Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase.
Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.
Krishnamoorthy
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Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Hinkle
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PC
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001974:Bromosuccinimide / Q000494:pharmacology; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D007202:Indicators and Reagents; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D008962:Models, Theoretical; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000235:genetics*
D007202:Indicators and Reagents; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D012795:Sialoglycoproteins; C057692:apopolysialoglycoprotein, rainbow trout eggs; D012402:Rotenone; D009243:NAD; D004024:Dicyclohexylcarbodiimide; D002731:4-Chloromercuribenzenesulfonate; D004139:Dinitrofluorobenzene; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D001974:Bromosuccinimide