Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast.
A study of the steady-state kinetics of NADH(NADPH)-cytochrome c reductase (FMN-containing) from ale yeast (M. S. Johnson and S. A. Kuby (1985) J. Biol. Chem. 260, 12341-12350) has led to a postulated three-substrate random-ordered hybrid mechanism, where NAD(P)H and FMN add randomly and very likely in a steady-state fashion, followed by an ordered addition of cytochrome c. Kinetic parameters have been derived from this mechanism. Arrhenius plots showed large differences between NADH and NADPH, as the substrate-reductant. Menadione accelerated cytochrome c reduction and also O2 uptake, but vitamin K1 and coenzyme Q10 were ineffective as electron mediators, possibly as a result of their insolubility. With NADPH as the substrate-reductant, the order of the rate of reduction of electron acceptors was ferricyanide greater than DCIP greater than cytochrome c greater than oxygen; with menadione, the specificity sequence was cytochrome c greater than ferricyanide greater than DCIP greater than oxygen. With NADH, the order was ferricyanide greater than cytochrome c greater than oxygen greater than DCIP, which changed to cytochrome c greater than ferricyanide greater than oxygen greater than DCIP on addition of menadione. Cytochrome b5 was also reduced in the absence of oxygen. No transhydrogenase activity was observed, but the reduced thionicotinamide analogs of NADH and NADPH acted as substrates. Superoxide dismutase inhibited cytochrome c reduction in air by 50%, but O2-. was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O2. The product of the reaction with oxygen appeared to be H2O2.
Johnson
M S
MS
Kuby
S A
SA
D002384:Catalysis; D004789:Enzyme Activation; D005285:Fermentation; D005486:Flavin Mononucleotide / Q000032:analysis; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis*; D010084:Oxidation-Reduction / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014812:Vitamin K / Q000494:pharmacology
D014812:Vitamin K; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase