生物化學(xué)：chapter 19 Oxidative Phosphorylation

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1、Chapter 19 Oxidative PhosphorylationOxidation of NADH and FADHOxidation of NADH and FADH2 2 by O by O2 2, , with energy released coupled to ATP with energy released coupled to ATP synthesis.synthesis. “Anyone who is not confused about oxidative phosphorylation just doesnt understand the situation” E

2、fraim Racker, 1970s The most efficient energy transformation process found on the earth!“The formation and use of ATP is the principle net chemical reaction occurring in the whole world!”P(pán)aul BoyerOxidative phosphorylation is the final stage of nutrient oxidationA 1.14-volt potential difference (E0)

3、 between NADH and O2 drives electron flow through the respiratory chain with a Go of 220 kJ/Mol.The respiratory chain: the battery (exergonic)Electric motor(endergonic)1. Redox reactions (electron transferring) carried out by a series of protein complexes within cell membranes;2. Proton transport ac

4、ross cell membrane;3. ATP synthesis using the energy stored in the proton gradient.Photophosphorylation in chloroplast is highly comparable to oxidativephosphorylation in mitochondria!e-e-Battery (exergonic)Electric motor(endergonic)Historical events in unveiling Historical events in unveiling molec

5、ular mechanism of oxidative molecular mechanism of oxidative phosphorylation (1)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 oxidation and ATP generation (Herman Ka

6、lckar, 1940s) Coenzyme NADH linked metabolic pathways to mitochondrial synthesis of ATP (Lehninger, 1949)HardenLipmannKalckarHistorical events in unveiling Historical events in unveiling molecular mechanism of molecular mechanism of oxidative phosphorylation (2)oxidative phosphorylation (2) Electroc

7、hemical concentration gradient of protons across 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 Historical events in unvei

8、ling molecular mechanism of molecular mechanism of oxidative phosphorylation (3)oxidative phosphorylation (3) Binding change mechanism (1973) and rotational catalysis (1982) proposed for ATP synthase (Boyer) Structure of ATP synthase partially determined (1994, Walker) Rotation of the ATP synthase m

9、olecule observed in vitro (Masasuki Yoshida, 1997).Masasuki Yoshida, 1997).YoshidaThe mitochondrial electron-transfer chain was separated into four major protein complexesElectrons of NADH and FADH2 are transferred to O2 via many intermediate electron carriers making up the respiratory chain.NADH de

10、hydrogenase (complex I): 34-46 subunits, 1000 kDNature, 2010, 466:441-447.Science, 2006, 311:1430-1436Involving FMN and a seriesof iron-sulfur centers as electron carriers, with ubiquinone taking away the electrons at the iron-sulfurprotein N-2.Hydrophilic domainFe-SAt least eight different types of

11、 iron-sulfur centers (first revealed by Helmut Beinert) act in the respiratory chain (in complex I, II and III): iron atoms cycle between Fe2+ (reduced) and Fe3+ (oxidized).2Fe-2S4Fe-4SInorganic sulfurUbiquinone (or coenzyme Q10)is the only e- carrier that is not bound to a protein and is able to di

12、ffuse freely in the lipid bilayer .(or dihydroubiquinone) Isoprenoid tailComplete reduction of Q requires two electrons and two protons, occurring in two steps via a semiquinone radical Intermediate. Fredrick Crane, 1957 FADH2 of other flavoproteins also transfer their electrons to ubiquinone (Q) vi

13、a a Fe-S, but with no H+ pumped.SuccinatedehydrogenaseSuccinate dehydrogenase (complex II) transfers electrons from succinate to ubiquinoneComplex II (of the citric acid cycle)Involving FAD andmultiple Fe-S centersas electron carrier.The heme group is believed to help decrease production of ROS.Cyto

14、chrome bc1 complex (Complex III) transfers electrons from QH2 to cytochrome c (Cyt c)Cyt c Coenzyme Q : cytochrome c oxidoreductase (Encoded by mitochondrial genome) (John Rieske, 1964) Cytochromes were discovered as heme-containing respiratory pigments(MacMunn, 1884, David Keilin, 1925)KeilinThree

15、types of heme groups serve as prosthetic groups of the cytochrome proteinsExist in Complex IIIExist in cytochrome cand complex IVThe iron interconverts between its reduced (Fe2+) and oxidized (Fe3+) forms in cytochromes.Cytochrome reduction can be detected by light absorption at unique visible wavel

16、engthsThe reduced (Fe2+) state of cytochromes a, b, and c has unique light absorption near 600, 560, and 550 nm respectively.This allows cytochromes (hemoproteins)action to be detecteddirectly in mitochondria via spectroscopy.c c b ba aCytochromes are classified on the basis of position of their low

17、est energy absorption band in the reduced state. e- transferring & H+ pumping in Complex III proposed to occur via the Q cycle the Q cycleFrom: http:/en.wikipedia.org/wiki/Image:Theqcycle.gifCytochrome c oxidase (Complex IV ) transfers electrons from Cyt c to O2.Heme a and heme a3 has identical stru

18、ctures but different reduction potential.4 Fe2+-cytochrome c + 8 H+in + O2 4 Fe3+-cytochrome c + 2 H2O + 4 H+outCyanide, sulfide, azide, and CO all inhibit cytochrome c oxidase! Complexes III and IV might form supercomplexes (respirasome) on the membraneElectrons flow from NADH (or succinate) to O2

19、is coupled to transmembrane proton pumping 10 protons per NADH and 6 protons per FADH2 oxidized are pumped across the inner membrane and the electron motive force is converted to an proton motive force.(Estimated by titrations upon the presence of specific inhibitors)Reactive oxygen species (ROS) mi

20、ght 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 (Chemical reactions coupled to osmotic gradients.)osmotic gradients.) Facts not yet explained:Facts not ye

21、t explained:？“Hight-energy”intermediates elusive to identification;“Hight-energy”intermediates elusive to identification; ？ Phosphorylation closely associated with membrane; Phosphorylation closely associated with membrane; ？ Uncoupling caused by agents of various structures; Uncoupling caused by ag

22、ents 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 chemiosmotic theory of Mitchell:e- flow and ATP synthesis are separate

23、 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 bacterial flagella.ATP synthase ( (EC 3.6.3.14)EC 3.6.3.14)

24、was first identified by dissociation and reconstitution studies (Efraim Racker, 1960s)Knob-like structures were seen with inside-out vesicle of mitochondria, thylakoid membrane of chloroplasts and inside-out E. coli plasma membrane. The mitochondrial ATPase is the ATP synthase!Mitochondria-catalyzed

25、 18O, 32P exchange data suggests that oxidative phosphorylation is dynamically revesible (Mildred Cohn, 1953)Covalent intermediates was proposed to be formed.Later such a phosphorylated protein was detected, but later found to be succincyl CoA synthetase (by Paul Boyer; “We were reaching for a gold

26、but got a bronze instead”)The energy from electron transport was proposed to be needed for releasing preformed ATP from ATP synthase, not needed for the formation of ATP (Boyer et al., 1973, PNAS, 70:2837-9. Pi HOH exchange is considerably less sensitive to uncouplers than the Pi ATP and ATP HOH exc

27、hanges.The uncoupler-insensitive Pi HOH exchange is inhibited by oligomycin. Similar exchange was also observed for myosin.This release could logically involve energy-requiring protein con-formational change.(Not a single word was mentioned about the chemiosmotic hypothesis!)Catalytic cooperativity

28、revealed: removal of ADP stopped ATP HOH exchange and removal 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)(st

29、ator) F1: Fo: ab2c10-14The rotorThe rotor: :the the c- c-ring 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 sy

30、nthase (Paul Boyer, 1980) 1. The subunit found to interact with the catalytic subunits; 2. Distribution of 18O in Pi (with 1, 2 or 3 O exchanged) formed from 18O-ATP suggested identical behavior of all catalytic sites. 3. Mild cross-linking stopped catalysis and cleavage of the cross-linker restored

31、 activityBinding Binding ADP + PADP + Pi iSynthesizing Synthesizing ATPATPReleasing Releasing ATPATPThe binding-changeMechanism or rotational catalysis(Paul Boyer, 1980s) Each Each subunit subunitwill take threewill take threedifferent conformationsdifferent conformationsin turn during each in turn

32、during each cycle of action.cycle of action.http:/en.wikipedia.org/wiki/Image:ATPsyn.gifThe binding-change mechanism of ATP synthase:The binding-change model was elegantly supported by X-ray crystallography analysis of F1-ATPase.The three catalytic subunitsdiffer in conformation and the nucleotide b

33、ound;The three catalytic subunitsare in different statesof the catalytic cycle atany instant;Interconversion of the state may be achieved by rotationof the subunit in the center. AMP-PNPAMP-PNPAMP-PNPAMP-PNPADPNature, 1994, 370:621-628.Rotation of c-ring or c-ring/ subunit directly observed using fl

34、uorescence microscopy (1997)Mechanically driven ATP synthesis by F1-ATPase demonstrated (2004).Model of the action of E. coli ATP synthase:the proton gradient drives the rotation of the c ring using two half-channels on the a subunit.Protonation/deprotonation of an Asp is believed to be essential fo

35、r rotating the c ring and the subunit.10-14 protons needed for every3 ATP synthesized.Asp-COO- Asp-COOHThus 4 protons per ATP synthesizedhttp:/www.mrc-dunn.cam.ac.uk/research/atp_synthase/ The energy stored in the proton gradient can be used to do other work.The rotary motion of the bacterial flagel

36、la is energized directly by the proton gradient present across the cytoplasmic membrane.The proton-motive force is used foractive transport through the inner membrane of the mitochondria.Heat is generated in Brown fat through the action of thermogenin,an uncoupling protein:to produce heat to maintai

37、n body temperature for animals in hibernation, of newly born and adapting to the cold (thermogenesis).Fatty acids seem to activate and nucleotides inhibit UCP1UCP1Electrons in NADH generated in cytosol are shuttled onto the respiratory chain. The malate-aspartate shuttle system: The malate-aspartate

38、 shuttle system: Malate Malate t translocates electrons produced during glycolysis across inner membrane of mitochondrion for oxidative phosphorylationsemipermeableIrreversibleIrreversibleThe glycerol-3-phosphate shuttle systemThe pathways leading to ATP synthesis are coordinately regulated.Interloc

39、king regulation of all these pathways might be realized by the relative levels of ATP, NADH, ADP, AMP, Pi, and NAD+. 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

40、 by the availability of ADP (“acceptor control”) No ATP consumption, no electron flow!Pyruvate oxidationSome respiratory proteinsare encoded by the humanmitochondrial genomeComplexes I, III, and IV and ATP synthaseare assembled by usingsubunits made in both thecytosol and mitochondria.How was the mo

41、lecular mechanism of oxidative phosphoryation revealed?Kalckar HM (1974). Origins of the concept oxidative phosphorylation. Mol. Cell. Biochem. 5 (12):55-62.1. Glucose degradation (fermentation, Glycolysis)2. Biological oxidation (O2 consumption, citric acid cycle)3. Energy supply of muscle contract

42、ion4. Understanding the role of ATP as the universal energy currency5. Subcellular location and protein isolation and characterization.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, 35, 1

43、63 Wieland, O. (1912) Ber. Dtsch. Chem. Ges. 45, 45, 484499; 26062615 The oxidation of a large number of organic 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

44、the key for biological oxidation by WarburgWarburg, O. (1924) Warburg, O. (1924) Biochem. Z. Biochem. Z. 177, 471177, 471486.486. Iron-containing respiratory enzymes, oxidases activate oxygen. Used inhibitors (e.g., NCN, CO) and spectroscopy to indirectly detect the behavior of O2-transferring ferme

45、nt of respiration in living cells.Warburg(1883-1970)Nobel Prize, 1931Cyt a?Cyt c?600 nm560 nmHeme-like pigments found to Heme-like pigments found to exist widely in animals (1886)exist widely in animals (1886) MacMunn, C. A. (1886) Researches on MacMunn, C. A. (1886) Researches on myohaematin and th

46、e histohaematin, myohaematin and the histohaematin, Phil. Trans. Phil. Trans. Roy. Soc.Roy. Soc. , 177:267-298. , 177:267-298. Examined organs and tissues of vertebrates and invertebrates, using microspectroscope. Discovered the presence of the histohaematins and myohaematin (having light absorption

47、 in reduced state). Cyt a?Cyt b?Cyt c?Cytochromes (Cytochromes (“cellular pigments) cellular pigments) rediscoveredrediscovered Keilin, D. (Keilin, D. (19251925) On cytochrome, a respiratory ) On cytochrome, a respiratory pigment, common to animals, yeast, and higher pigment, common to animals, yeas

48、t, and higher plants, plants, Proc. R. Soc. B Biol. Sci. Proc. R. Soc. B Biol. Sci. 98:31298:312339.339. Miscrospectroscope showed 4 absorption bands when reduced, assumed to be made of three types of haeme groups (a, b, c). Its a common intracellular respiratory catalyst, the reduction and oxidatio

49、n of which can be specifically inhibited.David Keilin(1887-1963)An oxidase was demonstrated to be a respiratory catalyst in yeast D. KEILIN (1927) D. KEILIN (1927) Influence of Carbon Monoxide and Light on Indophenol Oxidase of Yeast Cells, NatureNature 119:670-671. 119:670-671. A respiratory chain

50、concept was proposed (1929) Keilin, D. (1929) Cytochrome and respiratory Keilin, D. (1929) Cytochrome and respiratory enzymes, enzymes, Proc. Roy, SocProc. Roy, Soc, B104:206-252., B104:206-252.The activities of indophenol oxidase is highly correlated to the oxidation of the cytochromes.Succinate (o

51、rganic substrate) leads to the reduction of the cytochromes effectively.Cytochromes act as carriers of hydrogen between the dehydrase (i.e., dehydrogenase)and the oxidase (i.e., cytochrome c oxidase).Activation of hydrogen atoms(Wieland)Activation of O2(Warburg)Cytochromes(Keilin)The respiratory cha

52、in proposed by Keilin was highly regarded by future scientistsCoupling between phosphorylations and oxidations observed (1939) Lipmann, F. (1939) Coupling between Pyruvic Acid Dehydrogenation and Adenylic Acid Phosphorylation, Nature, 143: 281. NADH oxidation by ONADH oxidation by O2 2 is linked to

53、is linked to phosphorylation (1948)phosphorylation (1948) Friedkin & Lehninger (1948) Phosphorylation coupled to electron transport between DPNH2 and O2 J. Biol. Chem. 174, 757-758; 178178: 61123. Rat liver particulate; 32P-labeled Pi as tracer. The respiratory chain was The respiratory chain was se

54、parated into four membrane separated into four membrane complexes (1948)complexes (1948)WAINIO et al. (1948) The preparation of a soluble WAINIO et al. (1948) The preparation of a soluble cytochrome oxidase. J Biol Chem. 173:145-52. cytochrome oxidase. J Biol Chem. 173:145-52. Green, D. E. (1966) in

55、 Green, D. E. (1966) in Comprehensive Biochemistry Comprehensive Biochemistry (Florkin, (Florkin, M., and Stotz, E. H., eds) Vol. 14, pp. 309M., and Stotz, E. H., eds) Vol. 14, pp. 309326, Elsevier 326, Elsevier Science Publishers B.V., Amsterdam.Science Publishers B.V., Amsterdam. Using deoxycholat

56、e and cholate to disperse the membrane and allow its components to be separated by conventional ammonium sulfate fractionation. Catalyzing the reduction of ubiquinone by NADH (Complex I) or succinate (Complex II), the reduction of ferricytochrome c by ubiquinol (Complex III), and the oxidation of fe

57、rrocytochrome c by oxygen (Complex IV).Chemical coupling hypothesis on respiratory chain phosphorylation proposed (1953) Slater, EC (1953) Mechanism of phosphorylation Slater, EC (1953) Mechanism of phosphorylation in the respiratory chain. in the respiratory chain. Nature.Nature. 172:975-8. 172:975

58、-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.Uncoupling agentsLipmanns scheme (1946)Slaters scheme (1953)Photophosphorylation concept proposed

59、(1954) Arnon, D. I., F. R. Whatley, and M. B. Allen. Arnon, D. I., F. R. Whatley, and M. B. Allen. (1954) Photosynthesis by isolated (1954) Photosynthesis by isolated chloroplasts. II. Photosynthetic chloroplasts. II. Photosynthetic phosphorylation, the conversion of light phosphorylation, the conve

60、rsion of light into phosphate bond energy. into phosphate bond energy. J. Am. Chem. J. Am. Chem. Soc.Soc. 76: 6324-6328. 76: 6324-6328. A quinone was found to be A quinone was found to be needed for respiration (1957)needed for respiration (1957) Crane et al. (1957). Isolation of a quinone from beef

61、 heart mitochondria. Biochimica et Biophysica Acta 2525: 2201. Iron-sulfur proteins are Iron-sulfur proteins are involved in respiration (1960)involved in respiration (1960) Beinert, H., and Sands, R. H. (1960) Studies on succinic and DPNH dehydrogenase preparations by paramagnetic resonance (EPR) s

62、pectroscopy Biochem. Biophys. Res. Commun. 3:3:4146ATP synthase was purified ATP synthase was purified and charaterized (1960-1973)and charaterized (1960-1973) Pullman et al & Racker E. (1960-1973). Partial Pullman et al & Racker E. (1960-1973). Partial Resolution of the Enzymes Catalyzing Oxidative

63、 Resolution of the Enzymes Catalyzing Oxidative Phosphorylation. I -XXV Phosphorylation. I -XXV J. Biol. Chem.J. Biol. Chem. 235: 3322; 235: 3322; 241:2475; 248:676. 241:2475; 248:676. A chemi-osmotic mechanism was proposed to explain the coupling Mitchell, P. (1961). Coupling of phosphorylation Mit

64、chell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-to electron and hydrogen transfer by a chemi-osmotic type of mechanism. osmotic type of mechanism. NatureNature 191:144. 191:144. Unexplained factsUnexplained facts: : “high-energy intermediateshigh-energy int

65、ermediates” yet identified; membrane structure essential; yet identified; membrane structure essential; uncoupling agents differ greatly in structure. uncoupling agents differ greatly in structure. acid-bath dark phosphorylation acid-bath dark phosphorylation by chloroplasts observed (1966)by chloro

66、plasts observed (1966) Jagendorf and Uribe (1966) ATP formation Jagendorf and Uribe (1966) ATP formation caused by acid-base transition of spinach caused by acid-base transition of spinach chloroplasts; chloroplasts; PNAS, PNAS, 55:17055:170177.177. Without illumination or oxygen; Made first acid, then basic.Reconstituted light-activated Reconstituted light-activated proton pump catalyzes ATP proton pump catalyzes ATP formation (1974)formation (1974) Racker and Stoeckenius (1974) Reconstitution