Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH.
The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5.
D000818:Animals; D002417:Cattle; D003579:Cytochrome Reductases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D009243:NAD; D009249:NADP; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase; D014357:Trypsin