Interconversion of nitrate reductase from Ankistrodesmus braunii related to redox changes

Reversible inactivation of homogeneous nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii has been carried out by aerobic incubation of the enzyme with reduced pyridine nucleotide. The involvement of superoxide radicals in the inactivation process is inferred from the fact that it does not take place in the absence of oxygen or in the presence of superoxide dismutase. On the other hand, cyanide also causes the inactivation of the enzyme under reducing conditions. The inactivation of A. braunii nitrate reductase takes place in two steps; the first is the one-electron reduction of the enzyme probably involving the molybdenum centers, and the second, and rate-limiting step, results from the interaction of the reduced enzyme with a nucleophylic agent such as superoxide or cyanide. The mean potential value, at pH 7.5, of the inactivation process, measured by reductive titration with dithionite in the presence of cyanide, was −50 mV. Inactive nitrate reductase, previously dialyzed to remove the inactivating agents, can be immediately reactivated by treatment with ferricyanide in a process requiring the removal of only one electron. This process showed a mean potential value, measured by oxidative titration with ferricyanide, of +230 mV at pH 7.5, independent of the system used to inactivate the enzyme.