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1、Chapter 19 Oxidative PhosphorylationOxidation of NADH and FADH2 by O2, with energy released coupled to ATP synthesis. “Anyone who is not confused about oxidative phosphorylation just doesnt understand the situation” Efraim Racker, 1970sThemostefficientenergytransformationprocessfoundontheearth!“Thef
2、ormationanduseofATPistheprinciplenetchemicalreactionoccurringinthewholeworld!”PaulBoyerOxidative phosphorylation is the final stage of nutrient oxidationA1.14-voltpotentialdifference(E0)betweenNADHandO2driveselectronflowthroughtherespiratorychainwithaGoof220kJ/Mol.Therespiratorychain:thebattery(exer
3、gonic)Electricmotor(endergonic)1.Redoxreactions(electrontransferring)carriedoutbyaseriesofproteincomplexeswithincellmembranes;2.Protontransportacrosscellmembrane;3.ATPsynthesisusingtheenergystoredintheprotongradient.Photophosphorylationinchloroplastishighlycomparabletooxidativephosphorylationinmitoc
4、hondria!e-e-Battery(exergonic)Electricmotor(endergonic)Historical events in unveiling molecular mechanism of oxidative phosphorylation (1)Vital role of Pi and coferment in yeast fermentation (Arthur Harden, 1906)Central role of ATP in energy transformation (Fritz Lipmann, 1941)Link between sugar oxi
5、dation and ATP generation (Herman Kalckar, 1940s)Coenzyme NADH linked metabolic pathways to mitochondrial synthesis of ATP (Lehninger, 1949)HardenLipmannKalckarHistorical events in unveiling molecular mechanism of oxidative phosphorylation (2)Electrochemical concentration gradient of protons across
6、a membrane proposed to be harnessed to make ATP (chemi-osmotic theory, Peter Mitchell, 1961)Electron transport protein complexes and ATP synthase isolated (David Green and Efraim Racker, 1960s)Historical events in unveiling molecular mechanism of oxidative phosphorylation (3)Binding change mechanism
7、 (1973) and rotational catalysis (1982) proposed for ATP synthase (Boyer)Structure of ATP synthase partially determined (1994, Walker)Rotation of the ATP synthase molecule observed in vitro (Masasuki Yoshida, 1997).YoshidaThe mitochondrial electron-transfer chain was separated into four major protei
8、n complexesElectrons of NADH and FADH2 are transferred to O2 via many intermediate electron carriers making up the respiratory chain.NADHdehydrogenase(complexI):34-46subunits,1000kDNature,2010,466:441-447.Science, 2006, 311:1430-1436InvolvingFMNandaseriesofiron-sulfurcentersaselectroncarriers,withub
9、iquinonetakingawaytheelectronsattheiron-sulfurproteinN-2.Hydrophilic domainFe-SAtleasteightdifferenttypesofiron-sulfurcenters(firstrevealedbyHelmutBeinert)actintherespiratorychain(incomplexI,IIandIII):ironatomscyclebetweenFe2+(reduced)andFe3+(oxidized).2Fe-2S4Fe-4SInorganicsulfurUbiquinone(orcoenzym
10、eQ10)istheonlye-carrierthatisnotboundtoaproteinandisabletodiffusefreelyinthelipidbilayer.(or dihydroubiquinone)Isoprenoid tailCompletereductionofQrequirestwoelectronsandtwoprotons,occurringintwostepsviaasemiquinoneradicalIntermediate. FredrickCrane,1957FADH2ofotherflavoproteinsalsotransfertheirelect
11、ronstoubiquinone(Q)viaaFe-S,butwithnoH+pumped.SuccinatedehydrogenaseSuccinate dehydrogenase (complex II) transfers electrons from succinate to ubiquinoneComplexII(ofthecitricacidcycle)InvolvingFADandmultipleFe-Scentersaselectroncarrier.ThehemegroupisbelievedtohelpdecreaseproductionofROS.Cytochromebc
12、1complex(ComplexIII)transferselectronsfromQH2tocytochromec(Cytc)CytcCoenzymeQ:cytochromec oxidoreductase(Encodedbymitochondrialgenome)(JohnRieske,1964)Cytochromeswerediscoveredasheme-containingrespiratorypigments(MacMunn,1884,DavidKeilin,1925)KeilinThreetypesofhemegroupsserveasprostheticgroupsofthec
13、ytochromeproteinsExistinComplexIIIExistincytochromecandcomplexIVTheironinterconvertsbetweenitsreduced(Fe2+)andoxidized(Fe3+)formsincytochromes.CytochromereductioncanbedetectedbylightabsorptionatuniquevisiblewavelengthsThereduced(Fe2+)stateofcytochromesa,b,and chasuniquelightabsorptionnear600,560,and
14、550nmrespectively.Thisallowscytochromes(hemoproteins)actiontobedetecteddirectlyinmitochondriaviaspectroscopy.c c b ba aCytochromesareclassifiedonthebasisofpositionoftheirlowestenergyabsorptionbandinthereducedstate.e-transferring&H+pumpinginComplexIIIproposedtooccurviatheQcycletheQcycleFrom:http:/en.
15、wikipedia.org/wiki/Image:Theqcycle.gifCytochromec oxidase(ComplexIV)transferselectronsfromCytctoO2.Hemeaandhemea3 hasidenticalstructuresbutdifferentreductionpotential.4Fe2+-cytochromec+8H+in+O24Fe3+-cytochromec+2H2O+4H+outCyanide,sulfide,azide,andCOallinhibitcytochromecoxidase!Complexes III and IV m
16、ight form supercomplexes (respirasome) on the membraneElectrons flow from NADH (or succinate) to O2 is coupled to transmembrane proton pumping10protonsperNADHand6protonsperFADH2oxidizedarepumpedacrosstheinnermembraneandtheelectronmotiveforceisconvertedtoanprotonmotiveforce.(Estimatedbytitrationsupon
17、thepresenceofspecificinhibitors)Reactive oxygen species (ROS) might be produced from the respiratory chainThe chemiosmotic model was proposed by Peter Mitchell in 1961 to explain the coupling of electron flow and ATP synthesis (Chemical reactions coupled to osmotic (Chemical reactions coupled to osm
18、otic gradients.)gradients.)Facts not yet explained:Facts not yet explained:？“Hight-energyHight-energy”intermediates elusive to identification;intermediates elusive to identification; ？ Phosphorylation closely associated with membrane; Phosphorylation closely associated with membrane; ？ Uncoupling ca
19、used by agents of various structures; Uncoupling caused by agents of various structures; ？ Swelling and shrinkage phenomena. Swelling and shrinkage phenomena.Mitchell, P. 1961, Nature, 191:144-148.A “paradigm” shift in understanding bioenergetics!Vectorial metabolism vsScalar metabolismThe chemiosmo
20、tic theory of Mitchell:e- flow and ATP synthesis are separate events, coupled via a transmembrane H+ gradient!The chemiosmotic theory unified the apparently disparate energy transduction processes as oxidative phosphorylation, photophosphorylation, active transport across membrane and the motion of
21、bacterial flagella.ATP synthase (EC 3.6.3.14) was first identified by dissociation and reconstitution studies (Efraim Racker, 1960s)Knob-likestructureswereseenwithinside-outvesicleofmitochondria,thylakoidmembraneofchloroplastsandinside-outE. coliplasmamembrane.ThemitochondrialATPaseistheATPsynthase!
22、Mitochondria-catalyzed 18O, 32P exchange data suggests that oxidative phosphorylation is dynamically revesible (Mildred Cohn, 1953)Covalentintermediateswasproposedtobeformed.Latersuchaphosphorylatedproteinwasdetected,butlaterfoundtobesuccincylCoAsynthetase(byPaulBoyer;“We were reaching for a gold bu
23、t got a bronze instead”)TheenergyfromelectrontransportwasproposedtobeneededforreleasingpreformedATPfromATPsynthase, not needed for the formation of ATP (Boyer et al., 1973, PNAS, 70:2837-9. PiHOHexchangeisconsiderablylesssensitivetouncouplersthanthePiATPandATPHOHexchanges.Theuncoupler-insensitivePiH
24、OHexchangeisinhibitedbyoligomycin.Similarexchangewasalsoobservedformyosin.Thisreleasecouldlogicallyinvolveenergy-requiringproteincon-formationalchange.(Not a single word was mentioned about the chemiosmotic hypothesis!)Catalytic cooperativity revealed: removal of ADP stopped ATP HOH exchange and rem
25、oval of ATP stopped Pi HOH exchange (Boyer, 1975)At lower ATP level, more O in Pi is exchanged with HOHSubunit composition of ATP synthase characterized, for the the ATPase (F1) and proton channel (Fo) Stationary unitStationary unit(stator)(stator)F1: Fo: ab2c10-14The rotorThe rotor: :the the c- c-r
26、ing and ring and the the stalk; stalk;the statorthe stator: :the remainder.the remainder.Proton-conductingProton-conductingCatalyticCatalyticA rotational binding change (or flip-flop) mechanism was proposed to explain the catalysis of ATP synthase (Paul Boyer, 1980) 1. The subunitfoundtointeractwith
27、thecatalytic subunits;2.Distributionof18OinPi(with1,2or3Oexchanged)formedfrom18O-ATPsuggestedidenticalbehaviorofallcatalyticsites.3.Mildcross-linkingstoppedcatalysisandcleavageofthecross-linkerrestoredactivityBinding Binding ADP + PADP + Pi iSynthesizing Synthesizing ATPATPReleasing Releasing ATPATP
28、Thebinding-changeMechanismorrotationalcatalysis(PaulBoyer,1980s)Each Each subunit subunitwill take threewill take threedifferent conformationsdifferent conformationsin turn during each in turn during each cycle of action.cycle of action.http:/en.wikipedia.org/wiki/Image:ATPsyn.gifThebinding-changeme
29、chanismofATPsynthase:The binding-change model was elegantly supported by X-ray crystallography analysis of F1-ATPase.Thethreecatalytic subunitsdifferinconformationandthenucleotidebound;Thethreecatalyticsubunitsareindifferentstatesofthecatalyticcycleatanyinstant;Interconversionofthestatemaybeachieved
30、byrotationofthe subunitinthecenter.AMP-PNPAMP-PNPAMP-PNPAMP-PNPADPNature,1994,370:621-628.Rotationofc-ringorc-ring/ subunitdirectlyobservedusingfluorescencemicroscopy(1997)MechanicallydrivenATPsynthesisbyF1-ATPasedemonstrated(2004).ModeloftheactionofE. coliATPsynthase:theprotongradientdrivestherotat
31、ionofthecringusingtwohalf-channelsontheasubunit.Protonation/deprotonation of an Asp is believed to be essential for rotating the c ring and the subunit.10-14 protons needed for every3 ATP synthesized.Asp-COO- Asp-COOHThus4protonsperATPsynthesizedhttp:/www.mrc-dunn.cam.ac.uk/research/atp_synthase/ Th
32、e energy stored in the proton gradient can be used to do other work.Therotarymotionofthebacterialflagellaisenergizeddirectlybytheprotongradientpresentacrossthecytoplasmicmembrane.Theproton-motiveforceisusedforactivetransportthroughtheinnermembraneofthemitochondria.HeatisgeneratedinBrownfatthroughthe
33、actionofthermogenin,anuncouplingprotein:toproduceheattomaintainbodytemperatureforanimalsinhibernation,ofnewlybornandadaptingtothecold(thermogenesis).FattyacidsseemtoactivateandnucleotidesinhibitUCP1UCP1Electrons in NADH generated in cytosol are shuttled onto the respiratory chain. The malate-asparta
34、te shuttle system: Malate translocates electrons produced during glycolysis across inner membrane of mitochondrion for oxidative phosphorylationsemipermeableIrreversibleIrreversibleTheglycerol-3-phosphateshuttlesystemThe pathways leading to ATP synthesis are coordinately regulated.Interlockingregula
35、tionofallthesepathwaysmightberealizedbytherelativelevelsofATP,NADH,ADP,AMP,Pi,andNAD+.ATP/(ADPPi) fluctuates only slightly in most tissues due to a coordinated regulation of all the pathways leading to ATP production.The rate of the respiration assumed to be controlled by the availability of ADP (“a
36、cceptor control”)No ATP consumption, no electron flow!PyruvateoxidationSomerespiratoryproteinsareencodedbythehumanmitochondrialgenomeComplexesI,III,andIVandATPsynthaseareassembledbyusingsubunitsmadeinboththecytosolandmitochondria.How was the molecular mechanism of oxidative phosphoryation revealed?K
37、alckarHM(1974).Originsoftheconceptoxidativephosphorylation.Mol. Cell. Biochem.5(12):55-62.1.Glucosedegradation(fermentation,Glycolysis)2.Biologicaloxidation(O2consumption,citricacidcycle)3.Energysupplyofmusclecontraction4.UnderstandingtheroleofATPastheuniversalenergycurrency5.Subcellularlocationandp
38、roteinisolationandcharacterization.The activation of hydrogen atoms was thought to be the key for biological oxidation (Thunberg and Wieland, 1910s)Thunberg, T. (1917) Skand. Arch. Physiol. 35, 163Wieland, O. (1912) Ber. Dtsch. Chem. Ges. 45, 484499; 26062615The oxidation of a large number of organi
39、c compounds (e.g., succinic acid) can be catalyzed by specific dehydrogenases in the presence of artificial hydrogen acceptors (e.g., methylene blue).The activation of O2 was thought to be the key for biological oxidation by WarburgWarburg, O. (1924) Biochem. Z. 177, 471486.Iron-containing respirato
40、ry enzymes, oxidases activate oxygen.Used inhibitors (e.g., NCN, CO) and spectroscopy to indirectly detect the behavior of O2-transferring ferment of respirationin living cells.Warburg(1883-1970)NobelPrize,1931Cyta?Cytc?600nm560nmHeme-like pigments found to exist widely in animals (1886)MacMunn, C.
41、A. (1886) Researches on myohaematin and the histohaematin, Phil. Trans. Roy. Soc. , 177:267-298. Examined organs and tissues of vertebrates and invertebrates, using microspectroscope. Discovered the presence of the histohaematins and myohaematin (having light absorption in reduced state). Cyta?Cytb?
42、Cytc?Cytochromes (“cellular pigments) rediscoveredKeilin, D. (1925) On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants, Proc. R. Soc. B Biol. Sci. 98:312339. Miscrospectroscope showed 4 absorption bands when reduced, assumed to be made of three types of haeme groups (a
43、, b, c).Its a common intracellular respiratory catalyst, the reduction and oxidation of which can be specifically inhibited.DavidKeilin(1887-1963)An oxidase was demonstrated to be a respiratory catalyst in yeastD. KEILIN (1927) Influence of Carbon Monoxide and Light on Indophenol Oxidase of Yeast Ce
44、lls, Nature 119:670-671. A respiratory chain concept was proposed (1929)Keilin, D. (1929) Cytochrome and respiratory enzymes, Proc. Roy, Soc, B104:206-252.Theactivitiesofindophenoloxidaseishighlycorrelatedtotheoxidationofthecytochromes.Succinate(organicsubstrate)leadstothereductionofthecytochromesef
45、fectively.Cytochromesactascarriersofhydrogenbetweenthedehydrase(i.e.,dehydrogenase)andtheoxidase(i.e.,cytochromecoxidase).Activationofhydrogenatoms(Wieland)ActivationofO2(Warburg)Cytochromes(Keilin)The respiratory chain proposed by Keilin was highly regarded by future scientistsCoupling between phos
46、phorylations and oxidations observed (1939)Lipmann,F. (1939) Coupling between Pyruvic Acid Dehydrogenation and Adenylic Acid Phosphorylation, Nature, 143: 281. NADH oxidation by O2 is linked to phosphorylation (1948)Friedkin & Lehninger (1948) Phosphorylation coupled to electron transport between DP
47、NH2 and O2 J. Biol. Chem. 174, 757-758; 178: 61123. Rat liver particulate; 32P-labeled Pi as tracer. The respiratory chain was separated into four membrane complexes (1948)WAINIO et al. (1948) The preparation of a soluble cytochrome oxidase. J Biol Chem. 173:145-52. Green, D. E. (1966) in Comprehens
48、ive Biochemistry (Florkin, M., and Stotz, E. H., eds) Vol. 14, pp. 309326, Elsevier Science Publishers B.V., Amsterdam.Using deoxycholate and cholate to disperse the membrane and allow its components to be separated by conventional ammonium sulfate fractionation.Catalyzing the reduction of ubiquinon
49、e by NADH (Complex I) or succinate (Complex II), the reduction of ferricytochrome c by ubiquinol (Complex III), and the oxidation of ferrocytochrome c by oxygen (Complex IV).Chemical coupling hypothesis on respiratory chain phosphorylation proposed (1953)Slater, EC (1953) Mechanism of phosphorylatio
50、n in the respiratory chain. Nature. 172:975-8. Respiration is compulsorily linked to phosphorylation.In intact mitochondria, passage of each pair of H atoms over the respiratory chain is coupled with the esterification of 1, 2 or 3 atoms of Pi.UncouplingagentsLipmannsscheme(1946)Slatersscheme(1953)P
51、hotophosphorylation concept proposed (1954)Arnon, D. I., F. R. Whatley, and M. B. Allen. (1954) Photosynthesis by isolated chloroplasts. II. Photosynthetic phosphorylation, the conversion of light into phosphate bond energy. J. Am. Chem. Soc. 76: 6324-6328. A quinone was found to be needed for respi
52、ration (1957)Crane et al. (1957). Isolation of a quinone from beef heart mitochondria. Biochimica et Biophysica Acta 25: 2201. Iron-sulfur proteins are involved in respiration (1960) Beinert, H., and Sands, R. H. (1960) Studies on succinic and DPNH dehydrogenase preparations by paramagnetic resonanc
53、e (EPR) spectroscopy Biochem. Biophys. Res. Commun. 3:4146ATP synthase was purified and charaterized (1960-1973)Pullman et al & Racker E. (1960-1973). Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation. I -XXV J. Biol. Chem. 235: 3322; 241:2475; 248:676. A chemi-osmotic mechanism
54、 was proposed to explain the couplingMitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144.Unexplained facts: “high-energy intermediates” yet identified; membrane structure essential; uncoupling agents differ greatly i
55、n structure. acid-bath dark phosphorylation by chloroplasts observed (1966)Jagendorf and Uribe (1966) ATP formation caused by acid-base transition of spinach chloroplasts; PNAS, 55:170177.Without illumination or oxygen;Made first acid, then basic.Reconstituted light-activated proton pump catalyzes A
56、TP formation (1974)Racker and Stoeckenius (1974) Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and ATP formation; J. Biol. Chem. 249:662663.Energy from electron transport was proposed to cause the release of preformed ATP from the catalytic sites (1973)Boyer et al.
57、 (1973) A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions. PNAS, 70:2837. (no citation of the Mitchell paper!)EffectofuncouplerS-13onexchangescatalyzedbymitochondriauncoupler-insensitiveuncoupler-sensitive32PlabelingofATP
58、A protonmotive Q cycle was hypothesized (1975)Peter Mitchell (1975) Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: Protonmotive ubiquinone cycle, FEBS Lett., 56:1-6. ATP synthase was proposed to exhibit a radical rotational catalysis (1982)Gresser, et al & Boye
59、r PD (1982). Catalytic site cooperativity of beef heart mitochondrial F1-ATPase. Correlations of initial velocity, bound intermediate, and oxygen exchange measurements with an alternating three-site model J. Biol. Chem. 257: 120308. Eadie-Hofsteeplotofv0vsATPBoundADPduringhydrolysisatlowATPCorrelati
60、onofexperimentaldatawithpredictionsfromanalternatingthree-sitemodel.The three -subunits of F1-ATPase differ in conformation (1994)Abrahams et al & Walker JE (1994) Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-8 ADPandAMP-PNParepresentinthecrystallization
61、medium;Piisabsent.The -subunitsinteractwiththe -subunitsinahighlyasymmetricmanner.The 3 3 was observed to rotate in vitro (1997)Noji et al. and Kinosita K Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature. 386:299-302.Attached a fluorescent actin filament to the -subunit to observe
62、motion directly. In presence of ATP, the filament rotated for more than 100 revolutions. SummaryElectronscollectedinNADHandFADH2arereleased(atdifferententeringpoints)andtransportedtoO2viatherespiratorychain,whichconsistsoffourmultiproteincomplexes(I,II,III,andIV)andtwomobileelectroncarriers(ubiquino
63、neandcytochromec).Aprotongradientacrosstheinnermembraneofmitochondriaisgeneratedusingtheelectronmotiveforcegeneratedbyelectrontransferringthroughtherespiratorychain.ThechemiosmotictheoryexplainsthecouplingofelectronflowandATPsynthesis.ATPsynthasecomprisesaprotonchannel(Fo)andaATPase(F1).Thebinding-changemodelwasproposedtoexplaintheactionmechanismofATPsynthase.Theenergystoredintheprotongradientcanbeusedtodootherwork.ElectronsinNADHgeneratedincytosolisshuttledintomitochondriatoentertherespiratorychain.ThepathwaysleadingtoATPsynthesisiscoordinatelyregulated.Summary
