Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: Steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast

Abstract

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 O 2 uptake, but vitamin K 1 and coenzyme Q 10 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 > DCIP > cytochrome c > oxygen; with menadione, the specificity sequence was cytochrome c > ferricyanide > DCIP > oxygen. With NADH, the order was ferricyanide > cytochrome c > oxygen > DCIP, which changed to cytochrome c > ferricyanide > oxygen > DCIP on addition of menadione. Cytochrome b 5 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 O ⪰ 2 was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O 2. The product of the reaction with oxygen appeared to be H 2O 2.