生物化學英文版課件：chapter 19 Oxidative Phosphorylation

Chapter19

OxidativePhosphorylationOxidationofNADHandFADH2byO2,withenergyreleasedcoupledtoATPsynthesis.

“Anyonewhoisnotconfusedaboutoxidativephosphorylationjustdoesn’tunderstandthesituation”EfraimRacker,1970s

Themostefficientenergytransformationprocessfoundontheearth!“TheformationanduseofATPistheprinciplenetchemicalreactionoccurringinthewholeworld!”—PaulBoyerOxidativephosphorylationisthefinalstageofnutrientoxidationA1.14-voltpotentialdifference(Eˊ0)betweenNADHandO2driveselectronflowthroughtherespiratorychainwithaΔGˊoof~220kJ/Mol.Therespiratorychain:thebattery(exergonic)Electricmotor(endergonic)1.Redoxreactions (electrontransferring) carriedoutbyaseries ofproteincomplexes withincellmembranes;2.Protontransportacross cellmembrane;3.ATPsynthesisusingthe energystoredinthe protongradient.Photophosphorylationinchloroplastishighlycomparabletooxidativephosphorylationinmitochondria!e-e-Battery(exergonic)Electricmotor(endergonic)Historicaleventsinunveilingmolecularmechanismofoxidativephosphorylation(1)VitalroleofPiandcofermentinyeastfermentation(ArthurHarden,1906)CentralroleofATPinenergytransformation(FritzLipmann,1941)LinkbetweensugaroxidationandATPgeneration(HermanKalckar,1940s)CoenzymeNADHlinkedmetabolicpathwaystomitochondrialsynthesisofATP(Lehninger,1949)HardenLipmannKalckarHistoricaleventsinunveilingmolecularmechanismofoxidativephosphorylation(2)ElectrochemicalconcentrationgradientofprotonsacrossamembraneproposedtobeharnessedtomakeATP(chemi-osmotictheory,PeterMitchell,1961)ElectrontransportproteincomplexesandATPsynthaseisolated(DavidGreenandEfraimRacker,1960s)Historicaleventsinunveilingmolecularmechanismofoxidativephosphorylation(3)Bindingchangemechanism(1973)androtationalcatalysis(1982)proposedforATPsynthase(Boyer)StructureofATPsynthasepartiallydetermined(1994,Walker)RotationoftheATPsynthasemoleculeobservedinvitro(MasasukiYoshida,1997).YoshidaThemitochondrialelectron-transferchainwasseparatedintofourmajorproteincomplexesElectronsofNADHandFADH2aretransferredtoO2viamanyintermediateelectroncarriersmakinguptherespiratorychain.NADHdehydrogenase(complexI):34-46subunits,~1000kDNature,2010,466:441-447.Science,2006,311:1430-1436InvolvingFMNandaseriesofiron-sulfurcentersaselectroncarriers,withubiquinonetakingawaytheelectronsattheiron-sulfurproteinN-2.HydrophilicdomainFe-SAtleasteightdifferenttypesofiron-sulfurcenters(firstrevealedbyHelmutBeinert)actintherespiratorychain(incomplexI,IIandIII):ironatomscyclebetweenFe2+(reduced)andFe3+(oxidized).2Fe-2S4Fe-4SInorganicsulfurUbiquinone(orcoenzymeQ10)

istheonlye-carrierthatisnotboundtoaproteinandisabletodiffusefreelyinthelipidbilayer.

(ordihydroubiquinone)?IsoprenoidtailCompletereductionofQrequirestwoelectronsandtwoprotons,occurringintwostepsviaasemiquinoneradicalIntermediate.

FredrickCrane,1957

FADH2ofotherflavoproteinsalsotransfertheirelectronstoubiquinone(Q)viaaFe-S,butwithnoH+pumped.

SuccinatedehydrogenaseSuccinatedehydrogenase(complexII)transferselectronsfromsuccinatetoubiquinoneComplexII(ofthecitricacidcycle)InvolvingFADandmultipleFe-Scentersaselectroncarrier.ThehemegroupisbelievedtohelpdecreaseproductionofROS.Cytochromebc1complex(ComplexIII)transferselectronsfromQH2tocytochromec(Cytc)Cytc

CoenzymeQ

:cytochromecoxidoreductase

(Encodedbymitochondrialgenome)(JohnRieske,1964)Cytochromeswerediscoveredasheme-containingrespiratorypigments(MacMunn,1884,DavidKeilin,1925)KeilinThreetypesofhemegroupsserveasprostheticgroupsofthecytochromeproteinsExistinComplexIIIExistincytochromecandcomplexIVTheironinterconvertsbetweenitsreduced(Fe2+)andoxidized(Fe3+)formsincytochromes.Cytochrome

reductioncanbedetectedbylightabsorptionatuniquevisible

wavelengths

Thereduced(Fe2+)?

stateofcytochromesa,b,andchasuniquelightabsorptionnear600,560,and550nmrespectively.Thisallowscytochromes(hemoproteins)actiontobedetecteddirectlyinmitochondriaviaspectroscopy.cbaCytochromesareclassifiedonthebasisofpositionoftheirlowestenergyabsorptionbandinthereducedstate.e-transferring&H+pumpinginComplexIIIproposedtooccurviatheQcycle

theQcycleFrom:/wiki/Image:Theqcycle.gifCytochromecoxidase(ComplexIV)transferselectronsfromCytctoO2.Hemeaandhemea3

hasidenticalstructuresbutdifferentreductionpotential.4Fe2+-cytochromec+8H+in+O2→4Fe3+-cytochromec+2H2O+4H+outCyanide,sulfide,azide,andCOallinhibitcytochromecoxidase!ComplexesIIIandIVmightformsupercomplexes(respirasome)onthemembraneElectronsflowfromNADH(orsuccinate)toO2iscoupledtotransmembraneprotonpumping~10protonsperNADHand~6

protonsperFADH2oxidized

arepumpedacrosstheinnermembraneandtheelectronmotiveforceisconvertedtoanprotonmotiveforce.(Estimatedbytitrationsuponthepresenceofspecificinhibitors)Reactiveoxygenspecies(ROS)mightbeproducedfromtherespiratorychainThechemiosmoticmodelwasproposedbyPeterMitchellin1961toexplainthecouplingofelectronflowandATPsynthesis(Chemicalreactionscoupledtoosmoticgradients.)?

Factsnotyetexplained:

？“Hight-energy”intermediateselusivetoidentification;

？Phosphorylationcloselyassociatedwithmembrane;

？Uncouplingcausedbyagentsofvariousstructures;

？Swellingandshrinkagephenomena.Mitchell,P.1961,Nature,191:144-148.A“paradigm”shiftinunderstandingbioenergetics!VectorialmetabolismvsScalarmetabolismThechemiosmotictheoryofMitchell:

e-flowandATPsynthesisareseparateevents,coupledviaatransmembraneH+gradient!

Thechemiosmotictheoryunifiedtheapparentlydisparateenergytransductionprocessesasoxidativephosphorylation,photophosphorylation,activetransportacrossmembraneandthemotionofbacterialflagella.

ATPsynthase(EC4)wasfirstidentifiedbydissociationandreconstitutionstudies(EfraimRacker,1960s)Knob-likestructureswereseenwithinside-outvesicleofmitochondria,thylakoidmembraneofchloroplastsandinside-outE.coliplasmamembrane.ThemitochondrialATPaseistheATPsynthase!!!Mitochondria-catalyzed18O,32Pexchangedatasuggeststhatoxidativephosphorylationisdynamicallyrevesible(MildredCohn,1953)Covalentintermediateswasproposedtobeformed.Latersuchaphosphorylatedproteinwasdetected,butlaterfoundtobesuccincylCoAsynthetase(byPaulBoyer;“Wewerereachingforagoldbutgotabronzeinstead”)TheenergyfromelectrontransportwasproposedtobeneededforreleasingpreformedATPfromATPsynthase,notneededfortheformationofATP(Boyeretal.,1973,PNAS,70:2837-9.

PiHOHexchangeisconsiderablylesssensitivetouncouplersthanthePiATPandATPHOHexchanges.Theuncoupler-insensitivePiHOHexchangeisinhibitedbyoligomycin.

Similarexchangewasalsoobservedformyosin.Thisreleasecouldlogicallyinvolveenergy-requiringproteincon-formationalchange.(Notasinglewordwasmentionedaboutthechemiosmotichypothesis!)Catalyticcooperativityrevealed:removalofADPstoppedATPHOHexchangeandremovalofATPstoppedPiHOHexchange(Boyer,1975)

AtlowerATPlevel,moreOinPiisexchangedwithHOHSubunitcompositionofATPsynthasecharacterized,forthetheATPase(F1)

andprotonchannel(Fo)

Stationaryunit(stator)?F1:

Fo:ab2c10-14Therotor:thec-ringandthe

stalk;thestator:theremainder.?Proton-conductingCatalyticArotationalbindingchange(orflip-flop)mechanismwasproposedtoexplainthecatalysisofATPsynthase(PaulBoyer,1980)

1.

The

subunitfoundtointeractwiththecatalyticbsubunits;

2.Distributionof18OinPi(with1,2or3Oexchanged)formedfrom18O-ATPsuggestedidenticalbehaviorofallcatalyticsites.

3.Mildcross-linkingstoppedcatalysisandcleavageofthecross-linkerrestoredactivityBindingADP+PiSynthesizingATPReleasingATPThebinding-changeMechanismorrotationalcatalysis(PaulBoyer,1980s)?Each

subunitwilltakethreedifferentconformationsinturnduringeachcycleofaction./wiki/Image:ATPsyn.gifThebinding-changemechanismofATPsynthase:Thebinding-changemodelwaselegantlysupportedbyX-raycrystallographyanalysisofF1-ATPase.Thethreecatalyticbsubunitsdifferinconformationandthenucleotidebound;Thethreecatalyticsubunitsareindifferentstatesofthecatalyticcycleatanyinstant;Interconversionofthestatemaybeachievedbyrotationofthegsubunitinthecenter.

AMP-PNPAMP-PNPAMP-PNPAMP-PNPADPNature,1994,370:621-628.Rotationofc-ringorc-ring/

subunitdirectlyobservedusingfluorescencemicroscopy(1997)MechanicallydrivenATPsynthesisbyF1-ATPasedemonstrated(2004).ModeloftheactionofE.coliATPsynthase:theprotongradientdrivestherotationofthecringusingtwohalf-channelsontheasubunit.Protonation/deprotonationofanAspisbelievedtobeessentialforrotatingthecringandthe

subunit.10-14protonsneededforevery3ATPsynthesized.Asp-COO-Asp-COOHThus~4protonsperATPsynthesizedhttp://www.mrc-dunn.cam.ac.uk/research/atp_synthase/

Theenergystoredintheprotongradientcanbeusedtodootherwork.Therotarymotionofthebacterialflagellaisenergizeddirectlybytheprotongradientpresentacrossthecytoplasmicmembrane.Theproton-motiveforceisusedforactivetransportthroughtheinnermembraneofthemitochondria.HeatisgeneratedinBrownfatthroughtheactionofthermogenin,

anuncouplingprotein:

toproduceheattomaintainbodytemperatureforanimalsinhibernation,ofnewlybornandadaptingtothecold

(thermogenesis).

FattyacidsseemtoactivateandnucleotidesinhibitUCP1UCP1ElectronsinNADHgeneratedincytosolareshuttledontotherespiratorychain.

Themalate-aspartateshuttlesystem:MalatetranslocateselectronsproducedduringglycolysisacrossinnermembraneofmitochondrionforoxidativephosphorylationsemipermeableIrreversibleTheglycerol-3-phosphateshuttlesystemThepathwaysleadingtoATPsynthesisarecoordinatelyregulated.Interlockingregulationofallthesepathwaysmightberealizedbythe

relativelevelsofATP,NADH,ADP,AMP,Pi,andNAD+.

[ATP]/([ADP][Pi])fluctuatesonlyslightlyinmosttissuesduetoacoordinatedregulationofallthepathwaysleadingtoATPproduction.

TherateoftherespirationassumedtobecontrolledbytheavailabilityofADP(“acceptorcontrol”)?NoATPconsumption,noelectronflow!PyruvateoxidationSomerespiratoryproteinsareencodedbythehumanmitochondrialgenomeComplexesI,III,andIVandATPsynthaseareassembledbyusingsubunitsmadeinboththecytosolandmitochondria.Howwasthemolecularmechanismofoxidativephosphoryationrevealed?KalckarHM(1974)."Originsoftheconceptoxidativephosphorylation".Mol.Cell.Biochem.

5(1–2):55-62.1.Glucosedegradation(fermentation,Glycolysis)2.Biologicaloxidation(O2consumption,citricacidcycle)3.Energysupplyofmusclecontraction4.UnderstandingtheroleofATPastheuniversalenergycurrency5.Subcellularlocationandproteinisolationandcharacterization.Theactivationofhydrogenatomswasthoughttobethekeyforbiologicaloxidation(ThunbergandWieland,1910s)Thunberg,T.(1917)Skand.Arch.Physiol.35,163Wieland,O.(1912)Ber.Dtsch.Chem.Ges.45,484–499;2606–2615Theoxidationofalargenumberoforganiccompounds(e.g.,succinicacid)canbecatalyzedbyspecificdehydrogenasesinthepresenceofartificialhydrogenacceptors(e.g.,methyleneblue).TheactivationofO2wasthoughttobethekeyforbiologicaloxidationbyWarburgWarburg,O.(1924)Biochem.Z.177,471–486.Iron-containingrespiratoryenzymes,oxidasesactivateoxygen.Usedinhibitors(e.g.,NCN,CO)andspectroscopytoindirectlydetectthebehaviorofO2-transferringfermentofrespiration

inlivingcells.Warburg(1883-1970)NobelPrize,1931Cyta?Cytc?600nm560nmHeme-likepigmentsfoundtoexistwidelyinanimals(1886)MacMunn,C.A.(1886)Researchesonmyohaematinandthehistohaematin,Phil.Trans.Roy.Soc.,177:267-298.Examinedorgansandtissuesofvertebratesandinvertebrates,usingmicrospectroscope.Discoveredthepresenceofthehistohaematinsandmyohaematin(havinglightabsorptioninreducedstate).

Cyta?Cytb?Cytc?Cytochromes(“cellularpigments)rediscoveredKeilin,D.(1925)Oncytochrome,arespiratorypigment,commontoanimals,yeast,andhigherplants,Proc.R.Soc.BBiol.Sci.98:312–339.Miscrospectroscopeshowed4absorptionbandswhenreduced,assumedtobemadeofthreetypesofhaemegroups(a,b,c).It’sacommonintracellularrespiratorycatalyst,thereductionandoxidationofwhichcanbespecificallyinhibited.DavidKeilin(1887-1963)AnoxidasewasdemonstratedtobearespiratorycatalystinyeastD.KEILIN(1927)InfluenceofCarbonMonoxideandLightonIndophenolOxidaseofYeastCells,Nature119:670-671.Arespiratorychainconceptwasproposed(1929)Keilin,D.(1929)Cytochromeandrespiratoryenzymes,Proc.Roy,Soc,B104:206-252.Theactivitiesofindophenoloxidaseishighlycorrelatedtotheoxidationofthecytochromes.Succinate(organicsubstrate)leadstothereductionofthecytochromeseffectively.Cytochromesactascarriersofhydrogenbetweenthedehydrase(i.e.,dehydrogenase)andtheoxidase(i.e.,cytochromecoxidase).Activationofhydrogenatoms(Wieland)ActivationofO2(Warburg))Cytochromes(Keilin)TherespiratorychainproposedbyKeilinwashighlyregardedbyfuturescientistsCouplingbetweenphosphorylationsandoxidationsobserved(1939)Lipmann,

F.(1939)CouplingbetweenPyruvicAcidDehydrogenationandAdenylicAcidPhosphorylation,Nature,143:281.NADHoxidationbyO2islinkedtophosphorylation(1948)Friedkin&Lehninger(1948)PhosphorylationcoupledtoelectrontransportbetweenDPNH2andO2

J.Biol.

Chem.174,757-758;178:611–23.Ratliverparticulate;32P-labeledPiastracer.Therespiratorychainwasseparatedintofourmembranecomplexes(1948)WAINIOetal.(1948)Thepreparationofasolublecytochromeoxidase.JBiolChem.173:145-52.Green,D.E.(1966)inComprehensiveBiochemistry(Florkin,M.,andStotz,E.H.,eds)Vol.14,pp.309–326,ElsevierSciencePublishersB.V.,Amsterdam.Usingdeoxycholateandcholatetodispersethemembraneandallowitscomponentstobeseparatedbyconventionalammoniumsulfatefractionation.CatalyzingthereductionofubiquinonebyNADH(ComplexI)orsuccinate(ComplexII),thereductionofferricytochromecbyubiquinol(ComplexIII),andtheoxidationofferrocytochromecbyoxygen(ComplexIV).Chemicalcouplinghypothesisonrespiratorychainphosphorylationproposed(1953)Slater,EC(1953)Mechanismofphosphorylationintherespiratorychain.Nature.172:975-8.Respirationiscompulsorilylinkedtophosphorylation.Inintactmitochondria,passageofeachpairofHatomsovertherespiratorychainiscoupledwiththeesterificationof1,2or3atomsofPi.UncouplingagentsLipmann’sscheme(1946)Slater’sscheme(1953)Photophosphorylationconceptproposed(1954)Arnon,D.I.,F.R.Whatley,andM.B.Allen.(1954)Photosynthesisbyisolatedchloroplasts.II.Photosyntheticphosphorylation,theconversionoflightintophosphatebondenergy.J.Am.Chem.Soc.76:6324-6328.Aquinonewasfoundtobeneededforrespiration(1957)Craneetal.(1957)."Isolationofaquinonefrombeefheartmitochondria".BiochimicaetBiophysicaActa

25:220–1.Iron-sulfurproteinsareinvolvedinrespiration(1960)

Beinert,H.,andSands,R.H.(1960)StudiesonsuccinicandDPNHdehydrogenasepreparationsbyparamagneticresonance(EPR)spectroscopyBiochem.Biophys.Res.Commun.3:41–46ATPsynthasewaspurifiedandcharaterized(1960-1973)Pullmanetal&RackerE.(1960-1973).PartialResolutionoftheEnzymesCatalyzingOxidativePhosphorylation.I-XXVJ.Biol.Chem.235:3322;241:2475;248:676.Achemi-osmoticmechanismwasproposedtoexplainthecouplingMitchell,P.(1961)."Couplingofphosphorylationtoelectronandhydrogentransferbyachemi-osmotictypeofmechanism".Nature191:144.Unexplainedfacts:“high-energyintermediates”yetidentified;membranestructureessential;uncouplingagentsdiffergreatlyinstructure."acid-bath"darkphosphorylationbychloroplastsobserved(1966)

JagendorfandUribe(1966)ATPformationcausedbyacid-basetransitionofspinachchloroplasts;PNAS,55:170–177.Withoutilluminationoroxygen;Madefirstacid,thenbasic.Reconstitutedlight-activatedprotonpumpcatalyzesATPformation(1974)RackerandStoeckenius(1974)Reconstitutionofpurplemembranevesiclescatalyzinglight-drivenprotonuptakeandATPformation;J.Biol.Chem.249:662–663.EnergyfromelectrontransportwasproposedtocausethereleaseofpreformedATPfromthecatalyticsites(1973)Boyeretal.(1973)Anewconceptforenergycouplinginoxidativephosphorylationbasedonamolecularexplanationoftheoxygenexchangereactions.PNAS,70:2837.(nocitationoftheMitchellpaper!)EffectofuncouplerS-13onexchangescatalyzedbymitochondriauncoupler-insensitiveuncoupler-sensitive32PlabelingofATPAprotonmotiveQcyclewashypothesized(1975)PeterMitchell(1975)Protonmotiveredoxmechanismofthecytochromeb-c1complexintherespiratorychain:Protonmotiveubiquinonecycle,FEBSLett.,56:1-6.ATPsynthasewasproposedtoexhibit