The reaction of reduced cytochromes c with nitrous oxide reductase of Wolinella succinogenes

Abstract

Kinetic studies were carried out on the oxidation of dithionite-reduced, monoheme cytochromes c by nitrous oxide reductase from Wolinella succinogenes. These reduced cytochrome c-N 2O oxidoreductase systems showed second-order kinetics, first-order each in reduced cytochrome c and enzyme, at concentrations of reduced cytochrome c between 1 and 10 μM. The second-order rate constant at 25°C and pH 6.8, k 2, was 3.1 · 10 6, 9.3 · 10 4 and about 1 · 10 4 M −1 s −1 for cytochrome c from W. succinogenes, horse heart and Pseudomonas aeruginosa (cytochrome c-551), respectively, at enzyme concentrations ≦ 12, ≦3 and ≦ 11 nM, respectively. With horse-heart cytochrome c and cytochrome c-551, k 2 diminished substantially at higher enzyme concentrations. Evidence for reaction via an E-S (Michaelis) complex was not obtained. Unlike systems for which the radical cation of benzyl viologen (BV· +) served as reducing agent, nitrous oxide reductase failed to show turnover-dependent inactivation when a reduced cytochrome c was the reductant. The system thus mimicked the ability of nitrous oxide reductase to turnover in vivo without inactivation. Cytochrome c oxidase activity of nitrous oxide reductase was not observed when O 2 replaced N 2O. Similarly, BV· +-CO 2 oxidoreductase activity was not detected with bicarbonate buffer (CO 2 is isoelectronic with N 2O). The values for M r (9214), optical extinction coefficients and amino acid composition of the monoheme cytochrome c of W. succinogenes were found to be somewhat different from published values.