE NUCLEUS OF EUKAROYTES #YANOBACTERIAL #OX CAN BE CONVENIENTLY ASSAYED IN VITRO USING ISOLATED MEMBRANES AND HORSE HEART CYTOCHROME C AS THE ELECTRON DONOR )N CONTRAST !24/ SEE BELOW SHOWS NO DETECTABLE HORSE HEART CYTOCHROME C OXIDASE ACTIVITY DESPITE THE HIGH SE QUENCE SIMILARITY OF THE CORRESPONDING THREE SUBUNITS 4HIS PROPERTY HAS BEEN USED BY 0ILS AND COLLABORA TORS UNPUBLISHED TO SHOW THAT OF THE AT LEAST THREE SETS OF COX GENES IDENTIlED IN !NABAENA VARIABILIS !4## TWO ENCODE GENUINE CYTOCHROME C OXIDASES SINCE A DOUBLE MUTANT LACKING #OX AND #OX IN THIS STRAIN HAS NO HORSE HEART CYTOCHROME C OXIDASE ACTIVITY )N 3YNECHOCYSTIS SP 0## CYTOCHROME C OXI DASE IS PROBABLY LOCATED ONLY IN THE )#- 4HIS MAY NOT BE A UNIVERSAL FEATURE HOWEVER SINCE ISOLATED #- FROM OTHER STRAINS EG 0LECTONEMA BORYANUM 0## 0ESCHEK ET AL 3YNECHOCOCCUS SP 0## 3CHMETTERER UNPUBLISHED HAS BEEN FOUND TO CONTAIN IN VITRO HORSE HEART CYTOCHROME C OXIDASE ACTIVITY
$IBQUFS $ZBOPCBDUFSJBM3FTQJSBUJPO 4HE GENES ENCODING #OX CONSTITUTE AN OPERON AS HAS BEEN DEMONSTRATED FOR THE COX"!# GENES OF # 3YN ECHOCYSTIS SP 0## (OWITT AND 6ERMAAS # 3CHMETTERER ET AL AND COX"!# THE COX"!# 0ILS AND COLLABORATORS UNPUBLISHED GENES OF !NA BAENA VARIABILIS !4## AND THE COX"!# # GENES OF !NABAENA SP 0## 6ALLADARES ET AL (OWEVER THE PRIMARY TRANSCRIPT COULD BE PROCESSED IN !NABAENA STRAINS SINCE A STRONG SIGNAL IN .ORTHERN BLOTS WAS OBTAINED FOR AN M2.! CONTAINING ONLY THE lRST GENE OF THE OPERON THE COX" GENE 7HETHER THIS IS OF FUNCTIONAL SIGNIlCANCE IN THE CELLS CANNOT BE STATED CURRENTLY ESPECIALLY AS IT IS EXPECTED THAT THE THREE SUBUNITS ARE SYNTHESIZED IN EQUAL AMOUNTS IN VIVO #YTOCHROME C OXIDASE IS SUBJECT TO GENE REGULA TION IN RESPONSE TO THE GROWTH MEDIUM IN !NABAENA STRAINS )N ! VARIABILIS !4## THE COX"!# GENES ARE UPREGULATED BY FRUCTOSE 3CHMETTERER ET AL BUT THIS REGULATION OCCURS ONLY WITH ONE OF THE TWO TRANSCRIPTIONAL START SITES UPSTREAM OF COX" TSP WHILE THE OTHER ONE TSP IS CONSTITUTIVE )N # GENES AND !NABAENA SP 0## THE COX"!# ALSO THE COX"!# # GENES ENCODING THE !24/ SEE BELOW ARE UPREGULATED WHEN THE CELLS ARE GROWN IN THE ABSENCE OF COMBINED NITROGEN AND THUS CONTAIN HETEROCYSTS 6ALLADARES ET AL 4HE COX"!# GENES ARE EITHER PREFERENTIALLY OR POSSIBLY EVEN EX CLUSIVELY EXPRESSED IN HETEROCYSTS -UTANTS LACKING CYTOCHROME C OXIDASES HAVE BEEN CONSTRUCTED IN STRAINS 3YNECHOCYSTIS SP 0## 3CHMETTERER ET AL 0ILS ET AL !NABAENA SP 0## 6ALLADARES ET AL !NABAENA VARIABILIS !4## 3CHMETTERER ET AL 0ILS AND COLLABORATORS UNPUBLISHED AND 3YNECHOCOCCUS SP 0## .OMURA 4HESE MUTANTS HAVE ALLOWED TO DETERMINE THAT CYTOCHROME C OXIDASE IS THE MOST IMPORTANT RESPIRATORY TERMINAL OXIDASE WITH RESPECT TO BIOENERGETIC CAPACITY 7HEN THE GENES ENCODING CYTOCHROME C OXIDASE WERE IN ACTIVATED IN 3YNECHOCYSTIS SP 0## COX"!# # OR IN !NABAENA VARIABILIS COX"!# THE RESULTING # MUTANTS LOST THE CAPACITY FOR HETEROTROPHIC GROWTH 4HIS IS ESPECIALLY STRIKING FOR THE LATTER STRAIN SINCE IT CONTAINS AT LEAST ONE OTHER CYTOCHROME C OXIDASE COX"!# )N # 3YNECHOCYSTIS SP 0## THE LOSS OF #OX ALSO LED TO THE LOSS OF THE SO CALLED SALT RES PIRATION A SHORT TERM ENHANCEMENT OF RESPIRATORY ACTIVITY UPON THE ADDITION OF .A#L EVEN IF THE OTHER TWO RESPIRATORY TERMINAL OXIDASES WERE STILL PRESENT )N !NABAENA SP 0## THE COX LOCUS IS INVOLVED IN THE PROTECTION OF NITROGENASE IN HETEROCYSTS FROM THE
DAMAGE BY OXYGEN #URIOUSLY THIS PROTECTION CAN ALSO BE PERFORMED BY THE COX LOCUS CODING FOR !24/ SEE BELOW BUT NOT BY THE COX LOCUS THAT ENCODES A SECOND CYTOCHROME C OXIDASE ENZYME /NE POSSIBLE EXPLANATION IS THAT #OX AND THE !24/ BUT NOT #OX ARE LOCALIZED IN THE HETEROCYSTS !N IMPORTANT lNDING WAS THE OBSERVATION THAT CYTOCHROME C OXIDASE CANNOT BE INACTIVATED IN A 3YN ECHOCYSTIS SP 0## LACKING 0HOTOSYSTEM ) AND VICE VERSA -ANNA AND 6ERMAAS 4HEREFORE #OX IS A NECESSARY ELECTRON SINK FOR A 0HOTOSYSTEM ) MINUS STRAIN 6ERMAAS ET AL /NE MAY SPECU LATE THAT THIS MIGHT HAVE BEEN THE ORIGINAL FUNCTION OF CYTOCHROME C OXIDASE NAMELY REGULATING ELECTRON mOW BETWEEN 0HOTOSYSTEM )) AND 0HOTOSYSTEM ) C "MUFSOBUF3FTQJSBUPSZ5FSNJOBM0YJEBTFT "350
4HE SECOND TYPE OF ENZYMES OF THE HEME COPPER FAMILY WAS ORIGINALLY DISCOVERED BY SEQUENCING THE GENOME OF 3YNECHOCYSTIS SP 0## +ANEKO ET AL AND LATER CALLED !24/ 0ILS ET AL 4HE THREE PUTATIVE GENES ENCODING !24/ PROPOSED NOMENCLATURE CTA$ CTA# CTA% HAVE HIGH SEQUENCE SIMILARITY TO SUBUNITS ) )) AND ))) RESPECTIVELY OF MITOCHONDRIAL CYTOCHROME C OXIDASES BUT ARE EASILY DISCERNIBLE FROM THE GENUINE CYTOCHROME C OXIDASES BY TWO HIGHLY CHARACTERISTIC SEQUENCE DIFFERENCES AS lRST POINTED OUT BY (OWITT AND 6ERMAAS THE -G BINDING MOTIF IN CYTOCHROME C OXIDASE SUBUNIT ) (IS!SP AROUND AMINO ACID TO IS LACKING AND REPLACED BY 'LY!SN OR !SN!SN AND THE #U! BINDING MOTIF OF CYTOCHROME C OXIDASE SUBUNIT )) #YS 8 'LU 8 #YS AROUND AMINO ACID TO IS NOT OR ONLY PARTIALLY PRESENT IN THE CORRESPONDING !24/ SUBUNIT !N ENZYME RELATED TO !24/ IS THE CYTOCHROME BO QUINOL OXIDASE FROM %SCHERICHIA COLI BUT CURRENTLY THERE ARE NO DATA THAT SHOW A QUINOL OXIDASE ACTIVITY FOR CYANOBACTERIAL !24/ !S SHOWN IN 4ABLE NOT ALL CYANOBACTERIA CONTAIN AN !24/ AND THE GENE ARRANGEMENT IS NOT THE SAME IN THOSE STRAINS THAT DO 4HE MOST COMMON GENE AR RANGEMENT IS CTA#$% (OWEVER IN 3YNECHOCYSTIS SP 0## THE CTA# GENE IS LOCATED FAR AWAY FROM THE # CTA$% % GENES AND IN 'LOEOBACTER VIOLACEUS 0## THERE IS APPARENTLY NO CTA# GENE %SPECIALLY THE LAT #
TER OBSERVATION RAISES THE QUESTION WHETHER A #OX" PROTEIN SUBUNIT )) OF CYTOCHROME C OXIDASE IS ABLE TO FORM A FUNCTIONAL !24/ WITH THE #TA$ SUBUNIT ) AND #TA% SUBUNIT ))) PROTEINS FROM THE SAME STRAIN
)N NO CYANOBACTERIUM HAS THIS BEEN TESTED SO FAR 4HE ENZYMATIC REACTION CATALYZED BY !24/ IS STILL UNKNOWN 7HILE 0ILS AND 3CHMETTERER HAVE SHOWN THAT A 3YNECHOCYSTIS SP 0## MUTANT THAT CONTAINS !24/ AS THE ONLY TERMINAL RESPIRATORY OXIDASE DOES CONSUME / ALBEIT AT A VERY LOW RATE PRACTICALLY NO INFORMATION IS AVAILABLE ABOUT THE ELEC TRON DONORS (OWEVER 0ILS AND 3CHMETTERER WERE ABLE TO DEMONSTRATE THAT THE !24/ IS PROBABLY A BIOENERGETICALLY ACTIVE ENZYME 4HEY USED THE UPTAKE OF / METHYLGLUCOSE A NON METABOLIZABLE ANALOG OF GLUCOSE THAT IS ACTIVELY TRANSPORTED THROUGH THE #- TO MONITOR THE ENERGIZATION OF THE #- AND FOUND THAT DESPITE ITS LOW TOTAL RESPIRATORY ACTIVITY ABOUT OF THE WILD TYPE A MUTANT HAVING !24/ AS THE ONLY RESPIRATORY TERMINAL OXIDASE IS ABLE TO SUPPORT ACTIVE TRANSPORT ACROSS THE #- 4HIS ACTIVITY DEPENDS CRITICALLY ON THE PRESENCE OF CYTOCHROME C SO THAT AN INVOLVEMENT OF THIS PERIPHERAL INTERMEDIATE ELECTRON CARRIER IN THE RESPIRATORY BRANCH ENDING IN !24/ HAD TO BE POSTULATED 4HIS DOES NOT NECES SARILY IMPLY THAT !24/ IS A CYTOCHROME C OXIDASE ESPECIALLY AS THE IN VITRO CYTOCHROME C OXIDASE ASSAY GIVES NO DETECTABLE ACTIVITY WITH ISOLATED MEMBRANES FROM A MUTANT CONTAINING ONLY !24/ )N VIEW OF THE LOW RESPIRATORY ACTIVITY OF THIS MUTANT AND THE RATHER HIGH ACTIVE TRANSPORT CAPACITY OF THE STRAIN 0ILS AND 3CHMETTERER SUGGESTED THAT !24/ MIGHT BE THE TERMINAL RESPIRATORY OXIDASE OF THE RESPIRATORY CHAIN IN THE #- )NDEPENDENT EVIDENCE FOR THIS CAME FROM A PROTEOMICS PROJECT DESIGNED TO IDENTIFY PROTEINS IN THE PURIlED #- OF 3YNECHOCYSTIS SP 0## (UANG ET AL THE ONLY PEPTIDE FROM A RE SPIRATORY TERMINAL OXIDASE FOUND WAS SUBUNIT )) OF !24/ 4HE ACTIVITY OF !24/ IN THE WILD TYPE IS NOT NECESSARILY LOW HOWEVER )N A MUTANT CONTAINING BOTH 1OX AND !24/ THAT HAD ABOUT THE RESPIRATORY ACTIVITY OF THE WILD TYPE THE SPECIlC 1OX INHIBITORS (1./ HEPTYL HYDROXY QUINOLINE . OXIDE AND 0#0 PENTACHLOROPHENOL INHIBITED RESPIRATION BY ONLY AND RESPECTIVELY SO THAT APPRECIABLE CONTRIBUTIONS OF !24/ REMAINED )N 3YNECHOCOCCUS SP 0## A MUTANT LACKING #OX ALMOST HAS THE SAME LOW RESPIRATORY RATE AS A MUTANT LACKING BOTH #OX AND !24/ SO THAT IN THIS STRAIN !24/ ALSO APPEARS TO HAVE A LOW CONTRIBUTION TO TOTAL RESPIRATORY ACTIVITY )N !NABAENA SP 0## A DIFFERENT ASPECT OF !24/ ACTIVITY WAS DISCOVERED 6ALLADARES ET AL 4HIS lLAMENTOUS STRAIN DIFFERENTIATES ABOUT ONE IN EVERY n CELLS IN A SEMI REGULAR FASHION WHEN
(FPSH4DINFUUFSFSBOE%JFUNBS1JMT THE CELLS ARE GROWN IN A MEDIUM LACKING COMBINED NITROGEN 4HE RESULTING DIFFERENTIATED CELLS CALLED HETEROCYSTS ARE SPECIALIZED FOR NITROGEN lXATION 4HE KEY ENZYME OF NITROGEN lXATION NITROGENASE IS CONlNED TO THE HETEROCYSTS AND UNUSUALLY SENSITIVE TO / 2ESPIRATION AS A PROCESS REMOVING / FROM THE CYTOPLASM APPARENTLY PLAYS A DECISIVE ROLE IN THE PROTECTION OF NITROGENASE ,IKE #OX SEE ABOVE !24/ WAS FOUND TO BE PREFERENTIALLY OR POSSIBLY COMPLETELY EXPRESSED IN HETEROCYSTS -UTANTS LACKING BOTH !24/ AND #OX BUT NOT THE SINGLE MUTANTS WERE UNABLE GROW ON . AND lXED VERY LITTLE . IMPLYING THAT !24/ ALONE IS SUFlCIENT TO REMOVE / FROM THE CYTOPLASM OF HETEROCYSTS TO ALLOW DIAZOTRO PHIC GROWTH .O INFORMATION IS AVAILABLE ABOUT THE POSSIBLE HEME COFACTORS OF !24/ AND NOTHING IS KNOWN ABOUT THE INTRACELLULAR LOCATION OF !24/ IN HETEROCYSTS OF !NABAENA SP 0## $ZUPDISPNFCE5ZQF2VJOPM0YJEBTFT #YANOBACTERIAL GENES WITH HIGH SEQUENCE SIMILARITY TO THE TWO SUBUNITS OF % COLI CYTOCHROME BDD 1OX WERE lRST DETECTED IN THE PROCESS OF THE DETERMINATION OF THE TOTAL GENOMIC SEQUENCE OF 3YNECHOCYSTIS SP 0## +ANEKO ET AL !LL CYANOBACTERIA THAT HAVE SUCH AN ENZYME CONTAIN THE TWO SUBUNITS ENCODED BY THE CYD! AND CYD" GENES IN ADJACENT POSITIONS ON THE CHROMOSOME AS IN % COLI )N 3YN ECHOCYSTIS SP 0## (OWITT AND 6ERMAAS CONSTRUCTED A MUTANT THAT LACKS BOTH CYTOCHROME C OXIDASE AND !24/ SO THAT THE CYD!" GENES CODE FOR THE ONLY FUNCTIONAL RESPIRATORY TERMINAL OXIDASE "Y SHOWING THAT THIS STRAIN RESPIRED ALMOST AT THE SAME RATE OF THE WILD TYPE AND ADDITIONAL REMOVAL OF THE CYD!" GENES LED TO A NON RESPIRING STRAIN THEY PRODUCED CONCLUSIVE EVIDENCE THAT THE CYD!" GENES CODE FOR A RESPIRATORY TERMINAL OXIDASE 7HETHER IT IS A CYTOCHROME BDD TYPE ENZYME CANNOT BE STATED WITH CERTAINTY HOWEVER SINCE NO CONCLUSIVE EVIDENCE FOR THE EXISTENCE OF HEME DD EXISTS IN THE CYANOBACTERIAL LITERATURE 2ECENTLY A GOOD INDICATION OF THE FUNCTION OF CYA NOBACTERIAL 1OX WAS OBTAINED "ERRY ET AL 7HILE IT HAS NOT YET BEEN DEMONSTRATED DIRECTLY THAT THE ENZYMATIC REACTION OF 1OX IS A QUINOL DIOXYGEN OXIDOREDUCTASE AS IN % COLI THESE AUTHORS PROVIDE STRONG EVIDENCE FOR THIS BY SHOWING THAT IT CATALYSES THE OXIDATION OF THE QUINONE POOL WITHOUT USING THE CYTOCHROME BB F COMPLEX 4HE FUNCTION OF THE 1OX IS F THEREFORE PROBABLY THE PREVENTION OF THE OVERREDUC
$IBQUFS $ZBOPCBDUFSJBM3FTQJSBUJPO TION OF THE QUINONE POOL 3INCE THE PHOTOSYSTEMS ARE CONlNED TO THE )#- AND ARE NOT PRESENT IN THE #- 1OX IS PROBABLY LOCATED IN THE )#- ALTHOUGH ITS PRESENCE IN THE #- CANNOT BE COMPLETELY EXCLUDED SEE 6ERMAAS )N THE DARK 1OX APPARENTLY CONTRIBUTES CONSIDERABLY TO TOTAL RESPIRATION 5SING A 3YNECHOCYSTIS SP 0## MUTANT IN WHICH THE 1OX WAS THE ONLY REMAINING RESPIRATORY TERMINAL OXIDASE (OWITT AND 6ERMAAS 0ILS AND 3CHMETTERER IDENTIlED (1./ AND 0#0 AS SPECIlC INHIBI TORS OF 3YNECHOCYSTIS 1OX 4HUS IN CELLS WITH MORE THAN ONE RESPIRATORY TERMINAL OXIDASE THE (1./ OR 0#0 SENSITIVE PORTION OF TOTAL RESPIRATORY ACTIVITY COULD BE TAKEN TO REPRESENT THE ELECTRON mUX THROUGH THE ELECTRON TRANSPORT BRANCH ENDING IN 1OX )N WILD TYPE 3YNECHOCYSTIS SP 0## THIS CONTRIBUTION OF 1OX IS ABOUT n #URIOUSLY THE 3YNECHOCYSTIS SP 0## MUTANT CONTAINING ONLY 1OX AS THE RESPIRATORY TERMINAL OXI DASE DOES NOT DISPLAY AN ENHANCEMENT OF RESPIRATION UPON THE ADDITION OF GLUCOSE TO THE EXTERNAL MEDIUM IN CONTRAST TO ALL OTHER MUTANTS OF THIS STRAIN EXCEPT THE
ONE COMPLETELY DEVOID OF RESPIRATION OF COURSE 4HIS SEEMS TO IMPLY THAT THE RESPIRATORY BRANCH ENDING IN 1OX COULD BE LINKED TO A DIFFERENT ELECTRON TRANSPORT BRANCH AT THE REDUCING END OF THE RESPIRATORY CHAIN THAN THE BRANCHES ENDING IN THE OTHER TWO RESPIRATORY TERMINAL OXIDASES (OW THIS MIGHT WORK IS CURRENTLY UNKNOWN SINCE ALL RESPIRATORY BRANCHES CONTAIN THE QUINONE POOL AS INTERMEDIATE ELECTRON BUFFER )5FSNJOBM&MFDUSPO"DDFQUPS /NLY ONE TERMINAL ELECTRON ACCEPTOR OF THE CYANOBAC TERIAL RESPIRATORY ELECTRON TRANSPORT CHAINS IS KNOWN NAMELY / .EITHER PHYSIOLOGIC EXPERIMENTS NOR THE TOTAL GENOMIC SEQUENCES HAVE YIELDED EVIDENCE FOR THE EXISTENCE OF ANAEROBIC RESPIRATION IN CYANOBACTERIA *5IF3FTQJSBUPSZ$IBJOTPG4ZOFDIPDZTUJT TQ1$$ &IGURE SHOWS A MODEL FOR ELECTRON TRANSPORT IN 3YNECHOCYSTIS SP 0## $UE TO THE EARLY AVAIL
&IG -ODEL FOR THE RESPIRATORY ELECTRON TRANSPORT CHAINS IN 3YNECHOCYSTIS SP 0## &OR CLARITY 0HOTOSYSTEM )) DONATING ELEC TRONS DIRECTLY INTO THE QUINONE POOL OF THE )#- AND 0HOTOSYSTEM ) ACCEPTING ELECTRONS FROM EITHER CYTOCHROME C OR PLASTOCYANIN IN THE )#- HAVE BEEN OMITTED 4HERE IS ALSO AN ELECTRON TRANSPORT FROM 0HOTOSYSTEM ) BACK INTO THE QUINONE POOL CYCLIC PHOTOSYNTHETIC ELECTRON TRANSPORT WHOSE COMPONENTS HAVE ALSO BEEN OMITTED 0 INNER PERIPLASMIC SPACE # CYTOPLASM 4 INTRATHYLAKOID SPACE #- CYTOPLASMIC MEMBRANE )#- INTRACYTOPLASMIC MEMBRANES OR THYLAKOIDS 1 OXIDIZED QUINONES 1( REDUCED QUINONES
ABILITY OF THE TOTAL GENOMIC SEQUENCE THIS IS THE ONLY STRAIN IN WHICH SUCH A MODEL CAN BE CONSTRUCTED WITH SOME CONlDENCE !N APPLICATION OF THIS SCHEME TO OTHER CYANOBACTERIA SHOULD BE MADE ONLY WITH GREAT CAUTION %SPECIALLY IN STRAINS CAPABLE OF CELL DIFFERENTIATION EG HETEROCYSTS THE SITUATION IS CERTAINLY MUCH MORE COMPLICATED (OWEVER EVEN IN 3YNECHOCYSTIS THERE REMAIN SEVERAL UNCERTAINTIES #YTOCHROME C- IS NOT SHOWN BECAUSE BOTH ITS CEL LULAR LOCATION AND ITS POSITION IN THE ELECTRON TRANSPORT CHAINS IS UNCLEAR 3IMILARLY THE $RG! DEHYDROGENASE AND .!$( DEHYDROGENASE OF TYPE )) HAVE BEEN DE LIBERATELY OMITTED &URTHERMORE THE TYPE ) .!$0 ( DEHYDROGENASE IS NOT SHOWN WITH THE DETAILS OF ITS SUBUNITS ESPECIALLY SINCE THE SUBUNIT RESPONSIBLE FOR .!$0 ( OXIDATION IS NOT YET KNOWN 4HE ELECTRON TRANSPORT CHAIN OF THE #- IS LESS WELL CHARACTERIZED THAN THAT IN THE )#- ****OUFSBDUJPOCFUXFFO3FTQJSBUJPOBOE 1IPUPTZOUIFTJT 4HE QUESTION OF INTERACTION BETWEEN RESPIRATION AND PHOTOSYNTHESIS IN CYANOBACTERIA IS AN OLD ONE AND INDEED ONE OF THE MAIN REASONS THAT CYANOBACTERIAL RESPIRATION HAS ORIGINALLY BEEN STUDIED 4HE INTERAC TION IS VERY DIFFERENT FOR THE TWO DIFFERENT RESPIRA TORY CHAINS IN THE #- AND THE )#- 3INCE THE #- CONTAINS NO PHOTOSYSTEMS ITS RESPIRATORY CHAIN DOES NOT SHARE COMPONENTS WITH PHOTOSYNTHESIS AND THE INTERACTION IS OF NECESSITY INDIRECT 4HE ENERGIZATION OF THE #- IS A VERY IMPORTANT PROCESS HOWEVER AND MUST BE POSSIBLE IN ORDER TO ALLOW THE CELLS CONTROLLED TRANSPORT OF RELEVANT SUBSTANCES INTO OR OUT OF THE CELL 3INCE IT WAS POSSIBLE TO CONSTRUCT A MUTANT STRAIN OF 3YNECHOCYSTIS SP 0## THAT DOES NOT RESPIRE (OWITT AND 6ERMAAS ENERGIZATION OF THE #- IS EVIDENTLY POSSIBLE BY PRIMARY ENERGIZATION OF THE )#- BY PHOTOSYNTHESIS AND SUBSEQUENT TRANSFER OF ENERGY TO THE #- "IOCHEMICALLY THIS PROCESS INVOLVES PROBABLY DIFFUSION OF !40 GENERATED BY AN !40 SYNTHASE IN THE )#- TO AN !40ASE LOCATED IN THE #- #URIOUSLY CYANOBACTERIA CONTAIN ONLY ONE SET OF &O& !40ASE GENES SO THAT EITHER THIS !40ASE IS PRESENT IN BOTH MEMBRANES OR A COMPLETELY DIFFER ENT !40ASE IS ACTIVE IN ONE OF THEM )N THE lRST CASE WHICH ON CURRENT EVIDENCE SEEMS TO BE THE MORE LIKELY ONE THE IMPORTANT QUESTION REMAINS HOW THIS ENZYME IS TARGETED DURING BIOSYNTHESIS INTO BOTH
(FPSH4DINFUUFSFSBOE%JFUNBS1JMT MEMBRANES WHILE MOST MEMBRANE PROTEINS OCCUR ONLY IN #- OR )#- 4HE INTERACTION BETWEEN RESPIRATORY AND PHOTOSYN THETIC ELECTRON TRANSPORT CHAINS IN THE )#- IS CHARAC TERIZED BY THEIR SHARING OF COMMON COMPONENTS OF WHICH THE QUINONE POOL IS THE MOST IMPORTANT 4HE CONSTRUCTION OF MUTANTS THAT LACK DIFFERENT COMPO NENTS OF THIS LARGE BIOENERGETIC COMPLEX HAS CLEARLY DEMONSTRATED THAT ELECTRONS CAN ENTER FROM EITHER THE @RESPIRATORY SIDE IE THE PRIMARY DEHYDROGENASES OR THE @PHOTOSYNTHETIC SIDE IE 0HOTOSYSTEM )) AND END UP EITHER IN THE @RESPIRATORY OXYGEN CONSUMING TERMINAL OXIDASES OR 0HOTOSYSTEM ) 5NFORTUNATELY IT IS NOT YET KNOWN WELL TO WHAT EXTENT THE DIFFERENT BRANCHES OF ELECTRON TRANSPORT CONTRIBUTE TO TOTAL ELECTRON mOW UNDER NATURAL CONDITIONS IN WILD TYPE CELLS "ESIDES SOME TRIVIAL STATEMENTS SUCH AS THE INACTIVITY OF THE TWO PHOTOSYSTEMS IN DARKNESS THIS IS LARGELY DUE TO THE FACT THAT NO IN VIVO ASSAY EXISTS THAT ALLOWS TO FOLLOW THE REDOX REACTIONS OF ONE RE SPIRATORY BRANCH INDEPENDENT OF THE OTHERS THAT ARE ALSO PRESENT "DLOPXMFEHNFOUT 7E ARE GRATEFUL TO 0ROFS 3 4ABATA AND $ "RYANT FOR ACCESS TO UNPUBLISHED RESULTS 7ORK IN THE AUTHORS LABORATORY WAS PARTIALLY SUPPORTED BY (&30 (U MAN &RONTIERS FOR 3CIENCE 0ROGRAM PROJECT .O 2' AND !CCIONES )NTEGRADAS !USTRIA %SPA×A 4HE TECHNICAL ASSISTANCE OF -R / +UNTNER AND -R # +OLOWRAT IS GRATEFULLY ACKNOWLEDGED 3FGFSFODFT "ERGER 3 %LLERSIEK 5 3TEINMàLLER + #YANOBACTERIA CONTAIN A MITOCHONDRIAL COMPLEX ) HOMOLOGOUS .!$( DEHYDROGENASE &%"3 ,ETT n "ERRY 3 3CHNEIDER $ 6ERMAAS 7&* AND 2ÚGNER - %LECTRON TRANSPORT ROUTES IN WHOLE CELLS OF 3YNECHOCYSTIS SP D STRAIN 0## 4HE ROLE OF THE CYTOCHROME BD TYPE OXIDASE "IOCHEMISTRY n "IGGINS * 2ESPIRATION IN BLUE GREEN ALGAE * "ACTERIOL n "OISON ' "OTHE ( (ANSEL ! AND ,INDBLAD 0 %VIDENCE AGAINST A COMMON USE OF THE DIAPHORASE SUBUNITS BY THE BI DIRECTIONAL HYDROGENASE AND BY THE RESPIRATORY COMPLEX ) IN CYANOBACTERIA &%-3 -ICROBIOL ,ETT n "RODA % 4HE %VOLUTION OF THE "IOENERGETIC 0ROCESSES 0ERGAMON 0RESS /XFORD
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!UTHOR FOR CORRESPONDENCE EMAIL DAVIDEZANNONI UNIBOIT $AVIDE :ANNONI ED 2ESPIRATION IN !RCHAEA AND "ACTERIA 6OL $IVERSITY OF 0ROKARYOTIC 2ESPIRATORY 3YSTEMS PP n © Springer 0RINTED IN 4HE .ETHERLANDS
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THE 1A IS FULLY REDUCED UNDER ANAEROBIC CONDITIONS AND THIS MIGHT BE DUE TO THE LACK OF BOTH QUINOL OXIDASE AND )#- SYSTEM 4HE EXPRESSION OF PHOTOSYNTHETIC AND RESPIRATORY COMPONENTS IS CONTROLLED BY THE OXYGEN TENSION AND BY THE REDOX STATE OF THE SYSTEM 4HIS GENETIC COORDINATION MECHANISM DOES NOT NECESSARILY REQUIRE A DIRECT INTERAC TION OF THE TWO SETS OF COMPONENTS IN LINE WITH THEIR DIFFERENT SPATIAL MEMBRANE LOCATION 4HE SIGNALS TO WHICH THE SYSTEM RESPONDS ORIGINATE FROM EITHER SPECIlC RESPIRATORY COMPONENTS EG CBB OXIDASE AS IN THE CASE OF OXYGEN SENSING OR FROM REDOX CARRIERS INVOLVED IN BOTH OXIDATIVE AND PHOTOSYNTHETIC %4 AS FOR REDOX SENSING !LTHOUGH THE GENETIC CONTROL OF THE SUPRAMOLECULAR ARRANGEMENT OF THE %4#S IS AT PRESENT LARGELY UNDElNED THE WORKING SCHEME PRESENTED HERE SUGGESTS A TENTATIVE FRAMEWORK OF GENETIC REGULATORY CONNECTIONS IN 2B CAPSULATUS ANDOR 2B SPHAEROIDES
**OUSPEVDUJPO "#BDLHSPVOE8IBU*T*NQPSUBOUUP,OPX "CPVU1IZTJPMPHZ &DPMPHZ BOE#JPDIFNJTUSZ PG'BDVMUBUJWF1IPUPTZOUIFUJD#BDUFSJB &ACULTATIVE PHOTOSYNTHETIC BACTERIA ARE ANOXYGENIC PHOTOTROPHS IE DO NOT GENERATE OXYGEN DURING THEIR PHOTOSYNTHETIC GROWTH AS #YANOBACTERIA DO SEE #HAPTER BY ' 3CHMETTERER 6OL WHICH ARE ALSO CAPABLE TO OBTAIN ENERGY FROM AEROBIC AND ANAEROBIC METABOLISM IN DARKNESS 0RINCE :ANNONI )N GENERAL THEIR PHOTOSYNTHETIC APPARATUS BAC TERIOCHLOROPHYLLS AND CAROTENOIDS OF LIGHT HARVESTING SYSTEMS PHOTOCHEMICAL REACTION CENTER IS STRONGLY REPRESSED BY MOLECULAR OXYGEN $REWS AND 'OLECKI AN EXCEPTION TO THIS RULE IS A GROUP OF BACTE RIA THE SO CALLED @AEROBIC ANOXYGENIC PHOTOTROPHS PRIMARILY ISOLATED FROM MARINE ENVIRONMENTS GENERA %RYTHROBACTER AND R 2OSEOBACTER WHICH ARE UNABLE TO SYNTHESIZE THEIR PHOTOSYNTHETIC APPARATUS WITHOUT THE PRESENCE OF MOLECULAR OXYGEN 3HIMADA 9URKOV AND "EATTY 4HE METABOLIC OPTIONS AVAILABLE TO TYPICAL FACULTATIVE PHOTOTROPHIC BACTERIA EG GENERA 2HODOBACTER 2HODOFERAX 2HODOSPIRIL LUM 2HODOPSEUDOMONAS PUT THEM IN A POSITION TO SURVIVE IN QUITE DIFFERENT HABITATS AND MANY SPECIES CAN USE A VARIETY OF CARBON SOURCES SUCH AS ORGANIC ACIDS OR FATTY ACIDS BUT ALSO #/ AS SOLE CARBON SOURCE !BBREVIATIONS #- n CYTOPLASMIC MEMBRANE #/8 n CYTO CHROME C OXIDASE #YT n CYTOCHROME $-3 n DIMETHYLSULlDE $-3/ n DIMETHYLSULFOXIDE %4 n ELECTRON TRANSPORT %4#S n ELEC TRON TRANSPORT CHAINS (I0)0 n (IGH POTENTIAL )RON SULFUR 0ROTEIN )#- n INTRACYTOPLASMIC MEMBRANE ,( n LIGHT HARVESTING COMPLEX 1 POOL n UBIQUINONE POOL 1A n QUINONE PRIMARY AC CEPTOR 1B n QUINONE SECONDARY ACCEPTOR 1/8 n QUINOL OXIDASE 2# n PHOTOCHEMICAL REACTION CENTER 4-0$ n TETRAMETHYL P PHENYLENEDIAMINE
AND ( AS SOLE ELECTRON DONOR &ACULTATIVE PHOTOTROPHS CAN BE FOUND IN NATURAL HABITATS JUST BELOW THE OXIC ANOXIC INTERFACE OF LAKES AND THE CAPACITY TO GROW IN THE DARK BY RESPIRATION IS GENERALLY CONSIDERED A MECHANISM FOR TEMPORARY SURVIVAL IN TRANSIENTLY OXYGENATED ENVIRONMENTS OR AS A WAY FOR MAINTAIN ING THE ENERGYREDOX BALANCE -ADIGAN 4HIS LATTER REQUIREMENT IS HOWEVER THE @PRE REQUISITE FOR GROWTH IN THE CASE OF AEROBIC ANOXYGENIC PHOTOTROPHS 9URKOV AND "EATTY /N A PHYSIOLOGICALECOLOGICAL POINT OF VIEW 4RàPER AND 0FENNIG ALL ANOXYGENIC PHOTOTROPHS CAN BE DIVIDED INTO FOUR SUBGROUPS A 0URPLE SULFUR #HROMATIACEAE %CTOTHIORHODOSPIRACEAE B 0URPLE NONSULFUR 2HODOSPIRILLACEAE C 'REEN SULFUR #HLOROBIACEAE AND D 'REEN GLIDING BACTERIA #HLOROmEXACEAE 4HESE SUBGROUPS DO NOT INCLUDE STRICTLY ANAEROBIC SPORE FORMING PHOTOTROPHS OF THE GENUS (ELIOBACTERIUM 'EST AND &AVINGER WHICH ARE ALLOCATED APART ORDER #LOSTRIDIALES DUE TO THEIR PHYSIOLOGICAL PECULIARITIES EG PRESENCE OF BACTERIOCHLOROPHYLL G "ROCHMANN AND ,IPINSKI /N A PHYLOGENETIC BASIS 3 R2.! ANALYSES PURPLE BACTERIA AND THEIR RELATIVES ARE GROUPED IN A NEW CLASS THE 0ROTEOBACTERIA 3TACKEBRANDT ET AL WHICH IS FORMED BY SEVERAL SUBCLASSES NAMELY _ SUBCLASS CONTAINING MOST OF THE SPECIES OF THE 2HODOSPIRILLACEAE FAMILY ALONG WITH STRICTLY AEROBIC SPECIES OF THE GENERA %RYTHROBACTER ` SUBCLASS IN CLUDING NONSULFUR PURPLE GENERA SUCH AS 2HODOCYCLUS AND 2UBRIVIVAX a SUBCLASS WHICH INCLUDES PURPLE SULFUR BACTERIA #HROMATIACEAE AND %CTOTHIORHO DOSPIRACEAE 'REEN SULFUR #HLOROBIACEAE AND NONSULFUR BACTERIA #HLOROmEXACEAE RESULTED TO BE PHYLOGENETICALLY UNRELATED FORM TWO DISTINCT CLASSES 7OESE "EFORE GOING INTO DETAILS ABOUT THE METABOLIC
$IBQUFS *OUFSBDUJPO#FUXFFO1IPUPTZOUIFTJTBOE3FTQJSBUJPO OPTIONS CHARACTERIZING ANOXYGENIC FACULTATIVE PHO TOTROPHS SEE BELOW THE READER SHOULD BE AWARE OF A FEW BASIC NOTIONS ON GROWTH CAPACITY OF THE ABOVE REPORTED BACTERIAL GENERA 0HOTOSYNTHETIC BACTERIA WHICH ARE ABLE TO GROW BOTH IN THE LIGHT AND IN THE DARK UNDER AEROBIC CONDITIONS BELONG TO _ AND ` SUBCLASSES WHILE #HROMATIACEAE #HLOROBIACEAE AND %CTOTHIORHODOSPIRACEAE ARE RATHER STRICT LIGHT DEPENDENT ANAEROBES ALTHOUGH SOME SPECIES OF GREEN SULFUR BACTERIA MAY GROW CHEMOTROPHICALLY AT VERY LOW OXYGEN TENSION !S MENTIONED ABOVE FACULTATIVE PHOTOTROPHS ARE ENDOWED WITH SEVERAL METABOLIC OPTIONS THIS PECU LIARITY MAKES THIS GROUP ONE OF THE MOST mEXIBLE OF THE MICROBIAL WORLD 3PECIES SUCH AS 2HODOBACTER CAPSULATUS AND 2B SPHAEROIDES MAY GROW 2B AEROBICALLY AND PHOTOSYNTHETICALLY USING EITHER OR GANIC OR INORGANIC SUBSTRATES BUT ALSO ANAEROBICALLY WITH TRIMETHYLAMINE . OXIDE 4-!/ OR DIMETHYL SULFOXIDE $-3/ AS ELECTRON ACCEPTORS 4HE QUES TION WHETHER ANAEROBIC REDUCTION OF $-3/ OR 4-!/ IS A TRUE FERMENTATIVE PROCESS REQUIRING ACCESSORY OXIDANTS OR ANOTHER EXAMPLE OF ANAEROBIC RESPIRATION IS STILL A MATTER FOR DEBATE -ADIGAN AND 'EST -C%WAN ET AL *ONES ET AL :ANNONI SEE ALSO #HAPTER BY -C%WAN ET AL 6OL FOR DETAILS ON THE MOLECULAR STRUCTURE AND FUNCTION OF $-3/ AND 4-3/ REDUCTASES &URTHERMORE SOME STRAINS OF THE SPECIES 2HODOPSEUDOMONAS 2PS DENITRIlCANS AND 2B PALUSTRIS 2OSEOBACTER 2SB SPHAEROIDES CAN REDUCE NITRATE ./n INTO DINITROGEN . VIA NITRITE ./n AND IN SOME CASES ALSO NITRIC OXIDE ./ AND NITROUS OXIDE ./ &ERGUSON ET AL 2ICHARDSON ET AL SEE ALSO #HAPTER BY &ERGUSON AND 2ICHARDSON 6OL 4HESE ENERGY GEN ERATING PROCESSES ARE CATALYZED BY ELECTRON TRANSFER CHAINS %4#S WHICH REDOX COMPONENTS ARE INSERTED OR ASSOCIATED WITH THE CYTOPLASMIC MEMBRANE #- !PPARENTLY NOT ALL THE ABOVE SUMMARIZED METABOLIC OPTIONS ARE ACTIVATED OR CAN BE AVAILABLE SIMULTANE OUSLY IN A SINGLE SPECIES HOWEVER IN SOME CASES EG AEROBIC CONDITIONS IN THE LIGHT THE ELECTROCHEMICAL PROTON GRADIENT 6+( GENERATED BY A GIVEN ELECTRON TRANSPORT CHAIN MAY AFFECT DIRECTLY OR INDIRECTLY OTHER ENERGY REDOX TRANSDUCING APPARATUSES 0HOTOSYNTHETIC BACTERIA CONVERT THE LIGHT ENERGY INTO CHEMICAL ENERGY AT THE LEVEL OF THE PHOTOCHEMI CAL REACTION CENTER 2# 4HIS CHARGE SEPARATION IS FOLLOWED BY A CYCLIC ELECTRON TRANSFER WHICH INVOLVES QUINONE MOLECULES THE CYTOCHROME #YT BC COM PLEX AND THE SOLUBLE #YT C 0RINCE OR THE
MEMBRANE BOUND #YT CY IN THE CASE OF 2HODOBACTER CAPSULATUS *ENNEY AND $ALDAL 5NDER DARK AEROBIC CONDITIONS THESE BACTERIA USE A RESPIRATORY CHAIN RELATED TO THAT PRESENT IN MITOCHONDRIA 4HE LAST STEP OF / REDUCTION INTO (/ IS CATALYZED BY A #YT C OXIDASE AND ALSO BY THE SO CALLED ALTERNATIVE OXIDASE WHICH IS BRANCHED DIRECTLY AFTER THE QUINONE POOL :ANNONI ET AL 4HE PHOTOSYNTHETIC AND RESPIRATORY PROCESSES INVOLVE TRANSMEMBRANE COMPLEXES LOCATED ON THE CYTOPLASMIC MEMBRANE 2# #YT BC DEHYDROGENASE QUINOL OXIDASE CBB ANDOR AA OXIDASES AND ELECTRON CARRIERS #YT C (I0)0 #YT CY AND ENZYMES IN THE PERIPLASMIC SPACE NITRATE NITRITE ./ $-3/ REDUCTASES )N &IG A BLOCK SCHEME OF THE DIFFERENT ELECTRON TRANSPORT PATHWAYS OPERATING IN 2HODOBACTER SPHAEROIDES IS SHOWN SEE ALSO #HAPTERS OF 6OL FOR MOLECULAR DETAILS !LTHOUGH THIS METABOLIC mEXIBILITY ALLOWS THE BACTERIA TO PROSPER IN VARIOUS ENVIRONMENTS AND TO RESPOND TO CHANGES OF THEIR SURROUNDINGS THE ASSEM BLY OF THE DIFFERENT BIOENERGETIC PATHWAYS IS HIGHLY REGULATED TO PREVENT THE UNNECESSARY BIOSYNTHESIS OF SPECIlC ENZYMES AND ELECTRON TRANSFER COMPONENTS AS DESCRIBED IN 3ECTION 6 3HORT TERM REGULATIONS BETWEEN THE DIFFERENT BIOENERGETIC CHAINS ARE ALSO USED BY THE BACTERIA FOR AN EFlCIENT AND MAXIMAL UTILIZATION OF THE AVAILABLE ENERGY &OR EXAMPLE PHOTOSYNTHESIS STRONGLY SLOWS DOWN RESPIRATION DENITRIlCATION OR THE REDUCTION OF $-3/ OR 4-!/ .AKAMURA 3ATOH -C%WAN ET AL #OTTON ET AL 3ABATY ET AL 5NDER DARK CONDITIONS THE BACTERIA UTILIZE THE AVAILABLE ELECTRON ACCEPTOR WHICH POSSESSES THE HIGHEST MID POINT POTENTIAL TO OBTAIN THE MAXIMUM OF ENERGY /XYGEN /(/ %M M6 IS REDUCED IN PREFERENCE TO NITRATE ./n./n %M M6 OR $-3/ $-3/$-3 %M M6 4HE FREE ENERGY RECOVERED BY THE BACTERIA AT THE EXPENSE OF .!$( OXIDATION AND REDUCTION OF OXYGEN NITRATE OR $-3/ IS EQUAL TO AND K*MOL RESPECTIVELY (OWEVER THE MOST FAVORABLE BIOENERGETIC PROCESS IS PHOTOSYNTHESIS WHERE THE LIGHT ENERGY IS UTILIZED BY THE CELLS TO FORM !40 AND REDUCING POWER 4HE SHORT TERM REGULATIONS BETWEEN THE DIFFERENT BIOENERGETIC CHAINS ARE ILLUSTRATED IN &IG IN THE CASE OF 2HODOBACTER SPHAEROIDES SP DENITRIlCANS A STRAIN WHICH POSSESSES THE THREE BIO ENERGETIC PATHWAYS #ONTINUOUS ILLUMINATION PARTIALLY INHIBITS BOTH RESPIRATION AND DENITRIlCATION ACTIVITIES AND THIS LAST BIOENERGETIC PROCESS ONLY OCCURS WHEN ALL THE OXYGEN HAS BEEN CONSUMED BY THE CELLS ,IGHT
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&IG "LOCK SCHEME ILLUSTRATING THE COMPLEX NETWORK OF ELECTRON TRANSPORT CHAINS IN 2B SPHAEROIDES 3YMBOLS USED THICK BLACK ARROWS INDICATE THE INmUX OF REDUCING EQUIVALENTS WHILE THIN BLACK ARROWS SYMBOLIZE THE OUTPUT OF ELECTRONS LEADING TO THE MEMBRANE BOUND OXIDASES (ATCHED ARROWS INDICATE OUTPUT OF ELECTRONS INVOLVED IN ANAEROBIC RESPIRATION 'RAY COLORED REDOX COMPONENTS AND ARROWS ARE THOSE SPECIlCALLY INVOLVED IN PHOTOCYCLIC ELECTRON TRANSFER WHILE THOSE SHADED OFF ARE SHARED BY PHOTOSYNTHESIS AND RESPIRATION !BBREVIATIONS 2# PHOTOCHEMICAL REACTION CENTER Hi RADIANT ENERGY # SOLUBLE #YT C #Y MEMBRANE ATTACHED #YT CY 1/8 FUNC TIONAL UBIQUINOL OXIDASE QXT!" OPERON 1/8 NOT FUNCTIONAL UBIQUINOL OXIDASE QOX"! OPERON 3EE TEXT AND #HAPTERS AND BY #OOLEY ET AL AND BY /H AND +APLAN 6OL RESPECTIVELY AND #HAPTERS BY &ERGUSON AND 2ICHARDSON AND BY 6IGNAIS ET AL6OL RESPECTIVELY FOR FURTHER DETAILS
INHIBITS EACH STEP OF THE DENITRIlCATION PROCESS I E REDUCTION OF NITRITE INTO ./ OR REDUCTION OF ./ INTO . 3ABATY ET AL 4HE MODE OF INTERACTION BETWEEN THE DIFFERENT BIOENERGETIC CHAINS OF PHOTOSYNTHETIC BACTERIA HAS BEEN COMPREHENSIVELY STUDIED 4HE INTERACTIONS OCCUR BY TWO NON EXCLUSIVE MECHANISMS 4HE lRST MECHANISM IS AN INDIRECT EFFECT OF THE PROTON MOTIVE FORCE PRODUCED BY ONE BIOENERGETIC PROCESS ON TRANS MEMBRANE COMPLEXES INVOLVED IN THE OTHER CHAINS LIKE FOR EXAMPLE THE .!$( DEHYDROGENASE THE #YT BC COMPLEXES OR THE OXIDASES #OTTON ET AL 4HIS EXPLANATION IS AN EXTENSION OF THE RESPIRATORY CONTROL IN MITOCHONDRIA PROPOSED BY -ITCHELL .ICH OLLS AND &ERGUSON WHERE THE PROTON MOTIVE FORCE INDUCED BY RESPIRATION EXERTS A BACK PRESSURE ON THE PROTON TRANSLOCATING COMPLEXES 4HE SECOND MECHANISM SUPPOSES A DIRECT INTERACTION BETWEEN ELECTRON CARRIERS COMMON TO DIFFERENT BIOENERGETIC CHAINS :ANNONI ET AL 6ERMÏGLIO AND #ARRIER 3INCE SEVERAL ELECTRON CARRIERS THE #YT BC THE UBIQUINONE MOLECULES AND #YT C ARE ENGAGED
&IG )NHIBITION BY LIGHT OF AEROBIC RESPIRATION AND DENITRIlCATION #ELLS OF 2B SPHAEROIDES SP FORMA DENITRIlCANS WERE PLACED IN THE PRESENCE OF M- NITRATE 4HE CONCENTRATIONS OF DISSOLVED GASES / AND . IN THE SUSPENSION WERE MEASURED WITH A MASS SPECTROMETER )LLUMINATION OF THE SUSPENSION B INHIBITS THE / CONSUMPTION 0RODUCTION OF . FROM REDUCTION OF NITRATE OR DENITRIlCATION ONLY OCCURS WHEN ALL THE / HAS BEEN CONSUMED ;/= BY THE CELLS $ENITRIlCATION IS ALSO INHIBITED BY CON TINUOUS ILLUMINATION 4HE UPWARDS ARROWS INDICATE THE SWITCHING ON OF ILLUMINATION WHILE THE DOWNWARDS ARROWS CORRESPOND TO ITS CESSATION &IGURE ADAPTED FROM 3ABATY ET AL
$IBQUFS *OUFSBDUJPO#FUXFFO1IPUPTZOUIFTJTBOE3FTQJSBUJPO IN DIFFERENT BIOENERGETIC PROCESSES THE ACTIVITY OF A GIVEN BIOENERGETIC CHAIN AFFECTS THE REDOX STATE OF THESE COMPONENTS AND CONSEQUENTLY INmUENCES THE FUNCTIONING OF THE OTHERS CHAINS 4HE MAIN OBJECTIVE OF THIS #HAPTER IS TO OUTLINE ALL DIFFERENT LEVELS OF INTERACTION STRUCTURAL FUNCTIONAL ENERGETIC AND GENETIC BETWEEN PHOTOSYNTHETIC AND RESPIRATORY REDOX COMPONENTS WHEN SIMULTANEOUSLY PRESENT IN THE CYTOPLASMIC MEMBRANE SYSTEM #- )#- OF FACULTATIVE PHOTOSYNTHETIC BACTERIA 2ATHER THAN DESCRIBING IN DETAILS THE DATA AVAILABLE FOR THE NUMEROUS BACTERIAL SPECIES STUDIED REPRESENTATIVE CASES WILL BE ILLUSTRATED TO PROVIDE A GENERAL PICTURE &OR FURTHER DISCUSSION ALONG THE SAME RESEARCH LINES WE REFER THE READER TO THE BOOKS EDITED BY # !NTHONY !*" :EHNDER AND BY 2% "LANKEN SHIP ET AL **.PEFTPG*OUFSBDUJPO#FUXFFO1IPUPTZO UIFUJDBOE3FTQJSBUPSZ"DUJWJUJFT -OST OF THE ANOXYGENIC PURPLE PHOTOTROPHS CONTAIN A RATHER COMPLEX MEMBRANE SYSTEM WHICH IS FORMED BY AN EXTERNALLY LOCATED MEMBRANE SURROUNDING THE CELL NAMED CYTOPLASMIC MEMBRANE OR #- AND BY INTERNALLY LOCATED MEMBRANES WITH VARIOUS ARRANGE MENTS NAMED INTRACYTOPLASMIC MEMBRANES OR )#/ELZE AND $REWS #OLLINS AND 2EMSEN $REWS AND 'OLECKI ! CONSISTENT AMOUNT OF DATA TEND TO INDICATE THAT THE MAJOR COMPONENTS OF THE )#- ARE THE MEMBRANE BOUND COMPLEXES FORM ING THE PHOTOSYNTHETIC APPARATUS EG PHOTOCHEMICAL REACTION CENTER 2# OR LIGHT HARVESTING SYSTEMS ,( WHILE RESPIRATORY ENZYMES EG CYTOCHROME OXIDASES ARE THE MAJOR COMPONENTS OF THE #- 4HIS LATTER CONCLUSION IS DERIVED FROM THE OBSERVATION THAT THE SYNTHESIS OF THE PHOTOSYNTHETIC APPARATUS IS ASSOCIATED TO THE FORMATION OF LARGE AMOUNTS OF INTRACYTOPLASMIC MEMBRANES .EVERTHELESS THE CAROTENOID BAND SHIFT A SPECIlC INDICATOR OF THE MEMBRANE POTENTIAL DUE TO A 3TARK EFFECT ON THE LIGHT HARVESTING CAROTENOID MOLECULES IS IDENTICAL WHEN INDUCED EITHER BY THE PHOTOSYNTHETIC OR THE RESPIRATORY ACTIVITIES #HANCE AND 3MITH 7RAIGHT ET AL 4HIS DEM ONSTRATES THAT THE MEMBRANE POTENTIAL IS DELOCALIZED OVER THE ENTIRE INTERNAL MEMBRANE 4HEREFORE THE ELECTRON COMPONENTS OF THE DIFFERENT BIOENERGETIC CHAINS EXPERIENCE THE SAME MEMBRANE POTENTIAL 4HE INmUENCE OF THE PROTON MOTIVE FORCE GENERATED DURING PHOTOSYNTHETIC ACTIVITY ON THE INHIBITION OF THE RESPIRA
TORY ACTIVITY HAS BEEN DEMONSTRATED BY DETERMINING THE EFFECT OF UNCOUPLERS 4HE ADDITION OF UNCOUPLERS PREVENTS THE INHIBITION OF THE RESPIRATORY ACTIVITY BY CONTINUOUS ILLUMINATION 2AMIREZ AND 3MITH #OTTON ET AL 2ICHAUD ET AL 4HE LIGHT INDUCED PROTON MOTIVE FORCE INHIBITS THE RESPIRATORY CHAIN ESSENTIALLY AT THE COMPLEX ) LEVEL )NDEED THIS COMPLEX HAS BEEN SHOWN TO REVERSE ELECTRON mOW FROM THE QUINOL POOL TO .!$ IN THE PRESENCE OF A LIGHT INDUCED MEMBRANE POTENTIAL +LEMME 6IGNAIS ET AL +NAFF AND +ÊMPF $UPUIS ET AL (ERTER ET AL /N THE OTHER HAND THE PROTON MOTIVE FORCE APPEARS TO EXERT NO CONTROL ON THE OPERATION OF THE #YT BC OR THE TERMINAL OXIDASES INVOLVED IN THE RESPIRATION OR DENITRIlCATION 4HIS CAN BE INFERRED FROM THE OBSERVATION THAT NEITHER THE REDUCTION OF NITRITE ./ OR / CAN BE INHIBITED BY CONTINUOUS ILLUMINATION IN THE PRESENCE OF AN ARTIlCIAL ELECTRON DONOR LIKE 4-0$ ALTHOUGH A LARGE MEMBRANE POTENTIAL IS INDUCED UNDER THESE PARTICULAR CONDITIONS 3ABATY ET AL "ESIDES THIS INDIRECT INTERACTION DIRECT CONNECTION BETWEEN THE DIFFERENT BIOENERGETIC CHAINS HAS BEEN DEMONSTRATED BY SEVERAL APPROACHES 4HE INVOLVE MENT OF THE 1 POOL ACTING AS A BRANCHING POINT BETWEEN RESPIRATORY AND PHOTOSYNTHETIC CHAINS HAS BEEN CLEARLY ESTABLISHED BY THE STIMULATION OF THE UBIQUINOL OXIDASE ACTIVITY IN THE PRESENCE OF LIGHT AND EXOGENOUS ELECTRON DONORS EG HORSE HEART #YT C OR 4-0$ :ANNONI ET AL IN BOTH CHROMATOPHORES AND HYBRID MEMBRANES GENERATED FROM %4 MUTANTS :ANNONI ET AL 4HIS INTERACTION BETWEEN THE RESPIRATORY AND THE PHOTOSYNTHETIC CHAINS AT THE LEVEL OF THE 1 POOL HAS IMPORTANT IMPLICATIONS FOR THE OPERATION OF THE PHOTOSYNTHETIC APPARATUS 5NDER ANAEROBIC CONDITIONS AND IN THE PRESENCE OF REDUCED CARBON SUBSTRATES SUCH AS BUTYRATE OR SUCCINATE THE PHOTOCHEMICAL ACTIVITY IS PARTIALLY INACTIVATED DUE TO THE REDUCTION OF A FRACTION OF THE PRIMARY ELECTRON ACCEPTOR 1A OF THE 2# -C%WAN ET AL 4HIS IS CAUSED BY THE ENTRY OF ELECTRONS VIA THE DEHYDROGENASES WHICH ARE NOT REMOVED BY THE RE SPIRATORY ACTIVITIES 5NDER THESE REDUCING CONDITIONS CONTINUOUS ILLUMINATION COULD REACTIVATE ALL THE 2#S 6ERMÏGLIO AND *OLIOT UNPUBLISHED /NE POSSIBLE INTERPRETATION IS THAT THE PHOTOCHEMISTRY OF THE FRAC TION OF ACTIVE 2#S GENERATES A MEMBRANE POTENTIAL WHICH INDUCES A REVERSE ELECTRON mOW FROM THE QUINOL POOL TO .!$ +LEMME 6IGNAIS ET AL +NAFF AND +ÊMPF $UPUIS ET AL (ERTER ET AL 4HIS WOULD PARTIALLY REOXIDIZE
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THE 1 POOL AND THE 2#S ELECTRON ACCEPTORS AND FULLY REACTIVATE THEIR PHOTOCHEMICAL ACTIVITY 2EOXIDATION OF THE QUINOL POOL IS ALSO OBTAINED BY OXYGENATION OF THE BACTERIAL SUSPENSION OR BY ADDITION OF ALTER NATIVE ELECTRON ACCEPTORS SUCH AS NITRATE OR $-3/ -C%WAN ET AL 4AKAMIYA ET AL 4HIS TYPE OF INTERACTION IS PARTICULARLY CRUCIAL IN THE CASE OF AEROBIC PHOTOSYNTHETIC BACTERIA EG 2OSEOBACTER GENUS 4HESE BACTERIA CAN GROW PHOTOSYNTHETICALLY ONLY UNDER AEROBIC CONDITION OR IN THE PRESENCE OF ALTERNATIVE ELECTRON ACCEPTORS 4AKAMIYA ET AL 3HIMADA 9URKOV AND "EATTY 5NDER ANAEROBIC CONDITIONS 1A IS TOTALLY REDUCED AND NO PHOTOCHEMISTRY OCCURS 4HIS PARTICULAR BEHAVIOR COULD BE DUE TO THE MID POINT POTENTIAL %M OF THE PRIMARY QUINONE ACCEPTOR WHICH HAS BEEN REPORTED TO BE HIGHER THAN THAT OF ANAEROBIC 2HODOBACTER SPECIES 4AKAMIYA R ET AL +RAMER ET AL 9URKOV ET AL ALTHOUGH THIS LATTER HYPOTHESIS HAS BEEN RECENTLY CHAL LENGED BY 3CHWARZE ET AL &URTHER STUDIES ON THE RESPIRATORY APPARATUS OF 2SB DENITRIlCANS HAVE SHOWN THAT THE MOST STRIKING DIFFERENCE BETWEEN THIS PHOTOSYNTHETIC AEROBE AND TYPICAL PURPLE NON SULFUR BACTERIA IS THE LACK OF A QUINOL OXIDASE PATHWAY #ANDELA ET AL )N THIS RESPECT IT IS IMPORTANT TO REMARK THAT UNDER RESPIRATORY STEADY STATE CONDI TIONS CONTINUOUS ILLUMINATION DOES NOT PERTURB THE REDOX STATE OF THE 1 POOL WHEN THE QUINOL OXIDASE PATHWAY IS FUNCTIONING :ANNONI AND -OORE )NDEED AS SHOWN IN &IG THE MAJOR VARIATION OF THE 1 POOL REDOX STATE AS DETECTED BY A 1 ELECTRODE IN MEMBRANES FROM 2B CAPSULATUS -2 IS SEEN WHEN THE #/8 IS BLOCKED BY CYANIDE OR AFTER THE ONSET OF ANAEROBIOSIS :ANNONI AND -OORE 4HE LACK OF A 1 OXIDASE IN 2SB DENITRIlCANS HAS THEREFORE A GREAT IMPACT ON THE RATE OF PHOTOPHOSPHORYLATION BECAUSE AS MENTIONED ABOVE PHOTOCHEMISTRY DOES NOT OCCUR WHEN THE 1 POOL IN EQUILIBRIUM WITH THE SECONDARY ELECTRON ACCEPTOR 1B IS FULLY REDUCED #ROFTS )N FACT MEMBRANE VESICLES CHROMATOPHORES ISOLATED FROM A TYPICAL REPRESENTATIVE OF PURPLE NON SULFUR BACTERIA 2B CAPSULATUS AND FROM 2SB DENITRIlCANS PERFORM LIGHT INDUCED !40 SYNTHESIS ONLY UNDER OXIC CONDITIONS #ANDELA ET AL /N THE OTHER HAND IT HAS ALSO BEEN DEMONSTRATED THAT THE RATE OF PHOTOPHOSPHORYLATION STRONGLY DEPEND ON THE AMBIENT REDOX POTENTIAL ,OACH "ACCARINI -ELANDRI ET AL )T IS THEREFORE EVIDENT THAT LIGHT INDUCED PHOSPHORYLATION IS NOT AFFECTED BY OXYGEN PER SE BUT REQUIRES A SUITABLE REDOX POISE * M6 ) M6 WHICH IS CLOSE TO THE %M OF THE 1 POOL M6 )N
&IG 2EDOX LEVEL OF THE 1 POOL IN MEMBRANES FROM WILD TYPE STRAIN -2 TRACES A AND B AND MUTANT STRAIN -4 '3 C CELLS OF 2B CAPSULATUS 3TRAIN -4 '3 IS A DOUBLE MUTANT Cn Cn UNABLE TO GROW PHOTOSYNTHETICALLY 2ESPIRATION WAS INITIATED BY THE ADDITION OF M- SUCCINATE 3UCC #YANIDE #. ADDITION WAS AS INDICATED TRACE B AT A CONCENTRATION OF +- WHICH IS REQUIRED TO INHIBIT THE #YT C OXIDASE OF 2B CAPSULATUS CBB TYPE )N A AND C ILLUMINATION WAS SWITCHED /. AND /&& AS INDICATED ;/= SYMBOLIZES THE ONSET OF ANAEROBIOSIS 4HE VERTICAL BAR INDICATES THE OF THE TOTAL UBIQUINONE 1T WHICH CAN BE REDUCED BY SUCCINATE UNDER ANAEROBIC CONDITIONS 4HIS lGURE IS REDRAWN AND MODIlED FROM :ANNONI AND -OORE
THE CASE OF 2SB DENITRIlCANS A VERY ACTIVE RESPIRATORY PATHWAY LEADING TO A #/8 OF AA TYPE #ANDELA ET AL WOULD MAINTAIN THE OPTIMAL REDOX POISE TO KEEP THE 1 POOL PARTIALLY OXIDIZED ALSO UNDER CONTINUOUS ILLUMINATION 7HATEVER THE MOLECULAR EXPLANATION COULD BE IT IS APPARENT THAT RESPIRATION IS REQUIRED TO ALLOW PHOTOCHEMICAL ACTIVITY !NOTHER DEMONSTRATION OF THE DIRECT INTERACTION BETWEEN PHOTOSYNTHETIC AND RESPIRATORY CHAINS IN FACULTATIVE PHOTOTROPHS HAS BEEN PROVIDED BY ANALYSIS OF THE RESPIRATORY ACTIVITY AFTER mASH EXCITATION WITH A VERY SENSITIVE / ELECTRODE &IG 6ERMÏGLIO AND #ARRIER 2ICHAUD ET AL &OLLOWING A SERIES OF SHORT SATURATING mASHES THE RESPIRATORY ACTIVITY IS INHIBITED AFTER EACH mASH BUT STIMULATION IS OBSERVED AFTER AN EVEN NUMBER OF mASHES !DDITION OF UNCOUPLERS DOES NOT AFFECT THIS OSCILLATORY PATTERN 4HE LACK OF OSCILLATION PATTERN FOR A MUTANT OF 2B CAPSULATUS DElCIENT IN #/8 IMPLIES THAT THIS PHENOMENON IS DUE TO THE MODULATION OF THE ACTIVITY OF THIS ENZYME AND NOT TO THE ALTERNATIVE OXIDASE 2ICHAUD ET AL !FTER EACH mASH AN ELECTRON IS DIVERTED FROM THE RESPIRATORY CHAIN TO THE PHOTOXIDIZED 2# AT THE LEVEL OF #YT C "ECAUSE OF THE GATING MECHANISMS AT THE LEVEL OF THE SECONDARY ELEC TRON ACCEPTOR 1B6ERMÏGLIO 7RAIGHT RESPIRATION IS RESTORED ONLY AFTER AN EVEN NUMBER OF mASHES BY FORMATION OF A 1( MOLECULE
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&IG -ODULATION OF THE RESPIRATORY ACTIVITY BY SATURATING mASHES lRED EVERY SECOND FOR A SUSPENSION OF 2B SPHAEROIDES %ACH mASH INDUCES AN INHIBITION OF THE RESPIRATORY ACTIVITY THIS CORRESPONDS TO AN INCREASE IN / CONCENTRATION 4HE RESPIRATORY ACTIVITY IS RESTORED AFTER EVEN mASHES 0ART ! .O ADDITION 0ART " IN THE PRESENCE OF - OF THE UNCOUPLER ###0 &IGURE ADAPTED FROM 2ICHAUD ET AL
***.PEFTPG*OUFSBDUJPO#FUXFFO"FSPCJD BOE"OBFSPCJD3FTQJSBUPSZ"DUJWJUJFT !S ALREADY STATED PHOTOSYNTHETIC ACTIVITY INHIBITS EACH SINGLE STEP OF THE REDUCTION OF NITRATE INTO DINI TROGEN 3ABATY ET AL 4HESE INHIBITIONS ARE NOT PREVENTED IN THE PRESENCE OF UNCOUPLERS AND THEREFORE DO NOT REQUIRE THE FORMATION OF A TRANSMEMBRANE POTENTIAL )T HAS THEREFORE BEEN POSTULATED THAT THIS IS DUE TO DIVERSION OF ELECTRONS FROM THE DENITRIlCATION CHAIN TO THE PHOTOSYNTHETIC CHAIN AT THE LEVEL OF #YT BC AND #YT C TWO COMPONENTS WHICH ARE IN COMMON TO BOTH PATHWAYS 2ICHARDSON ET AL 3ABATY ET AL 4HE PRESENCE OF OXYGEN COMPLETELY INHIBITS THE ELECTRON TRANSFER TO ALTERNATIVE ACCEPTORS LIKE NITRATE OR 4-!/ -C%WAN ET AL +ING ET AL 3ABATY ET AL #ONTRARY TO THE LIGHT INHIBITION OF AEROBIC RESPIRATION INHIBITION OF DENITRIlCATION OR 4-!/ RESPIRATION BY OXYGEN IS NOT SUPPRESSED BY THE ADDITION OF UNCOUPLERS -C%WAN ET AL +ING ET AL 4HEREFORE THIS INHIBITION IS NOT A CON SEQUENCE OF THE THERMODYNAMIC CONTROL OF THE TRANS
MEMBRANE POTENTIAL RESULTING FROM THE RESPIRATORY ACTIVITY ON DIFFERENT ELEMENTS OF THE DENITRIlCATION OR 4-!/ REDUCTION CHAINS 4HIS IS RATHER DUE TO A DIRECT CHANGE IN THE REDOX STATE OF THE ELECTRON TRANSFER COMPONENTS 0OSSIBLE CANDIDATES ARE THE #YT C #YT BC COMPLEX IN THE CASE OF THE INHIBITION OF BOTH NITRITE AND ./ REDUCTION BY OXYGEN &OR NITRATE AND 4-!/ REDUCTION THE INHIBITION BY OXYGEN IS CERTAINLY MEDI ATED VIA THE REDOX STATE OF THE 1 POOL /NE POSSIBILITY IS THAT THE REDUCTION OF NITRATE OR 4-!/ IS ATTAINABLE ONLY WHEN THE 1 POOL IS LARGELY REDUCED 4HE PARTIAL OXIDATION OF THE 1 POOL BY THE RESPIRATORY ACTIVITY MAY PREVENT THE NITRATE OR 4-!/ REDUCTION -ODULATION OF THE ACTIVITY OF OXIDO REDUCING ENZYMES BY THE REDOX STATE OF THE 1 POOL HAS ALREADY BEEN DEMONSTRATED IN THE CASE OF THE ALTERNATIVE OXIDASE OR 1/8 :ANNONI AND -OORE 4HIS OXIDASE IS NOT FUNCTIONAL UNTIL THE QUINONE POOL IS AT LEAST REDUCED /N THE OTHER HAND THE RATE OF THE #/8 IS PROPORTIONAL TO THE REDUCTION LEVEL OF THE QUINONE POOL :ANNONI AND -OORE 4HIS IMPLIES A PREFERENTIAL INTERACTION IE A LOWER KKM BETWEEN QUINOL MOLECULES AND #YT BC THAN BETWEEN QUINOLS AND THE ALTERNATIVE OXIDASE 3UCH BEHAVIOR IS ILLUSTRATED IN &IG WHERE CELLS OF A MUTANT OF 2HODOBACTER CAPSULATUS DElCIENT IN THE CYTOCHROME OXIDASE HAVE BEEN PLACED IN THE PRESENCE OF THE EXOGENEOUS ELECTRON DONOR 4-0$ 5PON ILLU MINATION THE QUINONE POOL IS REDUCED AT THE EXPENSE OF 4-0$ PHOTOOXIDATION 4HIS STIMULATES THE OXYGEN UPTAKE BY THE ALTERNATIVE OXIDASE 5PON CESSATION OF ILLUMINATION THE OXYGEN UPTAKE IS STRONGLY INHIBITED AND IS ONLY RESTORED WHEN ALL THE PHOTOOXIDIZED 4-0$ IS RE REDUCED VIA THE #YT BC COMPLEX *7)FUFSPHFOFJUZBOE-BDLPG5IFSNPEZ OBNJD&RVJMJCSJVNCFUXFFO3FEPY$PNQP OFOUT 4HE RESULTS DESCRIBED ABOVE DEMONSTRATE A DIRECT INTERACTION BETWEEN DIFFERENT COMPONENTS OF THE PHOTOSYNTHETIC AND RESPIRATORY CHAINS 3INCE THESE CHAINS ARE LOCALIZED IN DIFFERENT PARTS OF THE INTERNAL MEMBRANE THIS IMPLIES DIFFUSION OF SOME OF ELECTRON CARRIERS LIKE THE QUINONE IN THE LIPID PHASE AND THE #YT C IN THE PERIPLASMIC SPACE 3EVERAL OBSERVATIONS INDICATE HOWEVER THAT THE DIFFUSION OF THESE ELEMENTS DOES NOT OCCUR ON THE ENTIRE INTERNAL MEMBRANE AND THAT THE DIFFERENT BIOENERGETIC CHAINS ARE NOT NECES SARILY IN THERMODYNAMIC EQUILIBRIUM 4WO DISTINCT FUNCTIONAL POOLS OF #YT C HAVE BEEN DEMONSTRATED IN
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&IG ,IGHT STIMULATION OF OXYGEN UPTAKE BY THE QUINOL OXIDASE IN THE PRESENCE OF M- 4-0$ FOR A BACTERIA SUSPENSION OF 2B CAPSULATUS DELETED IN THE #YT OXIDASE 4HE UPWARDS ARROWS INDICATE THE SWITCHING ON OF ILLUMINATION WHILE THE DOWNWARDS ARROWS COR R RESPOND TO ITS CESSATION !FTER ILLUMINATION THE RESPIRATORY ACTIVITY IS STRONGLY INHIBITED 2EACTIVATION OF RESPIRATION ONLY OCCURS WHEN ALL THE PHOTOOXIDIZED 4-0$ HAS BEEN REREDUCED VIA THE #YT BC COMPLEX &IGURE ADAPTED FROM 2ICHAUD ET AL
2HODOBACTER SPHAEROIDES AND 2HODOBACTER CAPSULA TUS 4HIS HAS CLEARLY BEEN SHOWN FOR CELLS GROWN IN THE PRESENCE OF NITRATE OR UNDER DARK AEROBIC CONDITION WHICH PRESENTS TWO KINETICALLY DISTINCT PHASES A FAST AND A SLOW PHASE FOR THE PHOTOOXIDATION OF #YT C BY CONTINUOUS ILLUMINATION AT HIGH P( OR IN THE PRESENCE OF GLYCEROL -ATSUURA ET AL 6ERMÏGLIO ET AL #ELLS GROWN UNDER ANAEROBIC PHOTOSYNTHETIC CONDITION EXHIBIT ONLY THE FAST PHOTOOXIDATION PHASE 4HIS CLEARLY ESTABLISHES THE OCCURRENCE OF TWO DISTINCT FUNCTIONAL POOLS OF #YT C 4HE lRST POOL OF #YT C WHICH IS RAPIDLY PHOTOOXIDIZED DURING THE FAST PHASE OR BY THE TWO lRST mASHES OF A SERIES CORRESPONDS TO #YT C CONNECTED TO 2#S AND #YT C 4HE SECOND POOL OF #YT C IS ONLY SLOWLY PHOTOOXIDIZED FOLLOWING CONTINUOUS ILLUMINATION AND REQUIRES A LARGE NUMBER OF EXCITATIONS WHEN CELLS ARE SUBJECTED TO A SERIES OF mASHES 4HIS IS LINKED TO A SMALL AMOUNT OF 2#S CON NECTED TO THIS POOL OF #YT C THE RATIO COULD BE AS HIGH AS #YT C PER 2# IN THE CASE OF DARK SEMIAEROBIC GROWN CELLS 'ROWTH OF BACTERIA UNDER DIFFERENT LIGHT
INTENSITIES AND DARK AEROBIC CONDITIONS SHOWS THAT THE RATIO BETWEEN THE FAST AND THE SLOW PHASES CORRELATES WITH THE AMOUNT OF )#- 6ERMÏGLIO ET AL -OREOVER THE SECOND POOL OF #YT C IS SPECIlCALLY OXIDIZED BY THE RESPIRATORY OR DENITRIlCATION ACTIVITIES 3ABATY ET AL 6ERMÏGLIO ET AL )N SUMMARY TWO POOLS OF #YT C ARE NOT IN THERMO DYNAMIC EQUILIBRIUM 4HE lRST POOL IS INVOLVED IN AN EFlCIENT CYCLIC PHOTOINDUCED ELECTRON TRANSFER 4HIS #YT C IS INTIMATELY CONNECTED TO 2#S AND #YT BC COMPLEX IN THE INTRACYTOPLASMIC PART OF THE MEMBRANE AND IT IS HARDLY OXIDIZED BY RESPIRATORY ACTIVITIES 4HE SECOND POOL INTERACTS WITH A SMALL NUMBER OF 2#S PRESENT IN THE CYTOPLASMIC PART OF THE MEMBRANE AND WITH THE ELEMENTS OF THE RESPIRATORY CHAINS LOCALIZED IN THIS MEMBRANE REGION 4HIS ALLOWS A DIRECT INTERAC TION BETWEEN THE TWO BIOENERGETIC CHAINS ,ACK OF THERMODYNAMIC EQUILIBRIUM HAS ALSO BEEN OBSERVED BETWEEN THE DIFFERENT PHOTOSYNTHETIC CHAINS IN THE CASE OF 2HODOBACTER SPHAEROIDES )N THIS SPECIES THE APPARENT EQUILIBRIUM CONSTANT BETWEEN #YT C #YT BC AND THE 2# MEASURED UNDER LOW IN TENSITY ILLUMINATION IS MUCH LESS THAN EXPECTED FROM THE MID POINT POTENTIALS OF THESE ELECTRON CARRIERS *OLIOT ET AL /NE POSSIBLE EXPLANATION OF THIS BEHAVIOR IS TO SUPPOSE THAT A RAPID THERMODYNAMIC EQUILIBRIUM IS ACHIEVED AT A LOCAL LEVEL WITHIN A DO MAIN OR SUPERCOMPLEX CONTAINING A SMALL NUMBER OF ELECTRON CARRIERS WHEREAS EQUILIBRATION AT A MACRO LEVEL BETWEEN THESE DOMAINS IS A MUCH SLOWER PROCESS ,AVERGNE ET AL )N OTHER WORDS FAST ELECTRON TRANSFER ONLY OCCURS IN THE SUPERCOMPLEX I E THE DIFFUSION OF #YT C IS CONlNED TO A SMALL DOMAIN INCLUDED ONLY 2#S AND #YT BC 3IMULATION OF THE DATA SUGGESTS THAT EACH SUPERCOMPLEX CONTAINS 2#S #YT C AND #YT BC COMPLEX *OLIOT ET AL 6ERMÏGLIO AND *OLIOT 4HE STOICHIOMETRY DETERMINED FOR THESE SMALL DOMAINS IS FULLY CONSISTENT WITH THE ONE MEASURED FOR THE FAST PHOTOOXIDATION PHASE OF #YT C ! SECOND EXPLANATION OF THE LOW EQUILIBRIUM CONSTANT HAS BEEN PROPOSED BY #ROFTS AND COWORK ERS #ROFTS ET AL )N THEIR MODEL THEY ASSUME FREE DIFFUSION OF THE #YT C IN THE AQUEOUS PHASE BUT HETEROGENEITY IN THE STOICHIOMETRY OF THIS ELECTRON TRANSFER COMPONENT AND THE MEMBRANE COMPLEXES ! FURTHER ARGUMENT WAS TAKEN FROM THE OBSERVATION THAT IN MUTANT STRAINS IN WHICH THE STOICHIOMETRIC RATIOS OF THE COMPONENTS OF THE SUPERCOMPLEXES HAVE BEEN CHANGED THE COMPONENTS OF THE PHOTOSYNTHETIC CHAINS PRESENT THE BEHAVIOR EXPECTED FOR THE DIF F
$IBQUFS *OUFSBDUJPO#FUXFFO1IPUPTZOUIFTJTBOE3FTQJSBUJPO FUSIONAL MODEL #ROFTS ET AL 7ITTHULN ET AL -OREOVER THE ADDITION OF A SUBSATURATING CONCENTRATION OF MYXOTHIAZOL A SPECIlC INHIBITOR OF THE #YT BC COMPLEX DECREASES THE RATE OF ELECTRON TRANSFER OF RE REDUCTION OF #YT C BUT NOT ITS AMOUNT IN ISOLATED CHROMATOPHORES &ERNËNDEZ 6ELASCO AND #ROFTS (OWEVER THIS BEHAVIOR IS NOT OBSERVED IN INTACT CELLS WHERE INHIBITION OF PART OF THE #YT BC COMPLEX DOES NOT AFFECT THE RATE OF ELECTRON TRANSFER FOR THE UNINHIBITED COMPLEXES &ERNËNDEZ 6ELASCO AND #ROFTS *OLIOT ET AL 4HIS IMPLIES A RESTRICTED DIFFUSION OF #YT C AT LEAST IN INTACT CELLS BUT ITS MOLECULAR EXPLANATION IS STILL A MATTER FOR DEBATE #ROFTS A B 6ERMÏGLIO AND *OLIOT ! STRONG ARGUMENT IN FAVOR OF A SUPRAMOLECULAR ORGANIZATION OF THE PHOTOSYNTHETIC CHAINS IN 2HO DOBACTER CAPSULATUS CAME FROM THE DISCOVERY OF A MEMBRANE BOUND CYTOCHROME NAMED #YT CY *ENNEY ET AL *ENNEY AND $ALDAL )N THIS SPECIES IN ADDITION TO #YT C THE #YT CY IS COMPETENT FOR THE PHOTOINDUCED ELECTRON TRANSFER -YLLYKALLIO ET AL 4HE MEMBRANE ATTACHMENT OF THIS CYTOCHROME CERTAINLY LIMITS ITS RAPID DIFFUSION AND THEREFORE IM PEDES POSSIBLE INTERACTIONS WITH A LARGE NUMBER OF DIFFERENT REDOX PARTNERS #ONSEQUENTLY ONE EXPECTS THAT THERMODYNAMIC EQUILIBRIUM BETWEEN THE 2#S INTERACTING WITH EITHER A #YT CY OR WITH A #YT C WILL BE SLOW OR EVEN INOPERATIVE 4HIS LACK OF EQUILIBRIUM HAS BEEN EXPERIMENTALLY DEMONSTRATED USING A MUTANT STRAIN LACKING #YT C )N THIS MUTANT ONLY ^ OF THE 2#S HAD THEIR PHOTOOXIDIZED PRIMARY DONOR RAPIDLY RE REDUCED BY #YT CY !LTHOUGH THIS CYTOCHROME IS RE REDUCED BY THE #YT BC COMPLEX IN THE MILLISECOND TIME RANGE IT REMAINED UNABLE TO CONVEY ELECTRONS TO THE LARGE AMOUNT OF UNCONNECTED 2#S WHICH REMAINED PHOTOOXIDIZED FOR SEVERAL SECONDS ALTHOUGH THE TWO TYPES OF 2#S ARE LOCALIZED IN THE SAME MEMBRANE REGION 6ERMÏGLIO ET AL -YLLYKALLIO ET AL 4HESE EXPERIMENTS NOT ONLY DEMONSTRATE THAT THE THERMODYNAMIC EQUILIBRIUM IS NOT REACHED BETWEEN THE DIFFERENT PHOTOSYNTHETIC ELECTRON TRANS PORT CHAINS BUT ALSO THAT 2#S #YT CY AND THE #YT BC COMPLEX ARE IN VERY CLOSE ASSOCIATION ! CY TYPE CYTOCHROME HAS ALSO BEEN EVIDENCED IN 2HODOBACTER SPHAEROIDES -YLLYKALLIO ET AL #ONTRARY TO THE #YT CY OF 2HODOBACTER CAPSULATUS THE 2HODOBACTER SPHAEROIDES #YT CY IS NOT FUNCTIONAL IN THE PHOTOINDUCED ELECTRON TRANSFER -YLLYKALLIO ET AL 4HIS HAS BEEN CLEARLY DEMONSTRATED BY THE NON PHOTOSYNTHETIC GROWTH OF A MUTANT DELETED IN #YT C $ONOHUE ET AL (OWEVER GENETIC
INTRODUCTION OF #YT CY FROM 2HODOBACTER CAPSULATUS IN A #YT C MINUS MUTANT OF 2HODOBACTER SPHAEROIDES READILY RESTORES THE PHOTOSYNTHETIC ACTIVITY *ENNEY AND $ALDAL *ENNEY ET AL 4HE DIFFERENT BEHAVIOR BETWEEN THE #YT CY OF 2HODOBACTER CAPSU LATUS AND OF 2HODOBACTER SPHAEROIDES IS PROBABLY LINKED TO VARIATIONS IN THE PRECISE LOCATION OF CHARGED RESIDUES IMPORTANT FOR THE INTERACTIONS OF THE HEME SUBDOMAIN WITH ITS REDOX PARTNERS BUT ALSO IT MIGHT DEPEND ON THE DIFFERENT LENGTH OF THEIR MEMBRANE ANCHORS -YLLYKALLIO ET AL #YTOCHROME CY IS ALSO INVOLVED IN THE RESPIRATORY ACTIVITY OF BOTH 2HODOBACTER SPHAEROIDES AND 2HODOBACTER CAPSU LATUS )NDEED THIS CYTOCHROME MEDIATES IN ADDITION TO THE SOLUBLE #YT C ELECTRON TRANSFER BETWEEN THE #YT BC COMPLEX AND THE CBB TYPE #YT OXIDASE IN 2HODOBACTER CAPSULATUS *ENNEY AND $ALDAL 'RAY ET AL AND BETWEEN THIS FORMER COMPLEX AND THE TWO TYPES OF CYTOCHROME OXIDASES CBB AND AA PRESENT IN 2HODOBACTER SPHAEROIDES $ALDAL ET AL #ONSEQUENTLY THE RESPIRATORY CHAINS EMBRACING A #YT CY COULD BE ORGANIZED IN SUPRAMO LECULAR COMPLEXES AND OPERATE INDEPENDENTLY OF THE REDOX STATE OF ELECTRON TRANSFER COMPONENTS OF THE PHOTOSYNTHETIC CHAINS OR OTHER RESPIRATORY PATHWAYS 3UPRAMOLECULAR ORGANIZATION OF PART OF THE RESPIRATORY CHAINS HAVE BEEN EVIDENCED IN DIFFERENT BACTERIAL SPE CIES MAINLY !RCHAEA SEE #HAPTER BY ' 3CHÊFER 6OL )) AND MORE RECENTLY IN MITOCHONDRIA 3CHÊGGER 3CHÊGGER AND 0FEIFFER 7(FOFUJD3FHVMBUJPO 4HE PHYSIOLOGICAL RELATIONSHIPS BETWEEN PHOTOSYN THESIS AND RESPIRATION AS DESCRIBED IN THE PRECEDING PARAGRAPHS ARE SUPERIMPOSED ON THE REGULATION AT THE GENETIC LEVEL /XYGEN IS THE KEY ELEMENT IN THE COORDINATED REGULATION OF PHOTOSYNTHETIC AND RESPIRATORY ACTIVITIES SINCE IT DIRECTLY OR INDIRECTLY PARTICIPATES IN DETERMINING THE LEVELS OF EXPRESSION OF ALL COMPONENTS INVOLVED 4HERE ARE lVE REGULATORY ELEMENTS IDENTIlED UP TO NOW IN 2B CAPSULATUS THAT ARE INVOLVED IN MEDIATING THE RESPONSE TO VARYING OXYGEN LEVELS NAMELY 2EG! (VR! #TR* !ER2 AND &NR, 4HIS PARAGRAPH WILL BE FOCUSED ON THE REGULA TORY ASPECTS WHICH ARE COMMON TO PHOTOSYNTHESIS AND RESPIRATION AND WILL TOUCH ONLY MARGINALLY THE ONES THAT ARE SPECIlC TO PHOTOSYNTHESIS FOR DETAILS ON THIS LATTER ASPECT SEE 0EMBERTON ET AL 'REGOR AND +LUG
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"3FHVMBUPSZ&MFNFOUT3FH" )WS" $US+ "FS3 BOE'OS2EG! 0RR! IN 2B SPHAEROIDES IS THE lRST ELEMENT IDENTIlED THAT ACTS AS AN ACTIVATOR OF REACTION CENTERS 2#S GENE EXPRESSION PUF F AND PUH IN RESPONSE TO LOW OXYGEN 3GANGA AND "AUER 4HIS PROTEIN IS THE EFFECTOR COMPONENT OF A REGULATORY COUPLE IN WHICH 2EG" 0RR" IN 2B SPHAEROIDES IS THE SENSOR PARTNER -OSLEY ET AL 4HE SENSOR EFFECTOR COUPLE IS FUNCTIONING ACCORDING TO THE CLASSICAL MODEL IN WHICH THE SENSOR 2EG" DETECTS CHANGES IN THE CELLULAR ENVIRONMENT IE VARIATIONS IN OXYGEN TENSION AND PHOSPHORYLATES THE EFFECTOR 2EG! THE LATTER IN ITS PHOSPHORYLATED FORM REGULATES THE EXPRESSION OF A NUMBER OF GENES 4HE GENES INITIALLY IDENTIlED AS RESPONDING TO 2EG! UNDER PHOTOSYNTHETIC ANAEROBIC CONDITIONS WERE ALL COMPONENTS OF THE PHOTOSYNTHETIC MACHINERY PUFF AND PUH OPERONS CODING FOR THE 2#S AND ,() SUBUNITS AND PUC OPERON REPRESENTING ,()) SUBUNITS 3GANGA AND "AUER 2ECENTLY IT HAS BEEN SHOWN THAT SEVERAL OTHER GENES CODING EITHER FOR ELEMENTS SHARED BY PHOTOSYNTHETIC AND RESPIRA TORY ELECTRONS TRANSPORT CHAINS %4# OR CODING FOR COMPONENTS UNIQUE TO THE RESPIRATORY APPARATUS ARE REGULATED BY 2EG! 3WEM ET AL 3WEM AND "AUER 4O THE lRST GROUP OF THESE GENES BELONGS THE PET!"# OPERON # BC COMPLEX WHICH EXPRESSION IS INCREASED BY 2EG! UNDER ANAEROBIC AND SEMIAEROBIC CONDITIONS AND SHOWS ONLY A SLIGHT INCREASE UNDER HIGH OXYGEN TENSION 4HE OTHER TWO ELEMENTS SHARED BY THE REDOX PATHWAYS IN 2B CAPSULATUS ARE #YT C CYT! AND #YT CY CYT9 #YT 9 C HAS A REGULATION PROlLE SIMILAR TO BC WITH 2EG! THAT HAS A GREATER IMPACT WITH LOW OR NO OXYGEN AND A LITTLE EFFECT WITH HIGH / #YT CY ON THE CONTRARY IS POORLY AFFECTED BY 2EG! UNDER ANAEROBIC CONDITIONS AND SIGNIlCANTLY INDUCED ONLY UNDER SEMIAEROBIC CONDITIONS 4O THE SECOND GROUP OF 2EG! REGULATED ELEMENTS BELONG THE TWO TERMINAL OXIDASES OF THE RESPIRATORY %4# 4HE CBB #YT C OXIDASE CCO./10 IS INCREASED UNDER AEROBIC AND SEMIAEROBIC CONDITIONS BUT IS LIMITED IN THE ABSENCE OF OXYGEN 4HE QUINOL OXIDASE CYD!" ON THE CONTRARY IS INDUCED BY 2EG! UNDER ALL GROWTH CONDITIONS 3WEM ET AL )N ADDITION TO ALL THE ABOVE DESCRIBED EFFECTS 2EG! EXERTS ITS ACTION IN RESPONSE TO CHANGES IN OXYGEN TENSION ON OTHER BASIC METABOLIC ACTIVITIES SUCH AS $-3/ REDUCTION +APPLER ET AL #/ ASSIMILATION 6ICHIVANIVES ET AL ( OXIDATION AND . lXATION %LSEN ET AL
4HE TRANSDUCING EVENTS THAT CONNECT THE SENSING OF CHANGES IN OXYGEN LEVELS TO THE PHOSPHORYLATIONDE PHOSPHORYLATION OF THE REGULATORY COUPLE 2EG"2EG! HAVE NOT BEEN YET IDENTIlED IN 2B CAPSULATUS 4HERE ARE HOWEVER A FEW CONSIDERATIONS THAT CAN BE MADE INDICATING THAT THE CBB #/8 COULD BE THE ELEMENT IN THE TRANSDUCING CHAIN CAPABLE OF SENSING THE LEVEL OF / VIA THE ELECTRON mOW GOING THROUGH IT &IRST IT IS LIKELY TO PRESUME THAT A REDOX COMPLEX WHICH IS BOTH EXPRESSED UNDER ALL CONDITIONS AND PLAYS A FUNDAMENTAL PART OF THE %4# MIGHT FUNCTION AS OXY GEN SENSOR 3ECOND #YT C OXIDASE DElCIENT MUTANTS SHOW AN INCREASED EXPRESSION OF 2#S PUF AND F PUH GENES UNDER ANAEROBIC AND SEMIAEROBIC CONDITIONS AS IF THE LACK OF A FUNCTIONAL CBB OXIDASE COULD BE SENSED AS A SIGNAL OF ANAEROBIOSIS EVEN UNDER AEROBIC CONDITIONS "UGGY AND "AUER 4HIRD IN 2B SPHAEROIDES A CLOSE RELATIVE OF 2B CAPSULATUS THE ROLE OF CBB IN SENSING THE OXYGEN AVAILABILITY AND TRANSDUCING THE SIGNAL TO THE GENES REGULATED BY THE 0RR"0RR! COUPLE 2EG"2EG! IN 2B CAPSULATUS HAS CLEARLY BEEN DEMONSTRATED /'ARA ET AL )N 2B SPHAEROIDES IT HAS BEEN SHOWN THAT THE REGU LATORY RELAY FROM / SENSING TO 03 GENE EXPRESSION INCLUDES 0RR# A PROTEIN ANCHORED IN THE MEMBRANE THAT TRANSMITS THE SIGNAL ORIGINATED FROM CBB TO 0RR" %RASO AND +APLAN SEE ALSO #HAPTER BY /H AND +APLAN 6OL ) 3EN# WHICH IS THE EQUIVALENT TO 0RR# IN 2B CAPSULATUS APPEARS TO HAVE A DIFFERENT AND LESS CLEAR ROLE IN THIS REGULATORY CASCADE "UGGY AND "AUER 2EG! IS THE ELEMENT THAT DIRECTLY INTERACTS WITH PHO TOSYNTHETIC AND RESPIRATORY GENES THAT ARE CONTROLLED BY THE ABOVE DESCRIBED REGULATORY CHAIN !CCORDING TO THIS SCHEME 2EG! NEEDS TO BE PHOSPHORYLATED IN THE PRESENCE OF LOW TO NO OXYGEN IN ORDER TO FUNCTION AS AN ACTIVATOR OF GENE EXPRESSION !S ILLUSTRATED IN &IG 2EG! CAN ALSO BEHAVE AS A REPRESSOR OF CBB OXIDASE CCO./10 TRANSCRIPTION UNDER ANAEROBIC CONDITIONS 3WEM ET AL 4HE ABILITY OF PHOSPHORYLATED 2EG! TO EXERT AN INHIBITORY ROLE ON TRANSCRIPTION HAS BEEN DESCRIBED FOR THE SEN# REG! HVR! OPERON $U ET AL SHOWING THAT 2EG! AUTO REGULATES ITS OWN EXPRESSION )N ADDITION TO ITS ACTIVATOR ANDOR INHIBI TORY ROLE UNDER ANAEROBIC OR SEMIAEROBIC CONDITIONS THAT IS IN THE PHOSPHORYLATED FORM 2EG! PARTICIPATES IN THE AEROBIC REGULATION OF THE TERMINAL OXIDASES COO./10 0 AND CYD!" 3WEM AND "AUER 5NDER THESE GROWTH CONDITIONS 2EG! IS SUPPOSED TO BE PRESENT IN THE NON PHOSPHORYLATED FORM WHICH HAS BEEN SHOWN TO HAVE A TIMES LOWER AFlNITY FOR ITS
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&IG 2EGULATORY CONNECTIONS BETWEEN RESPIRATORY AND PHOTOSYNTHETIC ELECTRON TRANSPORT CHAINS UNDER DIFFERENT GROWTH CONDITIONS ! AEROBIC " SEMIAEROBIC # ANAEROBIC 2EDOX COMPONENTS OF THE %4#S ARE GRAY 3QUARES INDICATE ELEMENTS SHARED BY BOTH %4#S OR SPECIlC TO PHOTOSYNTHESIS CIRCLES INDICATE ELEMENTS SPECIlC TO RESPIRATION $ASHED GRAY ARROWS INDICATE ELECTRON TRANSFER PATHWAYS BETWEEN REDOX COMPONENTS #ONTINUOUS BLACK ARROWS INDICATE GENETIC INDUCTION DOTTED BLACK LINES INDICATE GENETIC REPRESSION 4HE THICKNESS OF THE ARROWS INDICATE THE RELATIVE STRENGTH OF REGULATION 3TANDARD ABBREVIATIONS WERE USED EXCEPT FOR $( WHICH STANDS FOR .!$( DEHYDROGENASE 3EE TEXT FOR FURTHER DETAILS
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TARGET PROMOTERS THAN THE PHOSPHORYLATED COUNTERPART "IRD ET AL (VR! IS KNOWN TO BE PART OF A CLUSTER OF GENES INCLUDING 2EG! 2EG" AND 3EN# INVOLVED IN CONTROL LING THE EXPRESSION OF THE PHOTOSYNTHETIC APPARATUS OF 2B CAPSULATUS "UGGY ET AL (VR! HAS BEEN SHOWN TO INCREASE THE EXPRESSION OF 2# GENES UNDER ANAEROBIC CONDITIONS WHEN LIGHT INTENSITY IS LOWERED "UGGY ET AL )N ADDITION (VR! IS INVOLVED IN REGULATING . lXATION +ERN ET AL ! RECENT REPORT HAS DEMONSTRATED THAT (VR! PARTICIPATES IN THE ANAEROBIC REGULATION OF BOTH TERMINAL OXIDASES STIMULATES THE EXPRESSION OF 1/8 IN THE ABSENCE OF OXYGEN AND LIMITS THE EXPRESSION OF #/8 UNDER ANAEROBIC AND SEMIAEROBIC CONDITIONS 3WEM AND "AUER )T HAS BEEN HYPOTHESIZED THAT (VR! OR AN UPSTREAM REGULATORY ELEMENT MAY BIND A CHROMOPHORE AND TRANSDUCE ITS LIGHT ABSORBING PROPERTIES INTO REGULA TION OF PUF F AND PUH OPERONS "UGGY ET AL (OWEVER IN THE LIGHT OF RECENT DATA ABOUT THE EFFECT OF (VR! ON THE EXPRESSION OF THE TERMINAL OXIDASES OF THE %4# 3WEM AND "AUER WE ARE TEMPTED TO SUGGEST THAT THIS REGULATORY ELEMENT PARTICIPATES IN THE COORDINATED REGULATION OF PHOTOSYNTHETIC AND RESPIRATORY FUNCTIONS BY SENSING THE REDUCTION LEVEL OF THE 1 POOL )N THIS RESPECT THE !RC SYSTEM IN % COLI HAS BEEN SHOWN TO REGULATE A NUMBER OF GENE INVOLVED IN RESPIRATORY OR FERMENTATIVE METABOLISM BY DIRECTLY SENSING THE OXIDATION REDUCTION EQUILIBRIUM OF THE 1 POOL 'EORGELLIS ET AL )N 2B CAPSULATUS THE REDOX STATE OF THE 1 POOL WHICH IS LIKELY TO DEPEND ON THE LIGHT INTENSITY AS IN THE CLOSE RELATIVE 2B SPHAER R OIDES /H AND +APLAN AND ON THE RESPIRATORY ELECTRON TRANSPORT RATE :ANNONI AND -OORE COULD BE SENSED BY A RELAY REGULATORY SYSTEM WHICH INCLUDES (VR! THAT WOULD USE THIS SIGNAL TO OPTIMIZE THE EXPRESSION OF 2# AND TERMINAL OXIDASES 4WO REGULATORY ELEMENTS #RT* AND !ER2 ACT AS AEROBIC REPRESSORS OF GENES INVOLVED IN PHOTO SYNTHETIC PROCESSES AND GENES SPECIlC FOR AEROBIC RESPIRATION 4HESE REPRESSORS DIFFER IN THE WAY THEY ARE CONTROLLED BY OXYGEN #RT* RESPONDS DIRECTLY TO / BY THE FORMATION OF AN INTRAMOLECULAR DISULlDE BOND THAT ALTERS ITS BINDING TO TARGET PROMOTERS -ASUDA ET AL BUT ITS TRANSCRIPTION IS NOT REGULATED BY AEROBIOSIS OR ANAEROBIOSIS $ONG ET AL /N THE OTHER HAND !ER2 IS MORE EXPRESSED UNDER ANAEROBIC PHOTOSYNTHETIC CONDITIONS AND THIS TRANSCRIPTIONAL REGULATION DOES NOT DEPEND ON 2EG! #RT* OR !ER2 ITSELF SUGGESTING THAT THERE MIGHT EXIST AN ADDITIONAL
UNIDENTIlED OXYGEN RESPONDING REGULATOR $ONG ET AL 4HE TWO REPRESSORS ACT ON DIFFERENT SETS OF GENES WITH A SUBCLASS CONTROLLED BY BOTH REGULATORY ELEMENTS AS IS THE CASE OF PUC F OPERON ,()) ON WHICH THEY SHOW COOPERATIVITY $ONG ET AL &.2 IS A GLOBAL REGULATOR THAT RESPONDS TO SHIFTS 2 FROM AEROBIC TO ANAEROBIC CONDITIONS INDUCING OR REPRESSING ANAEROBICALLY DIFFERENT SETS OF GENES IN VOLVED IN THE TRICARBOXYLIC ACID CYCLE IN ANAEROBIC RESPIRATORY PATHWAYS AND IN AEROBIC RESPIRATION OF % COLI "AUER ET AL 4HE % COLI &.2 HOMO LOGUE IN 2B CAPSULATUS &NR, BEHAVES SIMILARLY IN THAT IT ACTS UNDER ANAEROBIC CONDITIONS AS AN INDUCER OF THE CBB OXIDASE AND AS A REPRESSOR OF THE QUINOL OXIDASE 3WEM AND "AUER &NR, IS REQUIRED FOR ANAEROBIC DARK GROWTH IN THE PRESENCE OF $-3/ BUT IT DOES NOT SEEM TO BE INVOLVED IN REGULATION OF PHOTOSYNTHETIC ACTIVITY IN 2B CAPSULATUS :EILSTRA 2YALLS ET AL #5IF$PPSEJOBUFE3FHVMBUJPO'SBNFXPSL" 1SPQPTBM ! SYNTHETIC DESCRIPTION OF THE REGULATORY SYSTEM USED BY 2B CAPSULATUS TO COORDINATE THE EXPRESSION OF PHOTOSYNTHETIC AND RESPIRATORY %4#S WHILE ADAPTING TO GROWTH MODE CHANGES IS HERE PROPOSED ACCORDING TO THE PRESENT KNOWLEDGE ALBEIT LARGELY INCOMPLETE OF THE GENETIC AND FUNCTIONAL NETWORKS THAT CON NECT PHOTOSYNTHESIS AND RESPIRATION 4HE REGULATORY SCHEME WE PROPOSE IS BY NO MEANS EXHAUSTIVE AND IS JUST INTENDED AS A PRELIMINARY PROPOSAL 4HE LEVEL OF OXYGEN AND THE REDOX STATE OF THE QUINONE POOL REPRESENT THE ENVIRONMENTAL AND THE PHYSIOLOGICAL INDICATORS THAT ARE SENSED BY THE CELL IN ORDER TO REGULATE THE RELATIVE EXPRESSION OF THE TWO %4#S COMPONENTS !S REPORTED IN THE PRECEDING PARAGRAPH THE DATA GATHERED ON THE GENETIC REGULA TION OF THE %4#S ELEMENTS EITHER SHARED BY THE TWO !40 GENERATING SYSTEMS OR SPECIlC TO THE RESPIRATORY CHAIN CONCERN MAINLY THE TERMINAL OXIDASES #/8 AND 1/8 AND ONLY MARGINALLY THE BC COMPLEX #YT C AND #YT CY 3WEM ET AL 3WEM AND "AUER )N OUR PROPOSAL THE ENVIRONMENTAL LEVEL OF OXYGEN IS SENSED BY THE CBB #YT C OXIDASE POSSIBLY VIA THE ELECTRON mOW GOING THOUGH IT AND THE SIGNAL IS RELAYED TO THE 2EG!2EG" COUPLE BY 3EN# 2EG! RESPONDS TO THIS SIGNAL BY REGULATING THE EXPRESSION OF THE %4#S ELEMENTS SHARED BY PHOTOSYNTHESIS AND RESPIRATION AND THE TERMINAL OXIDASES )N THIS SCHEME #/8 WOULD ESTABLISH A REGULATORY LOOP WITH 2EG!
$IBQUFS *OUFSBDUJPO#FUXFFO1IPUPTZOUIFTJTBOE3FTQJSBUJPO IN WHICH THE lRST COMPONENT AFFECTS THE ACTIVITY OF THE SECOND AS A FUNCTION OF THE OXYGEN LEVEL AND THE SECOND COMPONENT PARTICIPATES IN MODULATING THE EXPRESSION OF THE lRST IN A WAY THAT DEPENDS ON THE GROWTH CONDITIONS 5LTIMATELY IS THE LEVEL OF / THAT SETS THE EQUILIBRIUM POSITION OF THE LOOP 4HE OTHER REGULATORY ELEMENTS #RT* !ER2 (VR! AND &NR, RESPOND TO AEROBIOSISANAEROBIOSIS TRANSITIONS IN DIFFERENT WAYS 4HE QUINONE POOL PLAYS A FUNDAMENTAL ROLE IN THAT IT CONNECTS ALL ELECTRON TRANSPORT CHAINS &IG AND ITS CORRECT FUNCTIONING WHICH DEPENDS ON MAINTAINING AN OPTIMAL REDOX POISE IS OF PARAMOUNT IMPORTANCE UNDER ALL GROWTH MODES )N THE REGULATORY SCHEME WE PROPOSE IN ORDER TO MAINTAIN A CORRECT INTERAC TION BETWEEN PHOTOSYNTHESIS AND RESPIRATION (VR! WOULD HAVE TO RESPOND TO THE REDOX STATUS OF THE 1 POOL UNDER LOW TO NO OXYGEN AND TRANSLATE IT INTO THE REGULATION OF THE EXPRESSION OF THE TWO TERMINAL OXIDASES 4HE REDOX LEVEL OF THE 1 POOL IS DIRECTLY MODULATED BY THE OXIDATIVE ACTION OF 1/8 WHICH IN TURN IS CONTROLLED BY THE FORMER THROUGH (VR! 4HE INTERPLAY BETWEEN (VR! AND 1/8 MEDIATED BY THE REDOX LEVEL OF THE QUINONE POOL DElNES ANOTHER REGULATORY LOOP IN WHICH EACH COMPONENT REGULATES AND IS REGULATED BY THE OTHER 4HE @OXYGEN LOOP AND THE @1 POOL LOOP ARE CONSIDERED TO BE INTEGRATED BY THE 2EG!2EG" COUPLE WHICH RESPONDS TO BOTH SIGNALS "UGGY AND "AUER $U ET AL AND AT THE SAME TIME EXERTS ITS REGULATORY ACTION ON ALL %4#S COMPONENTS INVOLVED &IG 5NDER AEROBIC GROWTH CONDITIONS THE PRESENCE OF ABUNDANT OXYGEN IS SENSED BY THE CBB CYT OXIDASE AND THIS SIGNAL IS RELAYED TO THE 2EG!2EG" COUPLE 5NDER THESE CONDITIONS 2EG! IN ITS DEPHOSPHORYLATED FORM REGULATES POSITIVELY THE EXPRESSION OF SEVERAL COMPONENTS OF THE %4# HAVING A STRONG EFFECT ON THE EXPRESSION OF THE TERMINAL OXIDASES #/8 AND 1/8 AND A MILDER EFFECT ON BOTH #YT C AND BC COMPLEX 3WEM ET AL SEE &IG ! 4HE AEROBIC REPRES SORS #RT* AND !ER2 COUNTERACT THE ACTION OF 2EG! BY LIMITING THE EXPRESSION OF 1/8 3WEM AND "AUER !ER2 WORKS ALSO AS A REPRESSOR OF 2#S GENES EXPRESSION $ONG ET AL "ASED ON THE RELATIVE CONTRIBUTION OF 2EG! ON THE EXPRESSION OF THE OP ERONS INVOLVED IT IS LIKELY TO CONCLUDE THAT THE LEVEL OF THE CBB #YT OXIDASE COO./10 MAINLY DEPENDS ON THE PRESENCE OF 2EG! 3WEM ET AL 3WEM AND "AUER 4HE EXPRESSION OF 1/8 WOULD ALSO DEPEND MOSTLY ON THE ACTION OF 2EG! 3WEM ET AL BUT IN ADDITION ON THAT OF #RT* AND !ER2
THROUGH THE USE OF DIFFERENT MECHANISMS $ONG ET AL -ASUDA ET AL 3WEM AND "AUER 4HE INTERPLAY OF THESE REGULATORY EFFECTS GENERATES A lNE TUNING OF THE 1/8 EXPRESSION AS A FUNCTION OF THE CELL REDOX STATE !S THE OXYGEN CONCENTRATION DECREASES THE CELL REACTS ADJUSTING THE LEVEL OF THE %4#S COMPONENTS TO THE NEW GROWTH CONDITIONS 5NDER SEMIAEROBIOSIS MOST OF THE REDOX ELEMENTS EXCEPT THE BC COMPLEX REACH THEIR MAXIMUM LEVEL OF EXPRESSION THROUGH THE POSITIVE ACTION OF 2EG! &IG " 3WEM ET AL 7ITH LOW TO NO OXYGEN THE CBB OXIDASE SHOWS A COMPLEX REGULATION PATTERN THAT INCLUDES (VR! AND &NR, IN ADDITION TO 2EG! &IG " # 3WEM AND "AUER 3INCE THE EFFECT OF 2EG! ON THE EXPRESSION OF CBB IS LESS PRONOUNCED UNDER ANAEROBIC THAN AEROBIC CONDITIONS IT MIGHT BE SUG GESTED THAT AS OXYGEN DECREASES THE PROPORTION OF PHOSPHORYLATED 2EG! INCREASES AND IN THIS FORM THE REGULATORY ELEMENT WOULD HAVE LESS AFlNITY FOR THE CBB PROMOTER THEREBY LOWERING ITS POSITIVE ACTION 4HE HIGH LEVEL OF CBB EXPRESSION UNDER SEMIAEROBIC CONDITIONS ALSO DEPENDS ON &NR, WHICH HAS A REGU LATORY ROLE COMPARABLE TO 2EG! 3WEM AND "AUER !LTHOUGH THESE REGULATORS SHOW A MILD EFFECT WHEN LACKING SINGULARLY A DRAMATIC DECREASE OF CBB EXPRESSION IS SEEN WHEN BOTH ARE DELETED 3WEM AND "AUER 4HE THIRD ELEMENT PARTICIPATING IN THE REGULATION OF THE #YT C OXIDASE IS (VR! WHICH HAS A NEGATIVE FUNCTION 3WEM AND "AUER !S SUG GESTED ABOVE (VR! MIGHT RESPOND ANAEROBICALLY OR SEMIAEROBICALLY TO CHANGES IN THE REDOX EQUILIBRIUM OF THE 1 POOL )F THIS IS TRUE IT COULD BE ENVISAGED A REGULATORY SCHEME IN WHICH 2EG! AND &NR, INDUCE THE EXPRESSION OF CBB OXIDASE DEPENDING ON THE RELATIVE OXYGEN TENSION WHILE (VR! MAY CONTRAST THEIR ACTION BY SENSING THE REDOX LEVEL OF THE 1 POOL 7*$PODMVTJPOT 4HE PICTURE WE CAN PROPOSE PRESENTLY FOR THE INTER ACTION BETWEEN THE PHOTOSYNTHETIC AND RESPIRATORY CHAINS IN 2HODOBACTER SPHAEROIDES AND 2HODOBACTER CAPSULATUS COULD BE SUMMARIZED AS FOLLOWS 4HE ORGANIZATION OF THE BIOENERGETIC CHAINS SATISlES TWO APPARENTLY CONTRADICTORY REQUIREMENTS NAMELY THE PHOTOSYNTHETIC APPARATUS HAS TO INTERACT WITH THE RESPIRATORY CHAINS AEROBIC AND ANAEROBIC FOR A BETTER USE OF THE AVAILABLE ENERGY THIS LATTER INTERACTION MUST PRESERVE THE 2# FROM AN EXCESS OF
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REDUCING EQUIVALENTS )NDEED IF FULL THERMODYNAMIC EQUILIBRIUM IS ESTABLISHED BETWEEN THE RESPIRATORY AND PHOTOSYNTHETIC CHAINS THE PRIMARY ELECTRON ACCEPTOR SHOULD BE FULLY REDUCED DUE TO ITS MUCH HIGHER MID POINT POTENTIAL COMPARED TO THE .!$(.!$ COUPLE #ELLS FACE THESE CONTRADICTORY REQUIREMENTS BY ARRANG ING THE BIOENERGETIC CHAINS AT TWO DIFFERENT LEVELS IN 2HODOBACTER SPHAEROIDES AND 2B CAPSULATUS ! lRST LEVEL OF ORGANIZATION IS THE SPATIAL SEGREGATION OF THE PHOTOSYNTHETIC AND RESPIRATORY CHAINS IN TWO SEPARATE REGIONS OF THE INTERNAL MEMBRANE 4HE CYTOPLASMIC PART CONTAINS THE GREATER PART OF THE COMPONENTS OF THE RESPIRATORY CHAINS WHICH CAN GO INTO REDOX EQUILIBRATION WITH A SMALL PART OF THE 2#S 4HE MAJOR PART OF THE PHOTOSYNTHETIC CHAINS INCLUDING 2#S #YT C OR #YT CY AND #YT BC COMPLEX ARE LOCALIZED IN THE INTRACYTOPLASMIC PART OF THE MEMBRANE 4HE SECOND LEVEL OF ORGANIZATION IS THE ARRANGEMENT OF THESE PHOTOSYNTHETIC ELEMENTS IN SUPRAMOLECULAR COMPLEXES 4HIS TIGHT CONNECTION ALLOWS A FAST AND EFlCIENT CYCLIC ELECTRON TRANSFER BY LIMITING THE DIF F FUSION OF THE REACTANTS 4HESE TWO LEVELS OF ARRANGEMENT ARE NOT PRESENT IN ALL TYPES OF PHOTOSYNTHETIC BACTERIA 3PECIES LIKE "LASTOCHLORIS VIRIDIS OR 2UBRIVIVAX GELATINOSUS POSSESS A LARGE EXCESS OF 2#S OVER #YT BC COM PLEXES SO THAT THE FORMATION OF SUPERCOMPLEXES IS STOICHIOMETRICALLY HINDERED &OR SPECIES LIKE 2HO DOPSEUDOMONAS VIRIDIS OR 4HIOCAPSA PFENNIGII THE QUINONE POOL CAN HOWEVER BE MAINTAINED PARTIALLY OXIDIZED UNDER ANAEROBIC CONDITION DUE TO THE LACK OF THERMODYNAMIC EQUILIBRATION AT THE 1 POOL LEVEL BETWEEN THE PHOTOSYNTHETIC AND RESPIRATORY CHAINS DUE TO THE SPATIAL ISOLATION OF THESE CHAINS )NDEED THE HIGH ORDERING OF THE PHOTOSYNTHETIC APPARATUS IN THE INTRACYTOPLASMIC MEMBRANE OF THESE SPECIES CERTAINLY RENDERS DIFlCULT THE LONG RANGE DIFFUSION OF QUINONEQUINOL MOLECULES 2UBRIVIVAX GELATINO SUS POSSESS A VERY LOW AMOUNT OF INTRACYTOPLASMIC MEMBRANES AND NO SUPRAMOLECULAR ORGANIZATION OF THE PHOTOSYNTHETIC APPARATUS 4HE LACK OF REDOX EQUILIBRATION AT THE QUINONE LEVEL IN THIS SPECIES IS THEREFORE DUE TO A MECHANISM WHICH REMAINS TO BE REVEALED )N CONTRAST THE LACK OF INTRACYTOPLASMIC MEMBRANES IN AEROBIC PHOTOSYNTHETIC BACTERIA EG 2OSEOBACTER DENITRIlCANS COULD PARTIALLY EXPLAIN WHY ITS PRIMARY ELECTRON ACCEPTOR IS FULLY REDUCED UNDER ANAEROBIC CONDITION ! FURTHER LEVEL OF INTERACTION BETWEEN PHOTOSYN THESIS AND RESPIRATION IS BASED ON THE CAPABILITY TO COORDINATE THE EXPRESSION OF PHOTOSYNTHETIC AND
RESPIRATORY COMPONENTS IN RESPONSE TO THE OXYGEN TENSION AND TO THE REDOX STATE OF THE SYSTEM .OTA BLY THE GENETIC COORDINATION MECHANISM BETWEEN PHOTOSYNTHETIC AND RESPIRATORY CHAINS DOES NOT NEC ESSARILY REQUIRE A DIRECT INTERACTION OF THE TWO SETS OF COMPONENTS TO EXERTS ITS SENSING AND REGULATORY FUNCTIONS 4HE SIGNALS TO WHICH THE SYSTEM RESPONDS MAY BE ORIGINATED FROM COMPONENTS THAT ARE SPECIlC FOR RESPIRATION AS IN THE CASE OF OXYGEN SENSING OR DERIVE FROM COMPONENTS THAT MAY BE EITHER PART OF RESPIRATORY OR PHOTOSYNTHETIC %4#S AS FOR REDOX SENS ING 4HE PICTURE THAT HAS BEEN PRESENTED HERE WHICH IS FAR FROM BEING EXHAUSTIVE TEND TO DESCRIBE THE FRAMEWORK OF GENETIC REGULATORY CONNECTIONS EXISTING IN 2B CAPSULATUS ANDOR 2B SPHAEROIDES AND GIVES SUPPORT TO THE NOTION OF A TIGHT FUNCTIONAL RELATIONSHIP BETWEEN LIGHT AND DARK METABOLIC PATHWAYS /N THE OTHER HAND THE WAY THE SUPRAMOLECULAR ORGANIZATION OF THE %4#S MAY BE GENETICALLY CONTROLLED REMAINS AT PRESENT A RELATIVELY UNEXPLORED AREA "DLOPXMFEHNFOUT $: 2" AND !6 WERE SUPPORTED BY -)52 OF )TALY 02). AND #OMMISSARIAT Ë L%NERGIE !TOMIQUE DE &RANCE RESPECTIVELY 3FGFSFODFT !NTHONY # ED "ACTERIAL %NERGY 4RANSDUCTION !CADEMIC 0RESS ,ONDON "ACCARINI -ELANDRI ! -ELANDRI "! AND (AUSKA ' 4HE STIMULATION OF PHOTOPHOSPHORYLATION AND !40ASE BY ARTIlCIAL REDOX MEDIATORS IN CHROMATOPHORES OF 2HODOPSEUDOMONAS CAPSULATA AT DIFFERENT REDOX POTENTIALS * "IOENERG "IOMEMBR n "AUER #% %LSEN 3 AND "IRD 4( -ECHANISMS FOR REDOX CON TROL OF GENE EXPRESSION !NNU 2EV -ICROBIOL n "IRD 4( $U 3 AND "AUER #% !UTOPHOSPHORYLATION PHOS PHOTRANSFER AND $.! BINDING PROPERTIES OF THE 2EG"2EG! TWO COMPONENT REGULATORY SYSTEM IN 2HODOBACTER CAPSULATUS * "IOL #HEM n "LANKENSHIP 2% -ADIGAN -4 AND "AUER #% EDS !NOXYGENIC 0HOTOSYNTHETIC "ACTERIA +LUWER !CADEMIC 0UB LISHERS $ORDRECHT "ROCKMANN ( *R AND ,IPINSKI ! "ACTERIOCHLOROPHYLL G ! NEW BACTERIOCHLOROPHYLL FROM (ELIOBACTERIUM CHLORUM !RCH -ICROBIOL n "UGGY * AND "AUER #% #LONING AND CHARACTERIZATION OF SEN# A GENE INVOLVED IN BOTH AEROBIC RESPIRATION AND PHOTOSYN THESIS GENE EXPRESSION IN 2HODOBACTER CAPSULATUS * "ACTERIOL n "UGGY ** 3GANGA -7 AND "AUER #% #HARACTERIZATION OF
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# C TYPE CYTOCHROME 3EE ALSO CYTOCHROME C C TYPE CYTOCHROMES C TYPE CYTOCHROME MUTANTS CAA TYPE #C/ OF " SUBTILIS CALDARIELLAQUINONE CALDARIELLAQUINOL #ALVIN CYCLE QUINOL DEHYDROGENASE CARBON PRO 2 PRO 3 CARBON DIOXIDE #/ lXATION REDUCTION CARBON MONOXIDE #/ DEHYDROGENASE SENSOR CARBON STARVATION CATALYSIS MECHANISM OF CATALYTIC HEMES CBB CYTOCHROME C OXIDASE EXPRESSION OF #CC! #CC" #C/ CBB TYPE SOX " TYPE SUB TYPE #CO. #CO/ #CO0 ##0 3EE CYTOCHROME C PEROXIDASE ##0 CELL YIELD CHARGE TRANSLOCATION CHEMIOSMOSIS COUPLING IONS n PROTON STOICHIOMETRY n CHEMIOSMOTIC GRADIENT
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/ / / BOND SCISSION /CC GENE CLUSTER OPTICAL SPECTRO ELECTROCHEMISTRY OPTICAL SPECTRUM ORGANOHYDRAZINES ORIENTATION OF THE SUBSTRATE OVEROXIDATION OF ACETATE OXIDASE ALTERNATE COUPLED UNCOUPLED OXIDATION AMMONIA n OXIDATIVE FERMENTATION OXIDATIVE PHOSPHORYLATION
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2 RADICAL REBOUND MECHANISM REACTION BASED INACTIVATOR REACTION OF AMMONIA WITH HYDROXYLAMINE REBOUND REACTION REDOX COMPONENTS REDOX LADDER REDOX LOOP SYSTEM REDOX POTENTIALS HEME CENTERS n REDOX SENSOR REDUCING NITRITE REDUCING POWER REDUCTION $-3/ OR 4-!/ NITRITE TO HYDROXYAMINE PYRIDINE NUCLEOTIDES REDUCTIVE DECHLORINATION 2EG! 2EG"
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Advances in Photosynthesis Series editor: Govindjee, University of Illinois, Urbana, Illinois, U.S.A. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
D.A. Bryant (ed.): The Molecular Biology of Cyanobacteria. 1994 ISBN Hb: 0-7923-3222-9; Pb: 0-7923-3273-3 R.E. Blankenship, M.T. Madigan and C.E. Bauer (eds.): Anoxygenic Photosynthetic Bacteria. 1995 ISBN Hb: 0-7923-3681-X; Pb: 0-7923-3682-8 J. Amesz and A.J. Hoff (eds.): Biophysical Techniques in Photosynthesis. 1996 ISBN 0-7923-3642-9 D.R. Ort and C.F. Yocum (eds.): Oxygenic Photosynthesis: The Light Reactions. 1996 ISBN Hb: 0-7923-3683-6; Pb: 0-7923-3684-4 N.R. Baker (ed.): Photosynthesis and the Environment. 1996 ISBN 0-7923-4316-6 P.-A. Siegenthaler and N. Murata (eds.): Lipids in Photosynthesis: Structure, Function and Genetics. 1998 ISBN 0-7923-5173-8 J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds.): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. 1998 ISBN 0-7923-5174-6 H.A. Frank, A.J. Young, G. Britton and R.J. Cogdell (eds.): The Photochemistry of Carotenoids. 1999 ISBN 0-7923-5942-9 R.C. Leegood, T.D. Sharkey and S. von Caemmerer (eds.): Photosynthesis: Physiology and Metabolism. 2000 ISBN 0-7923-6143-1 B. Ke: Photosynthesis: Photobiochemistry and Photobiophysics. 2001 ISBN 0-7923-6334-5 E.-M. Aro and B. Andersson (eds.): Regulation of Photosynthesis. 2001 ISBN 0-7923-6332-9 C.H. Foyer and G. Noctor (eds.): Photosynthetic Nitrogen Assimilation and Associated Carbon and Respiratory Metabolism. 2002 ISBN 0-7923-6336-1 B.R. Green and W.W. Parson (eds.): Light-Harvesting Antennas in Photosynthesis. 2003 ISBN 0-7923-6335-3 A.W.D. Larkum, S.E. Douglas and J.A. Raven (eds.): Photosynthesis in Algae. 2003 ISBN 0-7923-6333-7 D. Zannoni (ed.): Respiration in Archaea and Bacteria. Diversity of Prokaryotic Electron Transport Carriers. 2004 ISBN 1-4020-2001-5 D. Zannoni (ed.): Respiration in Archaea and Bacteria. Diversity of Prokaryotic Respiratory Systems. 2004 ISBN 1-4020-2002-3 D. Day, A.H. Millar and J. Whelan (eds.): Plant Mitochondria. From Genome to Function. 2004 ISBN 1-4020-2399-5 Forthcoming.
19. G. Papageorgiou and Govindjee (eds.): Chlorophyll a Fluorescence. A Signature of Photosynthesis. 2004 ISBN 1-4020-3217-X
For further information about the series and how to order please visit our Website http://www.springeronline.com springeronline.com
