Reversible inactivation of wheat leaf nitrate reductase by NADH, involving superoxide ions generated by the oxidation of thiols and FAD

Abstract

The conversion of wheat leaf NADH-nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) to a reduced inactive form, by preincubation with NADH (in the absence of nitrate), occurred in the presence of either dithiothreitol and/or FAD but not with cysteine. This inactivation of nitrate reductase, unlike that by cyanide, was dependent on aerobic conditions and was prevented by EDTA and superoxide dismutase. Superoxide ions were produced by the auto-oxidation of dithiothreitol but not cysteine, at pH 7.5. Thus, superoxide ions can mediate the inactivation of NADH-reduced nitrate reductase in higher plants. Pretreatment of nitrate reductase with NADH alone (over-reduction) did not inactivate the enzyme. A nucleophilic agent, i.e., cyanide or superoxide is necessary to inhibit electron transfer by the enzyme to nitrate. Nitrite or azide were not effective. Data in the literature which suggest that NADH stabilizes nitrate reductase, rather than resulting in its inactivation, can now be explained. In these cases, cysteine was used as thiol and FAD was not added in the extraction or incubation media so that no superoxide ion was produced.