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-N2O 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, k2, was 3.1 · 106, 9.3 · 104 and about 1 · 104 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, k2 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 O2 replaced N2O. Similarly, BV·+-CO2 oxidoreductase activity was not detected with bicarbonate buffer (CO2 is isoelectronic with N2O). The values for Mr (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.