A nitrate reductase from Micrococcus denitrificans

Abstract

A nitrate reductase from Micrococcus denitrificans (N.C.I.B. 8944) was associated with cell membranes. This particulate system utilized NADH and succinate as electron donors for the reduction of nitrate to nitrite. Small amounts of nitric oxide, nitrous oxide and nitrogen gas were also produced. The K m for NADH using nitrate and oxygen as acceptors were 1.8 ·10 −5 M and 2·10 −5 M, respectively. The solubilized nitrate reductase purified 108-fold converts nitrate to nitrite stoichiometrically with reduced benzyl viologen as the electron donor. Reduced methyl viologen was also effective, but NADH, succinate and reduced cytochrome c were not utilized. The purified enzyme contained Mo as shown by isotope labelling. Its involvement in enzyme action is indicated by strong inhibition by KCNS and dithiol. Idoacetamide and p-Chloromercuribenzoate also inhibited the enzyme, and the effect in the latter case was reversed by reduced glutathione. The purified enzyme did not contain any cytochrome or flavin. Maximum enzyme activity was observed at pH 6.3 and a K m of 9.6·10 −4 M was recorded for nitrate when reduced benzyl viologen was the electron donor.