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Electron Transport Discovery Four Complexes Complex I: Nadhà Coqh2

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BI/CH 422/622 OUTLINE: Pyruvate pyruvate dehydrogenase Krebs’ Cycle How did he figure it out? Overview 8 Steps Citrate Synthase Aconitase Isocitrate dehydrogenase Ketoglutarate dehydrogenase Succinyl-CoA synthetase Succinate dehydrogenase Fumarase Malate dehydrogenase Energetics Regulation Summary Oxidative Phosphorylation Energetics (–0.16 V needed for making ATP) Mitochondria Transport (2.4 kcal/mol needed to transport H+ out) Electron transport Discovery Four Complexes Complex I: NADHà CoQH2 Complex II: Succinateà CoQH2 2+ Complex III: CoQH2à Cytochrome C (Fe ) 2+ Complex IV: Cytochrome C (Fe ) à H2O Electron Transport à O2 Inhibitors of Electron Transport Big Drop! • Inhibitors all stop ET and ATP synthesis: very toxic! Spectral work Big Drop! NADH Cyto-a3 Cyto-c1 Big Drop! Cyto-b Cyto-c Cyto-a Fully reduced Flavin Cyto-c + rotenone + antimycin A 300 350 400 450 500 550 600 650 700 1 Electron Transport Electron-Transport Chain Complexes Contain a Series of Electron Carriers • Better techniques for isolating and handling mitochondria, and isolated various fractions of the inner mitochondrial membrane • Measure E°’ • They corresponded to these large drops, and they contained the redox compounds isolated previously. • When assayed for what reactions they could perform, they could perform certain redox reactions and not others. • When isolated, including isolating the individual redox compounds, and measuring the E°’ for each, it was clear that an electron chain was occurring; like a wire! • Lastly, when certain inhibitors were added, some of the redox reactions could be inhibited and others not. Site of the inhibition could be mapped. Electron Transport Electron-Transport Chain Complexes Contain a Series of Electron Carriers • Better techniques for isolating and handling mitochondria, and isolated various fractions of the inner mitochondrial membrane • Measure E°’ • They corresponded to these large drops, and they contained the redox compounds isolated previously. • When assayed for what reactions they could perform, they could perform certain redox reactions and not others. • When isolated, including isolating the individual redox compounds, and measuring the E°’ for each, it was clear that an electron chain was occurring; like a wire! • Lastly, when certain inhibitors were added, some of the redox reactions could be inhibited and others not. Site of the inhibition could be mapped. 2 Electron Transport TABLE The Protein Components of the 19-3 Mitochondrial Respiratory Chain Reduction Enzyme Mass Number of Prosthetic Binding sites potential Inhibted by: complex/protein (kDa) subunitsa group(s) for: (DE’°V) I NADH dehydrogenase 850 45 (14) FMN, Fe-S 0.36 NADH, CoQ amytal, rotenone II Succinate Succinate, 140 4 FAD-E, Fe-S 0.09 malonate dehydrogenase CoQ III Ubiquinone: Hemes b, c , CoQ, cytochrome c 250 11 1 0.17 antimycin a Fe-S Cytochrome c oxidoreductaseb Cytochrome cc 13 1 Heme Hemes a, a ; Cytochrome c, Cyanide, azide, IV Cytochrome oxidaseb 204 13 (3–4) 3 0.57 CuA, CuB O2 CO aNumber of subunits in the bacterial complexes in parentheses. bMass and subunit data are for the monomeric form. cCytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. ‟DE°’ ” of ATP synthesis = –0.16 v Electron Transport NADH:Ubiquinone Oxidoreductase, Flavoprotein (FMNH2) a.k.a. Complex I N3(4Fe+24S) X2(4Fe+24S) 2H+ N2(4Fe+24S) Reduced +2 +2 +2 forms NADH FMNH2 N3Fe S X2Fe S N2-Fe S CoQH2 2e– 2x1e– 2x1e– 2x1e– 2x1e– +3 +3 +3 Oxidized NAD+ FMN N3Fe S X2Fe S N2-Fe S CoQ forms -0.38 V 2H+ -0.34 V Hates e–-0.32 V N3(4Fe+24S) -0.25 V Flavoprotein (FMNH ) NADH 2 X2(4Fe+24S) -0.10 V N2(4Fe+24S) +0.05 V Loves e– CoQH2 3 Electron Transport NADH:Ubiquinone Oxidoreductase, Flavoprotein (FMNH2) a.k.a. Complex I N3(4Fe+24S) X2(4Fe+24S) 2H+ N2(4Fe+24S) Reduced +2 +2 +2 forms NADH FMNH2 N3Fe S X2Fe S N2-Fe S CoQH2 2e– 2x1e– 2x1e– 2x1e– 2x1e– +3 +3 +3 Oxidized NAD+ FMN N3Fe S X2Fe S N2-Fe S CoQ forms -0.38 V 2H+ -0.34 V Hates e–-0.32 V N3(4Fe+24S) -0.25 V Flavoprotein (FMNH ) NADH 2 X2(4Fe+24S) -0.10 V N2(4Fe+24S) +0.05 V Loves e– CoQH2 Electron Transport: Complex I • One of the largest macro-molecular assemblies in the mammalian cell • Over 40 different polypeptide chains, encoded by both nuclear and 2x1e– mitochondrial genes • NADH binding site in the matrix side • Noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH. • Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site. 4 Electron Transport: Complex I NADH:Ubiquinone Oxidoreductase Is a Proton Pump • The transfer of two electrons from NADH to ubiquinone is accompanied by a 2x1e– transfer of protons from the matrix (N) to the intermembrane space (P). • Experiments suggest that about four protons are tare are are transported per one NADH. + + + NADH + Q + 5H N = NAD + QH2 + 4 H P • Additionally, the reduced coenzyme Q picks up two protons. • Protons are transported by proton “wires.” – a series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another Electron Transport: Complex I +2 +2 +2 NADH FMNH2 N3Fe S X2Fe S N2-Fe S CoQH2 2e– 2x1e– 2x1e– 2x1e– 2x1e– NAD+ FMN N3Fe+3S X2Fe+3S N2-Fe+3S CoQ 5 Electron Transport TABLE The Protein Components of the 19-3 Mitochondrial Respiratory Chain Reduction Enzyme Mass Number of Prosthetic Binding sites potential Inhibted by: complex/protein (kDa) subunitsa group(s) for: (DE’°V) I NADH dehydrogenase 850 45 (14) FMN, Fe-S 0.36 NADH, CoQ amytal, rotenone II Succinate Succinate, 140 4 FAD-E, Fe-S 0.09 dehydrogenase CoQ III Ubiquinone: Hemes b, c , CoQ, cytochrome c 250 11 1 0.17 antimycin a Fe-S Cytochrome c oxidoreductaseb Cytochrome cc 13 1 Heme Hemes a, a ; Cytochrome c, Cyanide, azide, IV Cytochrome oxidaseb 204 13 (3–4) 3 0.57 CuA, CuB O2 CO aNumber of subunits in the bacterial complexes in parentheses. bMass and subunit data are for the monomeric form. cCytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. Electron Transport: Complex II Succinate Dehydrogenase, • FAD accepts two electrons from succinate. a.k.a. Complex II • Electrons are passed, one at a time, via iron- sulfur centers to ubiquinone, which becomes reduced CoQH2. • Does not transport protons • Succinate dehydrogenase is a single enzyme with dual roles: • convert succinate to fumarate in the citric acid cycle • donate those electrons in the electron transport chain 6 Electron Transport TABLE The Protein Components of the 19-3 Mitochondrial Respiratory Chain Reduction Enzyme Mass Number of Prosthetic Binding sites potential Inhibted by: complex/protein (kDa) subunitsa group(s) for: (DE’°V) I NADH dehydrogenase 850 45 (14) FMN, Fe-S 0.36 NADH, CoQ amytal, rotenone II Succinate Succinate, 140 4 FAD-E, Fe-S 0.09 dehydrogenase CoQ III Ubiquinone: Hemes b, c , CoQ, cytochrome c 250 11 1 0.17 antimycin a Fe-S Cytochrome c oxidoreductaseb Cytochrome cc 13 1 Heme Hemes a, a ; Cytochrome c, Cyanide, azide, IV Cytochrome oxidaseb 204 13 (3–4) 3 0.57 CuA, CuB O2 CO aNumber of subunits in the bacterial complexes in parentheses. bMass and subunit data are for the monomeric form. cCytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. Hates e– +0.05 V +0.077 V CoQ Cytochrome b –0.1 to +0.4 V +0.20 V +0.22 V +0.254 V Reiske Protein([FeS]) Loves e– Cytochrome c1 Cytochrome c Electron Transport: Complex III Ubiquinone:Cytochrome c Oxidoreductase, a.k.a. Complex III • Uses two electrons from CoQH2 to reduce two molecules of cytochrome c • Additionally contains iron-sulfur clusters (Rieske protein), two different cytochrome b’s, and a cytochrome c1 • It’s a dimer of 11 subunits (22 proteins). Three main proteins for each of the redox centers. • Dimers create a central cavern with TWO CoQ binding sites • Problem: How do we take the 2-electron CoQH2 and get one electron at a time into cytochrome c without any flavin cofactors? 7 Electron Transport: Complex III 2H+ 4H+ +2 +2 +2 CoQH2 [FeS] Cyto-c1 Cyto-c – +3 +3 +3 •CoQ [FeS] Cyto-c1 Cyto-c CoQH2 QP site The Q Cycle – +2 +2 – •CoQ Cyto-bL Cyto-bH •CoQ QN site +3 +3 CoQ Cyto-bL Cyto-bH CoQ 2H+ +2 2 CoQH2 + CoQ à 2 CoQ + CoQH2 + 2 Cyto-c •CoQ– + + + 4H (matrix(N)) à 4H (intermembrane space (P)) – 2H (matrix(N)) Electron Transport: Complex III 2H+ 4H+ +2 +2 +2 CoQH2 [FeS] Cyto-c1 Cyto-c – +3 +3 +3 •CoQ [FeS] Cyto-c1 Cyto-c CoQH2 QP site The Q Cycle – +2 +2 – •CoQ Cyto-bL Cyto-bH •CoQ QN site +3 +3 CoQ Cyto-bL Cyto-bH CoQ 2H+ +2 2 CoQH2 + CoQ à 2 CoQ + CoQH2 + 2 Cyto-c •CoQ– + + + 4H (matrix(N)) à 4H (intermembrane space (P)) – 2H (matrix(N)) 8 Electron Transport: Complex III The Q Cycle • Clearance of electrons from the reduced quinones via the Q-cycle results in translocation of four protons to the inter- membrane space. • This cycle matches experimental evidence that four protons are transported across the membrane per two electrons that reach cyt c. • The Q cycle provides a good model that explains how two additional protons are picked up from the matrix. – Two molecules of CoQH2 become oxidized, releasing protons into the IMS.
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  • Consequences of the Structure of the Cytochrome B6 F Complex for Its Charge Transfer Pathways ⁎ William A

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    Biochimica et Biophysica Acta 1757 (2006) 339–345 http://www.elsevier.com/locate/bba Review Consequences of the structure of the cytochrome b6 f complex for its charge transfer pathways ⁎ William A. Cramer , Huamin Zhang Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA Received 17 January 2006; received in revised form 30 March 2006; accepted 24 April 2006 Available online 4 May 2006 Abstract At least two features of the crystal structures of the cytochrome b6 f complex from the thermophilic cyanobacterium, Mastigocladus laminosus and a green alga, Chlamydomonas reinhardtii, have implications for the pathways and mechanism of charge (electron/proton) transfer in the complex: (i) The narrow 11×12 Å portal between the p-side of the quinone exchange cavity and p-side plastoquinone/quinol binding niche, through which all Q/QH2 must pass, is smaller in the b6 f than in the bc1 complex because of its partial occlusion by the phytyl chain of the one bound chlorophyll a molecule in the b6 f complex. Thus, the pathway for trans-membrane passage of the lipophilic quinone is even more labyrinthine in the b6 f than in the bc1 complex. (ii) A unique covalently bound heme, heme cn, in close proximity to the n-side b heme, is present in the b6 f complex. The b6 f structure implies that a Q cycle mechanism must be modified to include heme cn as an intermediate between heme bn and plastoquinone bound at a different site than in the bc1 complex. In addition, it is likely that the heme bn–cn couple participates in photosytem + I-linked cyclic electron transport that requires ferredoxin and the ferredoxin: NADP reductase.
  • Regulation of Photosynthetic Electron Transport☆

    Regulation of Photosynthetic Electron Transport☆

    Biochimica et Biophysica Acta 1807 (2011) 375–383 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio Review Regulation of photosynthetic electron transport☆ Jean-David Rochaix ⁎ Department of Molecular Biology, University of Geneva, Geneva, Switzerland Department of Plant Biology, University of Geneva, Geneva, Switzerland article info abstract Article history: The photosynthetic electron transport chain consists of photosystem II, the cytochrome b6 f complex, Received 14 September 2010 photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation Received in revised form 11 November 2010 events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain Accepted 13 November 2010 with the oxidation of water followed by electron flow along the electron transport chain and concomitant Available online 29 November 2010 pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO assimilation. A Keywords: 2 Electron transport remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental Linear electron flow conditions by sensing light quality and quantity, CO2 levels, temperature, and nutrient availability. These Cyclic electron flow acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid Photosystem II protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in Photosystem I state transitions and in the regulation of electron flow as well as in thylakoid membrane folding.