19|OxidativePhosphorylationandPhotophosphorylation

2013 W. H. Freeman and Company

CHAPTER19OxidativePhosphorylationand

Photophosphorylation

Electrontransportchaininmitochondria Captureoflightenergyinphotosynthesis Buildinguptheprotonmotiveforce SynthesisofATPinmitochondriaandchloroplasts

Keytopics:

EnergyfromreducedfuelsisusedtosynthesizeATPinanimals

Carbohydrates,lipids,andaminoacidsarethemainreducedfuels forthecell

ElectronsfromreducedfuelsaretransferredtoreducedcofactorsNADH orFADH2

Inoxidativephosphorylation,energyfromNADH andFADH2 areusedtomakeATP

EnergyFlowinCellularRespiration

OxidativePhosphorylation

ElectronsfromthereducedcofactorsNADH andFADH2arepassedtoproteinsintherespiratorychain

Ineukaryotes,oxygen istheultimateelectronacceptorfortheseelectrons

EnergyofoxidationisusedtophosphorylateADP

OxidativePhosphorylation

Photophosphorylation Inphotosyntheticorganismslight causeschargeseparationbetweenapairchlorophyllmolecules

Energyoftheoxidizedandreducedchlorophyllmolecules isusedtodrivesynthesisofATP

Wateristhesourceofelectrons thatarepassedviaachainofproteintransporterstotheultimateelectronacceptor,NADP+

Oxygenisthebyproductofwateroxidation

Photophosphorylation

ChemiosmoticTheory

ADP+Pi ATP isHighlyThermodynamicallyUnfavorable Howdowemakeitpossible? PhosphorylationofADPisnotaresultofadirectreactionbetweenADPandsomehighenergyphosphatecarrier

EnergyneededtophosphorylateADPisprovidedbytheflowofprotonsdowntheelectrochemicalgradient

Theenergyreleasedbyelectrontransportisusedtotransportprotonsagainsttheelectrochemicalgradient

Chemiosmoticenergycouplingrequiresmembranes

TheprotongradientneededforATPsynthesiscanbestablyestablishedacrossamembranethatisimpermeabletoions Plasmamembraneinbacteria Innermembraneinmitochondria Thylakoidmembraneinchloroplasts

Membranemustcontainproteinsthatcouplethedownhillflowofelectrons intheelectrontransferchainwiththeuphillflowofprotonsacrossthemembrane

Membranemustcontainaproteinthatcouplesthedownhillflowofprotons tothephosphorylationofADP

ChemiosmoticTheory

StructureofaMitochondrionDoublemembraneleadstofourdistinctcompartments:1.OuterMembrane:

Relativelyporousmembraneallowspassageofmetabolites

2.IntermembraneSpace(IMS): similarenvironmenttocytosol higherprotonconcentration(lowerpH)

3.InnerMembrane Relativelyimpermeable,withprotongradientacrossit Locationofelectrontransportchaincomplexes ConvolutionscalledCristaeservetoincreasethesurfacearea

4.Matrix Locationofthecitricacidcycleandpartsoflipidandaminoacidmetabolism

Lowerprotonconcentration(higherpH)

StructureofaMitochondrion

Electrontransportchaincomplexescontainaseriesofelectroncarriers

Eachcomplexcontainsmultipleredoxcentersconsistingof: FlavinMononucleotide(FMN)orFlavinAdenineDinucleotide(FAD) InitialelectronacceptorsforComplexIandComplexII Cancarrytwoelectronsbytransferringoneatatime

Cytochromesa,borc

Ironsulfurcluster

Cytochromes Oneelectroncarriers Ironcoordinatingporphoryin ringderivatives a,borcdifferbyringadditions

IronSulfurClusters Oneelectroncarriers Coordinatingbycysteines intheprotein Containingequalnumberofironandsulfuratoms

CoenzymeQorUbiquinone

Ubiquinone isalipidsolubleconjugateddicarbonylcompoundthatreadilyacceptselectrons

Uponacceptingtwoelectrons,itpicksuptwoprotonstogiveanalcohol,ubiquinol

Ubiquinolcanfreelydiffuseinthemembrane,carryingelectrons withprotonsfromonesideofthemembranetoanotherside

CoenzymeQisamobileelectroncarriertransportingelectronsfromComplexesIandIItoComplexIII

CoenzymeQorUbiquinone

FreeEnergyofElectronTransport

ReductionPotential(E)Eo=Eo(e acceptor) Eo(e donor)

Go=nFEoFornegativeGneedpositiveE

E(acceptor) >E(donor)

Electronsaretransferredfromlower(morenegative)tohigher(morepositive)reductionpotential.

FreeEnergyreleasedisusedtopumpproton,storingthisenergyastheelectrochemicalgradient

FlowofElectronsfromBiologicalFuelsintotheElectronTransportChain

NADH:ubiquinoneoxidoreductase,a.k.a.ComplexI

Oneofthelargestmacromolecularassembliesinthemammaliancell

Over40differentpolypeptidechains,encodedbybothnuclearandmitochondrialgenes

NADHbindingsiteinthematrixside Noncovalentlyboundflavinmononucleotide(FMN)acceptstwoelectronsfromNADH

Severalironsulfurcenterspassoneelectronatatimetoward the ubiquinonebindingsite

ComplexI

NADH:Ubiquinoneoxidoreducaseisaprotonpump

TransferoftwoelectronsfromNADHtoubiquinoneisaccompaniedbyatransferofprotonsfromthematrix(N)totheintermembranespace(P)

Experimentssuggestthataboutfourprotons aretransportedperoneNADH

NADH+Q+5H+N =NAD+ +QH2 +4H+P

ReducedcoenzymeQpicksuptwoprotons Protonsaretransportedbyprotonwires

Aseriesofaminoacidsthatundergoprotonationanddeprotonationtogetanettransferofaprotonfromonesideofamembranetoanother

SuccinateDehydrogenase,a.k.a.ComplexII

FADacceptstwoelectronsfromsuccinate Electronsarepassed,oneatatime,viaironsulfurcenterstoubiquinone,whichbecomesreducedQH2

Doesnottransportprotons

ComplexII

Ubiquinone:Cytochromec Oxidoreductase,a.k.a.ComplexIII

UsestwoelectronsfromQH2 toreducetwomoleculesofcytochromec

Additionallycontainsironsulfurclusters,cytochromebs,andcytochromecs

TheQcycleresultsinfouradditionalprotonsbeingtransportedtotheIMS

ComplexIII

TheQCycle

Experimentally,fourprotons aretransportedacrossthemembranepertwoelectronsthatreachCytC

Twoofthefourprotons comefromQH2 TheQcycleprovidesagoodmodelthatexplainshowtwoadditionalprotonsarepickedupfromthematrix

Two moleculesofQH2 becomeoxidized,releasingprotonsintotheIMS

Onemoleculebecomesrereduced, thusaNettransferoffourprotonsperreducedCoenzymeQ

TheQCycle

TheQCycle:Cycle1

TheQCycle:Cycle2

Thesecondmobileelectroncarrier Asolublehemecontainingprotein in

theintermembranespace Hemeironcanbeeitherferrous

(Fe3+,oxidized)orferric(Fe2+,reduced)

Cytochromec carriesasingleelectronfromthecytochromebc1complextocytochromeoxidase

Cytochromec

Cytochromec absorbsvisiblelight

IntenseSoretbandnear400nmabsorbsbluelightandgivescytochromec anintenseredcolor

Cytochromesarenamedbythepositionoftheirlongestwavelength()peak

CytochromeOxidase,a.k.a.ComplexIV

Mammaliancytochromeoxidaseisamembraneproteinwith13subunits

Containstwohemegroups:aanda3 Containscopperions

CuA:twoionsthatacceptelectronsfromCytc CuB:bondedtohemea3 formingabinuclearcenterthattransfersfourelectronstooxygen

ComplexIV

CytochromeoxidasepasseselectronstoO2

Fourelectronsareusedtoreduce oneoxygenmoleculeintotwowatermolecules

Fourprotonsarepickedupfromthematrix inthisprocess Fouradditionalprotonsarepassedfromthematrixtotheintermembranespace

ElectronflowthroughComplexIV

Multiplecomplexesassociatetogethertoformarespirasome

SummaryoftheElectronFlowintheRespiratoryChain

SummaryofElectronTransport

ComplexI ComplexIV1NADH +11H+(N) +O2 >NAD+ +10H+(P) +H2O

ComplexII ComplexIVFADH2 +6H+(N) +O2 >FAD+6H+(P) +H2O

DifferenceinnumberofprotonstransportedreflectsdifferencesinATPsynthesized.

Reactiveoxygenspeciescandamagebiologicalmacromolecules

ProtonMotiveForce

Theproteinsintheelectrontransportchaincreatedtheelectrochemicalprotongradient byoneofthreemeans: activelytransportprotonsacrossthemembrane

ComplexIandComplexIV

Chemicallyremoveprotonsfromthematrix ReductionofCoQandreductionofoxygen

Releaseprotonsintotheintermembranespace OxidationofQH2

ProtonMotiveForce

ChemiosmoticModelforATPSynthesis Electrontransportsetsupaprotonmotiveforce EnergyofprotonmotiveforcedrivessynthesisofATP

Consequently,electrontransportiscoupledtoATPsynthesis

Asdescribed,ATPsynthesisrequireselectrontransportButelectrontransportalsorequiresATPsynthesis

MitochondrialATPSynthaseComplex

Containstwofunctionalunits: F1

Solublecomplexinthematrix IndividuallycatalyzesthehydrolysisofATP

F0 Integralmembranecomplex TransportsprotonsfromIMStomatrix,dissipatingtheprotongradient

EnergytransferredtoF1 tocatalyzephosphorylationofADP

MitochondrialATPSynthaseComplex

TheF1 catalyzesADP+Pi ATP Hexamerarrangedinthree dimers Dimerscanexistinthreedifferentconformations:

Open:empty Loose:bindingADPandPi Tight:catalyzesATPformationandbindsproduct

BindingChangeModel

CouplingProtonTranslocationtoATPSynthesis

ProtontranslocationcausesarotationoftheF0 subunitandthecentralshaft

Thiscausesaconformationalchange withinallthethree pairs

TheconformationalchangeinoneofthethreepairspromotescondensationofADPandPi intoATP

EvidenceofRotation

TransportofADPandPi intotheMatrix

MalateAspartateShuttle

Glycerol3PhosphateShuttle

RegulationofOxidativePhosphorylation

Primarilyregulatedbysubstrateavailability NADHandADP/Pi DuetocouplingbothsubstratesrequiredforelectrontransportandATPsynthesis

InhibitorofF1 (IF1) PreventshydrolysisofATPduringlowoxygen OnlyactiveatlowerpH,encounteredwhenelectrontransportitstalled(i.e.,lowoxygen)

InhibitionofOxPhosleadstoaccumulationofNADH CausesfeedbackinhibitioncascadeuptoPFK1inglycoysis

RegulationofOxidativePhosphorylation

LightEnergyisConvertedtoATPinPlantChloroplasts

VariousPigmentsHarvesttheLightEnergy

Photopigmentsabsorbdifferentwavelengthsoflight

Theenergyistransferredtothephotosyntheticreactioncenter

OrganizationofLightAbsorbingMoleculesinChloroplasts

FromLightEnergytoChargeSeparation

Chloroplastscouplethetwotypesofphotosystemsfoundinbacteria

PhotosystemIIevolvesoxygen

WaterSplitting

PhotosystemIresultsinreducedNADPH

StructureofPhotosystemI

Cytochromeb6f ComplexlinksPSIIandIandtranslocatesprotonsintothelumen

Organizationofphosyntheicmachineryinthethylakoidmembrane

Locationofphosyntheicmachineryinthethylakoidmembrane

Lightinducedredoxreactionscauseacidificationoflumen

TheprotonmotiveforceacrossthethylakoidmembranedrivesthesynthesisofATP.

FlowofProtons:Mitochondria,Chloroplasts,Bacteria

Accordingtoendosymbiotictheory, mitochondriaandchloroplastsarosefromentrappedbacteria

Bacterialcytosol becamemitochondrialmatrix andchloroplaststroma

Chapter19:Summary

Thereducedcofactorspasselectronsintotheelectrontransportchaininmitochondria

Theenergyofsunlightcreateschargeseparationinthephotosyntheticreactioncomplex

Stepwiseelectrontransportisaccompaniedbythedirectionaltransportofprotonsacrossthemembraneagainsttheirconcentrationgradient

TheenergyintheelectrochemicalprotongradientdrivessynthesisofATPbycouplingtheflowofprotonsviaATPsynthasetoconformationalchangesthatfavorformationofATPintheactivesite

Inthischapter,welearned: