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.