The Protein Components of the Mitochondrial Respiratory Chain

Electron Transport TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein I NADH dehydrogenase Mass (kDa) Number of subunits a Prosthetic group(s) Reduction potential ( E ’ V) ° Binding sites for: Inhibted by: 850 45 (14) FMN, Fe-S –0.36 NADH, CoQ amytal, rotenone II Succinate dehydrogenase 140 4 FAD-E, Fe-S 0.09 III Ubiquinone: cytochrome c oxidoreductase b 250 11 Hemes b, c 1 , Fe-S 0.17 13 1 Cytochrome c c IV Cytochrome oxidase b 204 13 (3–4) CoQ, Cytochrome c malonate antimycin a 0.25 (Cyt c) Heme Hemes a, a 3 ; Cu A , Cu B 0.05 Succinate, (CoQ) CoQ 0.57 Cytochrome c, O2 Cyanide, azide, CO 0.82 Number of subunits in the bacterial complexes in parentheses. (O2) 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. a bMass ‟DE°’ ” of ATP synthesis = –0.16 v DE°’ of Complex I = –0.41 v DE°’ of Complex III = –0.2 v DE°’ of Complex IV = –0.25 v D °’ “ needed for proton pumping = 0.053 V/proton “ E 1 Electron Transport Flavoprotein (FMNH2) NADH:Ubiquinone Oxidoreductase, a.k.a. Complex I N3(4Fe+24S) X2(4Fe+24S) 2H+ Reduced forms NADH N3Fe+2S FMNH2 2x1e– 2e– Oxidized forms N2(4Fe+24S) NAD+ –-0.32 V Hates e NADH X2Fe+2S 2x1e– N3Fe+3S FMN N2-Fe+2S 2x1e– X2Fe+3S N2-Fe+3S -0.38 V -0.34 V CoQ 2H+ -0.25 V N3(4Fe+24S) Flavoprotein (FMNH2) CoQH2 2x1e– X2(4Fe+24S) -0.10 V N2(4Fe+24S) +0.05 V CoQH2 Loves e– 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 mitochondrial genes 2x1e– • NADH binding site in the matrix side; on the flavoprotein subunit • Non-covalently bound flavin mononucleotide (FMN) accepts two electrons from NADH. • Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site. 2 Electron Transport: Complex I electrons FeS clusters FMN N3 X2 N2 FMNH2 NADH 2x1e– 2e– NAD+ FMN N3Fe+2S 2x1e– N3Fe+3S X2Fe+2S N2-Fe+2S 2x1e– X2Fe+3S CoQH2 2x1e– N2-Fe+3S CoQ Electron Transport: Complex I NADH:Ubiquinone Oxidoreductase Is a Proton Pump • The transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of multiple protons from the matrix (N) to the intermembrane space (P). Intermembrane space (P side) 2x1e– • Experiments suggest that about four protons are tare are are transported per one NADH. • Additionally, the reduced coenzyme Q picks up two protons. NADH + Q + 5H+N = NAD+ + QH2 + 4 H+P • 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. 3 subunits are related to known Na-H+ antiporters, the 4th seems to control them all and uses the energy from the oxidation of NADH 3 Electron Transport TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein I NADH dehydrogenase Mass (kDa) Number of subunits a Prosthetic group(s) Reduction potential (E°’ V) Binding sites for: Inhibted by: 850 45 (14) FMN, Fe-S –0.36 NADH, CoQ amytal, rotenone II Succinate dehydrogenase 140 4 FAD-E, Fe-S 0.09 Succinate, CoQ III Ubiquinone: cytochrome c oxidoreductase b 250 11 Hemes b, c 1 , Fe-S 0.17 CoQ, Cytochrome c antimycin a 13 1 0.57 Cytochrome c, O2 Cyanide, azide, CO Cytochrome c c IV Cytochrome oxidase b 204 Heme 13 (3–4) Hemes a, a 3 ; Cu A , Cu B aNumber of subunits in the bacterial complexes in parentheses. Mass and subunit data are for the monomeric form. Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. b c Electron Transport: Complex II Succinate Dehydrogenase, a.k.a. Complex II • FAD accepts two electrons from succinate. • Electrons are passed, one at a time, via ironsulfur 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 4 Electron Transport TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein Mass (kDa) Number of subunits a Prosthetic group(s) Reduction potential (E°’ V) Binding sites for: Inhibted by: amytal, rotenone I NADH dehydrogenase 850 45 (14) FMN, Fe-S –0.36 NADH, CoQ II Succinate dehydrogenase 140 4 FAD-E, Fe-S 0.09 Succinate, CoQ III Ubiquinone: cytochrome c oxidoreductase b 250 11 Hemes b, c 1 , Fe-S 0.17 CoQ, Cytochrome c antimycin a 13 1 0.57 Cytochrome c, O2 Cyanide, azide, CO Cytochrome c c IV Cytochrome oxidase b 204 Heme 13 (3–4) Hemes a, a 3 ; Cu A , Cu B aNumber bMass of subunits in the bacterial complexes in parentheses. and subunit data are for the monomeric form. c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. cCytochrome Hates e– +0.05 V CoQ +0.077 V Cytochrome b –0.1 to +0.4 V +0.20 V Loves e– Reiske Protein([FeS]) +0.22 V Cytochrome c1 +0.254 V 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 (gray, green, purple). • 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? Antimycin A binding site 5 Electron Transport: Complex III intermembrane space Inner membrane (outer leaflet) 2H 4H+ + [FeS]+2 Cyto-c1+2 Cyto-c+2 •CoQ– [FeS]+3 Cyto-c1+3 Cyto-c+3 QP site The Q Cycle CoQH2 1e– CoQH2 Inner membrane (matrix leaflet) •CoQ– Cyto-bL+2 Cyto-bH+2 Cyto-bL+3 Cyto-bH+3 •CoQ– Inner membrane (outer leaflet) 1e– CoQ QN site CoQ 2H+ 2 Cyto-c+3 + CoQH2 à CoQ + 2 Cyto-c+2 4H+(matrix(N)) à 4H+(intermembrane space (P)) – 2H+(matrix(N)) •CoQ– 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 intermembrane 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. – One molecule becomes re-reduced, thus a net transfer of four protons per reduced coenzyme Q, plus a loss of protons from the matrix into the re-reduced CoQ adds to the membrane potential. 6 Electron Transport: Complex III Electron Transport TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein I NADH dehydrogenase Mass (kDa) Number of subunits a Prosthetic group(s) Reduction potential (E°’ V) Binding sites for: Inhibted by: 850 45 (14) FMN, Fe-S –0.36 NADH, CoQ amytal, rotenone II Succinate dehydrogenase 140 4 FAD-E, Fe-S 0.09 Succinate, CoQ III Ubiquinone: cytochrome c oxidoreductase b 250 11 Hemes b, c 1 , Fe-S 0.17 CoQ, Cytochrome c antimycin a 13 1 0.57 Cytochrome c, O2 Cyanide, azide, CO Cytochrome c c IV Cytochrome oxidase b 204 13 (3–4) Heme Hemes a, a 3 ; Cu A , Cu B aNumber of subunits in the bacterial complexes in parentheses. Mass and subunit data are for the monomeric form. Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. b c 7 Electron Transport • Mobile electron carrier; a peripheral membrane protein – Cytochrome c moves through the intermembrane space (IMS). 2 Cytochrome c Fe+3 Complex IV CoQH2 Complex III CoQ 2 Cytochrome c Fe+2 2H+ Electron Transport TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein I NADH dehydrogenase Mass (kDa) Number of subunits a Prosthetic group(s) Reduction potential (E°’ V) Binding sites for: Inhibted by: 850 45 (14) FMN, Fe-S –0.36 NADH, CoQ amytal, rotenone II Succinate dehydrogenase 140 4 FAD-E, Fe-S 0.09 Succinate, CoQ III Ubiquinone: cytochrome c oxidoreductase b 250 11 Hemes b, c 1 , Fe-S 0.17 CoQ, Cytochrome c antimycin a 13 1 0.57 Cytochrome c, O2 Cyanide, azide, CO Cytochrome c c IV Cytochrome oxidase b 204 13 (3–4) Heme Hemes a, a 3 ; Cu A , Cu B aNumber of subunits in the bacterial complexes in parentheses. Mass and subunit data are for the monomeric form. Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. b c 8 Electron Transport: Complex IV Cytochrome Oxidase, a.k.a. Complex IV CuA • Uses electrons from FOUR reduced cytochrome c’s to reduce dioxygen (O2) to waters (2) from 2 NADH. CuB • This reaction is among the most difficult chemically. • The bovine complex is a dimer of 13 subunits (26 proteins). Three main proteins for each of the redox centers. • Contains two copper proteins: one with a Cu-Cys cluster, and one with two different cytochrome a’s (a & a3) and Cu. The third main protein moves protons from the matrix to the second Cucofactor. Function of the other 10 are unknown, although undoubtedly involved as a proton pump. Electron Transport: Complex IV 4H+ (matrix) Cytochrome Oxidase Passes Electrons to O2 [ [ CuA ]2+ 1+ 1.5+ 3+ ] CuB 4H+ (pumped into IMS) • Contains 6 copper ions per complex (3/monomer13) • There are two types/sites for the copper: – CuA: two ions that accept electrons from cyt c (looks like a 2Fe-2S cluster) – CuB: bonded to heme a3, forming a binuclear center that transfers four electrons to oxygen. • Four electrons are used to reduce one oxygen molecule into two water molecules. • Four protons are picked up from the matrix in this process. • Four additional protons are pumped from the matrix to the intermembrane space. This scheme is for 4e– coming down from 2 NADH or 2 FADH2 9 Electron Transport: Complex IV Electron Flow Through Complex IV 4H+ O2 4H+ 4H+ This scheme is for 4e– coming down from 2 NADH or 2 FADH2 2 Electron Transport: Complex IV Electron Flow Through Complex IV: Chemistry R Second water/ reduction of Fe FH (ferryl) OH | +2 +1 Fe •Cu •Tyr H2O Oxygen binding O=O OH | Fe+2•Cu+1•Tyr H+ e– from OH | +3 +1 Fe •Cu •Tyr H+ +4 e– from cyto-a+2 OH | Fe+3•Cu+2•Tyr O–2 Reduction of Fe O– | –OH O– | +4 +2 Fe •Cu •Tyr PR H+ Fe+4•Cu+2•Tyr OH PM Quenching radical O–2 cyto-a+2 –OH •O | +2 Fe •Cu •Tyr cyto-a+2 e– from O– H+ Splitting of dioxygen O–2 cyto-a+2 e– from – A (peroxy) (oxy) –OH Reduction of Cu EH O2 First water/from Cu H2O 10 Electron Transport Multiple Complexes Associate Together to Form a “Respirasome” Complex III and Complex IV Clinical Correlations Rare Fumarase Deficiency • Loss of activity in TCA enzymes is rare as it is lethal to the cell. However, a few very rare cases have been described. • One such case was a severe deficiency of fumarase. • Urine and blood have high levels of fumarate, succinate, a-ketoglutarate, citrate, and malate. • Humans with this disease have neurological impairment, encephalomyopathy (brain/muscle malady), dystonia (muscle teaching) 11 Clinical Correlations Cyanide poisoning • CN gas or KCN ingestion causes rapid loss of mitochondrial function and death. • Cyanide works by inhibition of cytochrome oxidase by binding tightly to the Fe3+ of heme-a3. Mitochondrial respiration, and energy production ceases, and cell death rapidly follows. – H C≡O C≡N 2–S 3+ • Cyanide is one of the most potent and rapidly acting poisons known. Other poisons do the same thing: CO, H2S, N3– • If detected early enough, the antidote is to offer the CN more Fe3+ sites to bind and titrate it off of the heme-a3. • Creation of “metHb” by oxidation of Hb using various nitrates (Fe2+ à Fe3+ ) can work due to vast amounts of Hb. 12