Studies on the enzymatic reduction of C-nitroso compounds. IV. Partial purification and kinetic properties of porcine heart C-nitrosoreductase.

Abstract

An NAD(P)H-dependent C-nitrosoreductase was purified 90 fold from porcine heart cytosol fraction by ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, and affinity chromatography on 5'-AMP-Sepharose. The enzyme had no aldehyde reductase activity and showed a pH optimum of 5.5. Its molecular weight estimated by gel filtration was about 65,000 and 70,000 daltons. In the reaction catalyzed by this enzyme, 2 mol of NADH were consumed per mol p-nitrosophenol (p-NSP) reduced to p-aminophenol (p-AmP). Nitrosobenzene and other aryl nitroso compounds were also reduced but neither phenylhydroxylamine nor hydroxylamine could serve as the electron acceptor. Kinetic measurements were also carried out and, based on the data obtained, the following scheme is proposed for the mechanism of the reaction: [Formula: see text], where E, E', and E" represent the active enzyme unit, the enzyme unit after two-electron reduction, and the enzyme unit after four-electron reduction, respectively, N in NADH, S is p-NSP, and P1 and P2 are NAD+ and p-AmP, respectively. Para-aminophenol showed an inhibition noncompetitive wih NADH and also one apparently noncompetitive with p-NSP. NAD+ showed an inhibition competitive with NADH and one uncompetitive with p-NSP. These results can also be accounted for by the proposed mechanism.