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Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine ¹

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Velázquez, 144, 28006 Madrid, Spain

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃^–. Since NO₃^–, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃^–), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN^–. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.