structures and proton-pumping strategies of mitochondrial respiratory enzymes

50 Pages·2005·0.69 MB·English

P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
Annu.Rev.Biophys.Biomol.Struct.2001.30:23–65
Copyright(cid:176)c 2001byAnnualReviews.Allrightsreserved
S P S
TRUCTURES AND ROTON-P UMPING TRATEGIES
M R E
OF ITOCHONDRIAL ESPIRATORY NZYMES
Brian E. Schultz1 and Sunney I. Chan1,2
g
or
s. 1ArthurAmosNoyesLaboratoryofChemicalPhysics,CaliforniaInstituteofTechnology,
w
vienly. Pasadena,CA91125,and
s.annualreonal use o e2Imnastiilt:[email protected],cAhc.eadduemiaSinica,Taipei11529,Taiwan;
nalers
m arjour5. For p KlienykWageo,rrdesspircartyosrtyalesletrcutrcotunretrsa,nfsrpeeoretncehragiyntransduction,mitochondrion,redox
ded fro09/08/0 unsinAgbtshteraecntergEynzmyamdeesaovfathileabmleitofcrohmonderleiacltrroenspitrraatnosrfyerchreaainctsieornvsetaosptrraontospnoprutmprpos-,
ownloaGY on tuosnesdafcorrotshsethperoidnuncetriomnitoofchAoTnPd.rTiahlemAeTmPbrsaynnethaansdecernezaytemaenieslreecvtreorscihbelmeaicnadlgcarandaielsnot
DO serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each
01.30:23-65. F TECHNOL oItehnnfezttsyhhemeisperrewsos.topeTriikrnha,setotahmrreyeecsedhtnirsazacnytueimsgsmsieeesssduaaisnnreedslidgeaefhfidstcciofrifeifebnrreceedycnetaonsnfttdrcparrttyheosgettoyasnltfrsoturtrracunptucsertlruaoflrocearbsmtaioosinfensgsaefprvoereroratatolhlsneorepasaucpnmtiairolapyntizonerogdyf.
0O
ct. 2TE in terms of the thermodynamics of the substrate transformations catalyzed by these
uU enzymes.
mol. StrNSTIT
hys. BioORNIA I CONTPEENRSTPSECTIVESANDOVERVIEW :::::::::::::::::::::::::::::::::: 24
BiopLIF ATPSYNTHASE:AReversibleRotarySteppingMotor::::::::::::::::::::: 26
v. CA EnzymologyofATPSynthase:::::::::::::::::::::::::::::::::::::: 27
Annu. Reby XXO--bRRseaaryyvaCCtrrioyynssttaaollfSSStturruubccuttnuuirrteeRooofftatthhtieeonFF01inCCAooTmmPppllSeeyxxnt::h::a::se:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 223890
CouplingofProtonTransferandATPSynthesis ::::::::::::::::::::::::: 30
Summary::::::::::::::::::::::::::::::::::::::::::::::::::::: 31
CYTOCHROMEbc1:ARedox-LinkedProtonPumpUsingaSmall
MoleculeasaProtonCarrier:::::::::::::::::::::::::::::::::::::::: 32
CrystalStructureofCytochromebc1 ::::::::::::::::::::::::::::::::: 33
TheQ-CycleMechanism 35
ThermodynamicsofRedoxLinkage ::::::::::::::::::::::::::::::::: 36
MechanismofBranchedElectronTransfer::::::::::::::::::::::::::::: 36
Summary::::::::::::::::::::::::::::::::::::::::::::::::::::: 37
1056-8700/01/0610-0023$14.00 23
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
24 SCHULTZ ¥ CHAN
CYTOCHROMEcOXIDASE:ARedox-LinkedProtonPumpUsing
TransmembraneIonChannels::::::::::::::::::::::::::::::::::::::: 38
X-RayCrystalStructures ::::::::::::::::::::::::::::::::::::::::: 39
ProtonChannelsInferredfromCrystalStructures :::::::::::::::::::::::: 40
MutagenesisStudiesandProtonTranslocation :::::::::::::::::::::::::: 41
ComparisonoftheCrystalStructuresofReducedandOxidized
CytochromecOxidase :::::::::::::::::::::::::::::::::::::::::: 41
Redox-LinkedChangesinMetalEnvironmentandProteinStructure :::::::::: 42
ReactionoftheEnzymewithDioxygen ::::::::::::::::::::::::::::::: 42
org Redox-CoupledProtonUptakebyCytochromeOxidase ::::::::::::::::::: 44
ws. ThermodynamicsofElectronTransfer :::::::::::::::::::::::::::::::: 45
vienly. ProspectsforFurtherUnderstandingoftheEnzyme :::::::::::::::::::::: 47
eo
s.annualronal use NUASnDtkrHunco:tUwuBrneIMQofUetchIhNeaOnCiNosmmEp:Ol:eX:xI:D::O::::R::E::D::U::C::::T::A::S::E:::::A::::P::ro::t::o::n::P::u::m::p::::w::it::h::a::n:::::::::::::::::::::: 4478
m arjournal5. For pers STTuhhbeeuIUnrobitniqD-SueiusnlcofrunipretCiBoliunnsdateninrdsgL:So:ict:eas:li::z::at::io::n::::o::f::P::ro::s::th::e::t::ic::G::::ro::u::p::s::::::::::::::::::::::::::::::::::::::::::::::::::::: 445990
ded fro09/08/0 TEhleecrtmroondTynraanmsficesro:f:P:r:o:to:n:T:r:a:n:s:lo:c:a:ti:on:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5511
wnloaY on SUDCeCtaIiNlsAoTfEP:rUoBtoInQPUuImNpOiNngEO::X:I:D:O:R:E::D:U:C:T:A::S:E:::A:R::es:p:i:ra:to:r:y::::::::::::: 51
DoOG EnzymeThatDoesNotPumpProtons::::::::::::::::::::::::::::::::: 52
01.30:23-65. F TECHNOL FINQTWAhFheRLyrmRICsoErndy’MysttnSAaalQRmSRKitcrSauscPo:truf:orE:teols:en::cPt::ruo::mn::::pT::?ra::n:::s:::fe:::r:::i:::n:::S:::Q:::R:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 55553556
0O
ct. 2TE
uU
mol. StrNSTIT PERSPECTIVESANDOVERVIEW
hys. BioORNIA I Tofhemionlneecrulmaritmocahcohninderisa,lthmeemenbzryamneesofofeuthkearryeostpeisraptolaryysehleocsttrotonatrfaansscpinoartticnhgasinet.
BiopLIF This set of enzymes, diagrammed in Figure 1, consists of NADH:ubiquinone
v. CA oxidoreductase (Complex I), succinate:ubiquinone oxidoreductase (Complex II,
u. Reby SQR),ubiquinone:cytochromecoxidoreductase(ComplexIII,cytochromereduc-
Ann tase,cytochromebc1),cytochromecoxidase(ComplexIV,cytochromeoxidase),
and ATP synthase (Complex V, F F -ATPase). Complexes I, III, and IV serve
1 0
asprotonpumps,usingtheenergyofelectrontransferstoperformtheelectrical
workoftranslocatingprotonsfromthematrixtothecytosolofthemitochondrion
to produce and sustain an electrochemical gradient across the inner membrane.
Undernormalphysiologicalconditions,ComplexVusesthefreeenergyreleased
fromtheflowofprotonsbackacrossthemembraneinordertoperformthechem-
ical work of producing ATP from ADP and inorganic phosphate. A steady state
is achieved in which the translocation of protons from the matrix into the inter-
membranespaceisbalancedbytheutilizationoftheprotonsforATPsynthesis.
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April14,2001 17:5 AnnualReviews AR128-02
MITOCHONDRIALRESPIRATORYENZYMES 25
However,underconditionsofahigh[ATP]/[ADP]ratioandlowmembraneener-
gization,complexVcanworkbackwardsandserveasaprotonpumpaswell.Each
oftheproton-pumpingenzymesutilizesadifferentstrategyforpromotingproton
translocation,astrategythatisdefinedbyandcanbeunderstoodintermsofthe
molecularstructuresoftheproteins.Recently,X-raycrystalstructureshavebeen
reportedforpartorallofComplexesII–V.Inlightofthesestructures,aglimpse
ofthemechanismsoftheseproteinscanbeobtained.
ComplexesI,III,andIVareredox-linkedprotonpumps.Thatis,theelectron
org transfers through the enzyme are coupled to an increase in the potential energy
ws. oftheproteinitself,sothattheassociatedconformationalchangesintheprotein
ey.
vinl drivetheunidirectional(vectorial)flowofprotonsacrossthemembrane.Asetof
eo
s.annualronal use cathoreemsdemifeofnenzryepnmhtyefssoicarareelap(carhi)norcenidpeol.exTshlairnpekpealingeeesc,te(obss)tiheteilesesectfproournmegpffiastc,iintehgno,tuapgnrhdott(ohcne)pctrhraoentmoslnioccgaaalttdiionengta.bilys
wnloaded from arjournalY on 09/08/05. For pers ctlathirunnaardnkTtsadchsfguheeeerarri:vnfinaesgrgnusetdtbstousrsoertdufrnqrabcouitsvoveiteroerebarrvmdtienoeiecndftntaritontath,ringeoias,ansfpleogrlmproeemrcteooetnamtriotntoiiensooittnnnratyer.dtaiduIacnttcasisetfnbhhetoceorevo.urmenaTl,dcfehotitbirsaroemeltnechnaseaftonatiitonctetevnthdoroaesrlltsvhircneiachvndtaaogobnnlxivtgnhcecedoedhsinnraitinengrndigtbootheuhfxesteaeectphlerteraeoncotdtttterooehorxicisnns--,
DoOG activesubstratetoanenzymeisinfactredox-linked,asthebindingfreeenergyof
5. OL thesubstrateisreflectedinachangeinthereductionpotentialofthesubstrate.
3-6HN Electrongatingreferstothenecessitythatwhenanelectronistransferredtoa
01.30:2F TEC gmivuestnorecdcuorxpcreinotrert,oatlhleofetlheectrreoqnuipsaitsesicnognftoortmheatnioenxatlccehnatnergeinsathnedpprroottoeinnt.raFnasilfuerres
0O
ct. 2TE ofthisrequirementleadstoadecouplingofelectrontransferandprotontransfer
hys. Biomol. StruORNIA INSTITU rpampenrrfaoodetttrroocirnxnoedntisrsfitaodomnremasofsoveafaertnidtoahernaeelagelmaacgibtenraaomsirnntrbitlderheareeasnckeeoe.l.uxeSIpicifsltmtertodhtiolc;iashtrphelcryimoes,vnipscedirantoiultttiaopottnhonioetgeinsabntiatnisincaorkglteflgfimseorrawetrhdte,eiodeefrntleotepq,cruaptosrirtroooaentnmepstirernotanotnstowtstrnhfauearscrdtltiuaaptrsnh.adeal
BiopLIF It is important to note that redox linkage is predominantly a thermodynamic
v. CA issue,whereaselectronandprotongatingarekineticinnature.Foragivenreac-
u. Reby tion intermediate in the proton-pumping reaction pathway, the protein structure
nn is defined by the oxidation state of all of the redox centers and the protonation
A
stateoftheprotein.Hence,redoxlinkagecanbedefinedcompletelythroughthe
structural description of all of the intermediates in the reaction cycle. Electron
leaksandprotonslipsremovetheproteinfromtheproton-pumpingreactionpath-
way. Since electron leaks and proton slips are thermodynamically favored, the
thermodynamically most favored outcome in the reaction of a proton-pumping
enzymewithitssubstratesiselectrontransferwithouttheassociatedtranslocation
ofprotons.Thus,thegatingrequirementsstatethattheprotonpumpingreaction
pathwaymustbethemostfavorablefromakineticstandpoint.Finally,itfollows
thattheremustbesufficientredoxlinkageateachreactionintermediatetoallow
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
26 SCHULTZ ¥ CHAN
theproteinreorganizationnecessarytoensurethevectorialnatureoftheoverall
proton-translocationprocess.
This review summarizes the current structural knowledge of the enzymes of
the respiratory chain and how these structures and associated spectroscopic and
enzymologicalstudieshavedefinedthemechanismofprotontranslocationinthe
proteins.Emphasisisplacedonthestructuralandthermodynamicaspectsofthese
enzymes.Otherbiophysicalandenzymologicaldataismentionedonlyasneces-
sarywithregardtotheprotontranslocationfunctionoftheenzymes.Moredetailed
org informationontheseaspectsofenzymefunctionmaybefoundelsewhere.Rather
ws. thanproceedingthroughthecanonicalorderofComplexItoComplexV,theen-
ey.
vinl zymesarepresentedintheorderofbest-understoodtoleast-understoodinterms
eo
s.annualronal use ocofnthsiedeprreodtobnriterflanysalotcthaetioenndmoefcthhainsiwsmor.kC.omplex II is not a proton pump and is
nalers
m arjour5. For p ATPSYNTHASE:AReversibleRotarySteppingMotor
ded fro09/08/0 eAlTecPtrsoycnhtehmasieca(lFg1rFa0d-AieTnPtaascer)o,sCsothmepilnexneVr moiftothcheornedspriiarlatmoreymcbhraainne, tuotilpizroems othtee
wnloaY on the phosphorylation of ADP in order to produce ATP, according to Equation 1,
oG wherenisintherangeof3–4(15,16,39,109).Intermsoffunction,thisprotein
DO
5. OL assemblyisanalogoustoaturbineorrotarymotor.Theflowofprotonsthrough
3-6HN the enzyme promotes the rotation of a large subunit assembly, and the energy
01.30:2F TEC finrotmhethoipsproostiatetiodnireisctuiotinlizuenddefrorcoAnTdPitisoynnsthoefsaish.iTghhe[AprToPte]/i[nAiDsPa]blreattioofaunndctlioown
0O
ct. 2TE membraneenergization(75).
uU
hys. Biomol. StrORNIA INSTIT bcoramTnpohlueesxeAcenoDsz.myPFmpCilgeeuxPcroieFnC12stiHhsstahCstoorwCefssanidaHmepCseami.rnctbyitarhtlaoenssmoetr-lua/btcor!tiuuxnrseAdpTosaufcPbetCh,caeonHmed2npOazleysxCmtaFlenk0H,tchaCoanlt.amnirsegacedttreieinsxxgc/trrtiahbmeedetw(m1ion)-
BiopLIF greaterdetailbelow.TheF0complexcontainsanionchannelthatallowstheflow
v. CA ofprotonsfromthecytoplasmictothematrixsideofthemitochondrialmembrane
u. Reby andcouplesthisflowofprotonstosubunitrotationwithintheenzymecomplex.
Ann ThemammalianF0complexcontainsninedistinctsubunits,termeda,b,c,d,e,f,
g,A6L,andF .Thereare9–12csubunitsinthecomplex,withonecopyeachof
6
subunitsb,d,andF (20,26,27).Thestoichiometryoftheremainingsubunitshas
6
notbeendeterminedtodate.AsimplerF complexfromEscherichiacolipossesses
0
an ab c subunit composition (42). This latter composition can be considered
2 9-12
theminimumnecessaryforafunctionalF complex.
0
ATPsynthesisandhydrolysisoccursintheF complex.Thiscomplexhasan
1
fi fl (cid:176)–" subunit composition with a molecular weight of 371,000. The active
3 3
sitesforATPsynthesis/hydrolysisresideonthefl subunits,allthreeofwhichare
involvedinoverallcatalysisinacooperativefashionasdescribedbelow.
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
MITOCHONDRIALRESPIRATORYENZYMES 27
ThestalkregionoftheF F -ATPaseconsistsofsubunitsfromboththeF and
1 0 0
F complexes.Inthemitochondrialenzyme,thesubunitOSCP(oligomycinsen-
1
sitivity conferring protein) is associated with this region. The stalk provides the
mechanicalcouplingbetweentheprotontranslocationintheF subunitandATP
0
synthesis/hydrolysis in the F complex. With respect to nomenclature, the mito-
1
chondrialsubunitsOSCPand–areequivalenttothe–and"subunitsinbacteria,
respectively,butthereisnoequivalenttothemitochondrial"subunitinbacteria.
org EnzymologyofATPSynthase
s.
w
ey. ThereactionmechanismforATPsynthesis,orinthereversedirection,hydrolysis,
vinl
s.annualreonal use o awTnhaiissnptmerogeprcaohtseaednditsarmismahebari;sn(sdbei)nvagesrcyahmlammnagejetormryfeoecfahtthaunereicssam:t(abalyy)ttPihcaefluflusBunobcyutienorintisno,tfehatehchleahtFea1v1-Ai9nT7g0Pdsais(f1efe5ar)s-.
nalers ent substrate binding and catalysis characteristics; (c) cooperativity between the
m arjour5. For p flthesuobthuenritssusbuucnhittshaantdthveicceonvfeorrsma;aatniodn(dat)oanneinstuebrucnointvdeircstiaotnesotfhtehecobninfodrinmgactihoanrsaca-t
ded fro09/08/0 twearisstpicosstouflaetaecdhtsouobcucnuirtathsrcoautgalhysroistaptrioocneaeldcsa.taTlhyesiisn,tienrcwohnivcehrstihoenflboewtwoefenprfootromnss
ownloaGY on tthhreopurgohpethrteieFs0osfutbhceoflmspulbeuxnciatsu.sTesheamsuebcuhnaintirsomtaitsiosnhoinwtnhescFh1ecmomatipclaelxlythinatFcihgaunrege3s.
5. DOLO The three conformations of the fl subunits are referred to as the tight, open,
01.30:23-6F TECHN andlooseforms.Inthereactioncycle,ADPandPibindtothesubunitintheloose
0O
ct. 2TE
uU
mol. StrNSTIT
ys. BioRNIA I
hO
BiopLIF
v. CA
Rey
u. b
n
n
A
Figure3 ThebindingchangemechanismofF -ATPase.Onlythesubstratetransforma-
1
tionsinthetopflsubunitareshown.Notethatinthismechanismonlythecentral(cid:176) subunit
rotates; the fl subunits change their binding characteristics as a result of the (cid:176) subunit
rotation.
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
28 SCHULTZ ¥ CHAN
form.Oncethisbindingoccurs,thissiteisconvertedtothetightconfiguration.In
thetightconfiguration,theflsubunitcatalyzesthereactionofADPandphosphate
to form tightly bound ATP. This step does not require energy transduction from
theprotongradient,asATPformationfromADPandphosphatecanbeobserved
in isolated F -ATPase. In the next step in the reaction cycle, the reaction site is
1
convertedtoitsopenformandATPisreleased.Thisisthestepthatiscoupledto
protontranslocation,asenergyisneededtoeliminatethestrongbindinginteraction
betweenATPandtheprotein.Inasubsequentstep,theopenformisconvertedto
org thelooseformtocompletethereactioncycle.Atanygivenpointinthereaction
ws. cycle, one subunit has ATP bound in the tight form, one subunit is in the open
ey.
evionl form,andthethirdsubunitisinthelooseformthatwillbindADPandPi.Only
s.annualronal use wlmohooeslenecusAulTebPuonfiisAtTrcePalneaantshdeedthsfeurobfmuonrmitthaeitniootepnreconofnsavuseberuscnioointndaonmcdcoulAercDtuoPleda.rnTidvheiPstihsaterriecretblreoeauqsnuediroeifnmoethnneet
wnloaded from arjournalY on 09/08/05. For pers fcritnaoeogghruPeaitpbhnrrldiieeitonsiftroigoosn.tr.towhaEinatsxhrdcdeeednrslezeloeryalincumvtttaiieotosiuncnzamaomotfimfoeAnaXTfrr-oiPoerfamssytyhocnecfltrahetyshanssseztieascyelaimslsswetetr,unuhdazcanyitteudmgsriemovhsleaoo,svrgteehtyhe,rbeeimeshcoeeoentnlcoothppleyaern,enzessiyxseittmcneihct-eeadditninirggseeehiclgsttsheeetwdtundwehmireeigutrshye-,
DoOG (15,16).Thefollowingsectionsprovidesalientresultsofrecentcrystallographic
5. OL and biophysical studies that reveal the details of the binding change mechanism
3-6HN andvalidatethishypothesis.
01.30:2F TEC
0O X-RayCrystalStructureoftheF Complex
ct. 2TE 1
hys. Biomol. StruORNIA INSTITU bsmTciolhsyow.esSrotceerrosvkyofeeslrtwrvaaselhl(dssi2,tct)rrhp.uurTccaotthrvuueiirrsdeevsesoatdorrfiufatahctnshtettuesrFruFe1oc-,1ftA-tuAhtTrheTaPelcParbosayaressisefgtirahsionlasfmavoflerob2bmro.ew8veeichnnA˚hiepcashuhntebriwsaultirceistctrh,ueweprgderiotr(hosp1owto,hl2svnea,eifil1dns13otflb,hf9y3e(cid:176)A6Wp,Tsr1uaPe1lbsk0seaye,nsr1nsc2eteahm3noe)d--f,
BiopLIF ADPandtheATPanalog50-adenylyl-imidodiphosphate(AMP-PNP),isthemost
v. CA illustrativewithrespecttothereactionmechanismoftheenzymeandservesasthe
u. Reby sourceoftheinformationthatfollows.
nn Theoverallshapeoftheproteincomplexisellipsoidal,withaheightof80A˚
A
andadiameterof100A˚.Thecomplexconsistsofapseudo-threefoldsymmetric
ringofalternatingfiandfl subunits,withthe(cid:176) subunitsittingasymmetricallyin
thecenterofthisring.Thefiandfl subunitshaveaveryhighsequencesimilarity
(20%identity)andhavenearlyidenticalfolds.Eachofsubunitsfiandfl consists
offl-barrel,nucleotide-binding,andhelixbundledomains.Oneofthefl subunits
containsamoleculeofAMP-PNPatthecatalyticsite.Asecondflsubunitcontains
bound ADP at its catalytic site. The third subunit has an empty catalytic site.
Thesesubunitsappeartocorrespondtothetight,loose,andopenconformations,
respectively,ofthefl subunits.
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April14,2001 17:5 AnnualReviews AR128-02
MITOCHONDRIALRESPIRATORYENZYMES 29
Thenucleotidebindingsiteofthefl subunitsconsistsofahydrophobicregion
in which the adenine moiety binds, and a binding region for the phosphate tail
ofthenucleotides.ThehydrophobicpocketincludesresiduesV164,Y345,F418,
A421,andF424.IntheAMP-PNP-boundform,showninFigure4,residuesG159
(amide NH), K162, R189, R373 from an adjacent fi subunit, and an Mg2C ion
formelectrostaticinteractions/hydrogenbondswiththephosphategroups.Inthe
ADP-bound form, residues 159–164, along with R373 from the fi subunit and
an Mg2C ion, serve to bind the diphosphate, while an ordered water molecule
org occupies the position that the terminal phosphate takes in the AMP-PNP-bound
ws. form.TheoverallstructuresoftheADP-boundandAMP-PNP-boundsubunitsare
ey.
vinl verysimilar.
eo
s.annualronal use PdoNmTPah-bienoeuomnppdentyfsoflurmpsut.hbAeuncdaiittsasrhluyoptwtiicossnaitmoef.aTtrhkheeedfldsi-stsrthauencecttuerssatbrlueccthwtauenreegneoFwf4tiht2he4rnaenuspdcelreceotsttiioddutehe-ebsiAKndM1i6nP2g-,
wnloaded from arjournalY on 09/08/05. For pers Rirabnede1ldteg8Twiao9thseii,oeeenanngaon,stfdfhcyrboemoRoum(cid:176)mu3pn7lsetidu3hntrbegAy(ufiToAnofPifsMtuptafhbrPrneoou-dtmnPflotiNhntst)ePhutirefibnafu3cflonflnrrseims3ftaeusrsrbitienousagnnba.tdiythBt.leA2eoa.Tet7hsmPthA˚pisynt,ydypn8rat.of6horgtereAmsi˚nni,s-.dbaurTonechndqeiduds1iin2rboeg.yp3seatthnAni˚geid,nh(cid:176)hgrteyissadnuplrtlbeeoocurpwatnhicsviotte.itbolhIiynnec,
DoOG interactionswereobservedbetweenthe(cid:176) subunitandspecificsubunitsonthering.
5. OL Specifichydrogen-bondinginteractionsincludethoseofR254andQ255ofthe(cid:176)
3-6HN subunitinteractingwithD316,T318,andD319ontheemptyfl subunit,R252of
01.30:2F TEC tshuebu(cid:176)nistu,baunnditKh8y7droofgtehneb(cid:176)osnudbinugnittoinDte3r3a3ctoinfganwfiithsuEb3u9n8itoafdtjhaeceAnMttoP-tPhNePem-bpotuynfld
0O
ct. 2TE flsubunit.These,incombinationwithseveralhydrophobicinteractionsoccurring
mol. StruNSTITU tbheetsweeseunbtuhneit(cid:176)s.subunit and the fi3fl3 subcomplex, provide the coupling between
ys. BioRNIA I X-RayCrystalStructureoftheF0Complex
hO
BiopLIF ArecentcrystalstructureofanF1c10complexfromSaccharomycescerevisiaeat
u. Rev. by CA A3.T9PA˚syrensthoalustei,oanshwaesllyaiesldsterducatupraarltiianlfosrtrmuacttiuornalredgeascrdriipntgiotnheolfinthkeagFe0bceotmwepelenxthoef
Ann Fth0eafind, flF,1(cid:176)co,manpdle–xessu(b1u1n6it)s.TohfeFstrwuecrteurperiessesnhto,wanloinngFwigituhre120.cInsuthbiusnsittrsuocftuFre.,
1 0
Thecsubunitsarearrangedinaring.Eachcsubunitconsistsoftwomembrane-
spanningfi-helicesconnectedbyaloopregionthatextendsintothematrixspace.
Thesubunitsareorientedsothattheyformacylindricalshell,withtheN-terminal
helicesontheinsideoftheshellandtheC-terminalhelicesontheoutside.Although
the resolution of the structure is low, it appears that the – subunit of F makes
1
contact with five c subunits, interacting with the loop regions of these subunits.
The(cid:176) subunitshowsinteractionswiththreecsubunits.The"subunit,whilenotyet
structurallyorspatiallydefined,isbelievedtobepresentinthestalkregionaswell.
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
30 SCHULTZ ¥ CHAN
EachofthecsubunitsofallknownF -ATPasescontainsacarboxylategroup
0
(AsporGlu)thatcanreactwithDCCDtoinactivatetheenzyme(39,108).This
residue, D61 in E. coli, was shown through NMR structures to reside near the
middle of the C-terminal helix of the subunit (47), which places the residue in
the middle of the mitochondrial membrane. The equivalent residue E59 in the
Saccharomyces cerevisiae enzyme was not resolved in the F c structure but
1 10
is predicted to lie in a similar position. Mutagenesis studies have shown that
residuesR210,E219,andH245intheE.coliasubunitarealsocrucialforproton
org translocation(60).Thus,itisassumedthattheprotonchannelliesintheinterface
ws. betweentheaandcsubunits,andthatinteractionsbetweentheaandcsubunits
ey.
vinl involvingtheaforementionedaminoacidsarecrucialforprotontranslocation.
eo
s.annualronal use ObservationofSubunitRotationinATPSynthase
nalers Directobservationofrotationofthe(cid:176) subunitofATPsynthasewithrespecttothe
m arjour5. For p fi(837fl)3.AsunbuFn1-itAcTlPusatseerfwroamsatchceotmheprlmisohpedhiilnicabtarcutleyrieulmegaBnatceixllpuesrPimS3enwtabsyeNxporjiesesteadl
ded fro09/08/0 rinesEid.ucoeliinatnroddmucoeddifioendthweit(cid:176)hspuobluynhiits.tTidhineeHtiasg-staognstwheerfleususebduntoitismanmdowbiiltihzeatchyesfite3iflne3
ownloaGY on s(cid:176)usbusbtruuncittuwreaosnbiaoctionvyelarstelidpacnodataetdtawchitehdntoicaknelancittirnilfioltarimaceenttatthea.tTwhaescmysotediinfieedonwtihthe
5. DOLO bothafluorophorandabiotinmolecule,usingstreptavidinasalinker.WhenATP
3-6HN wasintroducedtothesemolecules,hydrolysisoftheATPbytheF1complexcaused
01.30:2F TEC tchieen(cid:176)tlysulbounngi(t»an2d.5th„emat)ttahcahtetdheacrotitnatfiiolanmoefnthttiosfirolatamtee.nTthweaascotbinsefirlvaambelenbtwyeapsisfluuffio--
ct. 20TE O rAessciemniclaermexicpreorsicmoepnyt.dReomtaotinosntrwataesdotnhlayttohbese"rsvuebduinniaticsoaulnsoteprcalrotcokfwthiseerdoitroerc(t6io6n)..
uU
mol. StrNSTIT rotaUtisoinngotfhtehseaomliegmometehroodfoclosguybausnditessdcurirbinedgaAbToPveh,ySdarmolbyosnisgbiyettahledEem.coonlistFra1Fte0d-
ys. BioRNIA I AcyTsPteaisnee,(1a0n7d)t.hAisgclyusttaeminaetewianstuhseedcsausbaubnoivteotfothbeinFd0acflomuoprleesxcewnatlsyrleapblealceeddabcytina
hO
v. BiopCALIF fiuplaomneandtd.iAtiognaionf, AaTcoP.unterclockwise rotation of the actin filament was observed
u. Reby rotaTrhyemseottowro,sehxopweeridmtehnetslindkeamgoenbsetrtwateeednththeeoFpearantdioFn ocfomthpeleFx1eFs0,-aAnTdPraesveeaalseda
n 0 1
An atleastinparttherolethatthesubunitsplayinthefunctioningofthismachine.
Thebacterialc,(cid:176),and"subunitsareinvolvedintherotorportionofthemolecule,
whereasthea,b,fi,andfl subunitsremainstationaryduringenzymeturnover.It
shouldbenotedthatintheabsenceofatetherbetweenthe(fifl) trimerandtheF
3 0
subunit,rotationofthe(cid:176) subunitwouldrotatetheentireF subunit.Thebsubunit
1
appearstoprovidethistetherinthefunctioningholoenzyme.
CouplingofProtonTransferandATPSynthesis
Theenergycost1G–0 fortheformationofATPandwaterfromADP,phosphate,
andoneprotonis8.2kcal/mol.Theprotonmotiveforce(1ˆ-2.3RT1pH/F)across
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
MITOCHONDRIALRESPIRATORYENZYMES 31
anenergizedmembraneisapproximately200mV.Onthisbasis,theflowoftwo
protonsthroughtheF F -ATPasecomplex(1G»9.2kcal/mol)wouldbesuffi-
1 0
cient to produce one molecule of ATP under standard conditions. Experimental
measurementssuggestastoichiometryof3–4protonsperATPmoleculeformed
(30,117,124).Assumingthattheten-subunitstructureofthecassemblyofF is
0
correct,andthattherotationofthisassembly36– (360–/10)requiresoneproton,
3.3 protons would be required for the synthesis of one molecule of ATP. Thus,
theefficiencyofATPsynthesiswithrespecttofreeenergyconsumptionismuch
org less than unity. The transmembrane potential must provide an important driving
ws. forceandkineticdeterminantinthecouplingoftheprotongradienttothesubunit
ey.
evionl rotationoftheF1F0steppingmotorifthecouplingofATPhydrolysistothesubunit
s.annualronal use rboeteaTnthiaoesnsseitsrsesvneedgrtythhtroiogfuhtght.hetchoeudpilriencgtoobfsAeTrvPathioyndroofltyhseissutobu(cid:176)niatnrdotcatsiounbudneistcrroibtaetdioanbohvaes
wnloaded from arjournalY on 09/08/05. For pers (tt3otorh86fare7tqAkr,ieuoc1Tfena0oPl7or(/me2)fu.,–anotF5pdhlopeemfrroreoMraxsxcatt)iartmfianonudfadltalfiArerirllTvdoayiPmtnac4.gtoe0TinnofhptodnsiNrisotcwitnfeooimtnnrthhqseewucliAeeesasnTscasgopPastrrsrahroyosesdcestuiiopmnacotreoeotnhddttdoaeisrtwn.ertaoTittthnhhhaegeefeaparhreco1yeetfsdiGneer1nnofi–ceol4lyerfagsmo„i¡ysfme2roane4,fthq.sa3i6ugaimchrrkoeococnmsaloeseleni/cfsmncrutteoelonoenmls-ft;
DoOG thehigh[ATP]/[ADP]ratiousedintheexperiments.Becausetherotationofthe
5. OL actinfilamentcanbedrivenwithonlyasmallexcessofdrivingforce,thecoupling
3-6HN oftheATPhydrolysisandsubunitrotationmustbeboththermodynamicallyand
01.30:2F TEC kinAetticaalfluynvdearmyetnigtahlt.level, the energy transduction in ATP synthase is relatively
0O
ct. 2TE straightforward.Intheforwarddirection,electrostaticinteractionsbetweenthepro-
hys. Biomol. StruORNIA INSTITU totcaohllfelnaototshwtteaihdntpeehdrseo(cid:176)atbmroosinnuitnodbtorruatnatonocistti,frhdeoaesrtnaiaFndtne1td,hhcebeAonumTsctuePptbhltuseehynxenri.ottesIhtnnaoettsftiiihortseheniersowoFppti0oprllecro,veoseicimttnaheptneeldnrdeioxbrbterdycemrtatiiohkvoveenfe,atahvpAueopnTrrataPoirlbetohlntehryt.edreUelrpeqonrculcoytoirprsoueeisspmrtlacnientauningc-t
BiopLIF interactions within the F0 subcomplex or create unfavorable ones. Without the
v. CA uptakeandtransportofprotonstocounteracttheseunfavorableelectrostaticinter-
u. Reby actions,ATPhydrolysiswillnotprovideenoughenergytomovetherotor.Thus,
nn throughsimple,mechanicalcoupling,thereversibleenergytransductionbetween
A
anelectrochemicalgradientandaphosphatebondinATPcanbeachieved.
Summary
Thestructural,enzymological,andbiophysicalinformationgatheredtodatehas
led to a detailed understanding of the action of the F F -ATPase. The structural
1 0
basisforATPsynthesis/hydrolysishasbeencharacterized,theoperationoftheen-
zymeasarotarysteppingmotorhasbeenconfirmed,thesubunitsinvolvedinthe
rotorhaveforthemostpartbeenelucidated,andthecouplingofprotontransferto
ATPsynthesisisclearatleastataconceptuallevel.Severalquestionsstillremain,
P1:FXS
April14,2001 17:5 AnnualReviews AR128-02
32 SCHULTZ ¥ CHAN
however. The location of the remaining subunits in the overall structure of the
holoenzymeneedstobedefinedatamolecularlevel,andtherolesofthesubunits
thatarefoundinmitochondrialF F -ATPasebutnotinE.colineedtobeunder-
1 0
stood. The precise pathway of proton transfer through the F subunit is still un-
0
known,andtheinteractionsbetweentheaandthecsubunitsthatleadtorotationof
therotorarestillpoorlyunderstood.Also,whentheenzymeisperformingATPhy-
drolysis,themechanismbywhichthishydrolysisistightlycoupledtoprotontrans-
feragainsttheelectrochemicalgradientacrossthemembraneneedstobediscerned.
g
or
s.
w
ey.
vinl CYTOCHROMEbc :ARedox-LinkedProtonPump
eo 1
s.annualronal use UsingaSmallMoleculeasaProtonCarrier
nalers Cytochrome bc1 catalyzes the transfer of electrons from ubiquinol to ferricy-
m arjour5. For p tioncthhraotmboetch,oaxcicdoartdioinngotfouEbqiquuaitnioonl(2Q(H128),1an1d8,r1e1d9u)c.tiTohneorfeuabctiiqouniniosnceom(Qp)lioccacteudr
ded fro09/08/0 durPinrogttohnetrreaancstlioocnactiyocnlemaesdpiaatretdofbtyhethpisroetonnzy-tmraensolcoccuartisnignatpwpoarwatauys.s. First, the
wnloaY on opxlaisdmatiiconsidoefuobfitqhueimnoiltoacnhdotnhderaiaclcmomempabnryanineg,wrehleeareseasotfhperroetdouncstoiocncuorfounbitqhueicnyotnoe-
oG
DO byComplexesIandII(seebelow)involvesuptakeofprotonsfromthematrixspace.
5. OL
3-6HN Inthisway,ubiquinoneactsasashuttleforprotons,transportingtheminanun-
01.30:2F TEC ctrhaanrsgleodcaftoiormn iancvroolsvsesthaeQm-ictoycchleo,nadrbiaralnmchemedbrealencet.roTnhetrsaencsofenrdpmatehtwhoadyoinfpwrohtiocnh
0O one electron from the oxidation of ubiquinol is used to reduce ferricytochrome
ct. 2TE c on the cytoplasmic side of the membrane, while the other electron is used to
uU
mol. StrNSTIT raesdsouccieautebdiquupitnaokneeo(foprruobtiosnesmfirqoumintohneem)taotruixbisspemacieq.uTinwoonueb(oiqruuibnioqluminoolle)cwuliethstahree
ys. BioRNIA I ooxniedmizoeldecinuleeaochfurebaiqcutiionnoncye.cTleh,ereQd-uccyinclgetiwsothmeomleeccuhlaensisomffbeyrrwichyitcohchcryotmocehcroamnde
BiophLIFO bc1servesasaprotonpump.
v. CA 2QH C2cytc3CCQC2HC.matrix/!2QC2cytc2CCQH C4HC.cytosol/
Rey 2 2
u. b (2)
n
An Mitochondrialcytochromebc existsfunctionallyasadimer,whereeachmono-
1
mer consists of eleven subunits and has a molecular weight of 240,000. Three
subunits,cytochromeb,cytochromec ,andaniron-sulfurprotein,containallof
1
theredox-activemetalcentersoftheenzymeandconstitutethecatalyticcoreof
theprotein.Cytochromebcontainstwosix-coordinatelow-spinbhemes,b and
L
b (alsocalledb andb ),andtwoubiquinol/ubiquinonebindingsites,theP
H 565 560
centerandtheNcenter(alsoknownastheQ andQ sites).Theiron-sulfurprotein
o i
containsahigh-potentialFe S cluster,andcytochromec containsasix-coordinate
2 2 1
low-spincheme.Cytochromec servesastheelectrondonortocytochromec.
1