Inactivation by acetylene of spinach nitrate reductase

Abstract

The molybdoprotein NADH-nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1) from spinach can be inactivated by acetylene only when the enzyme is in its reduced state. Other gases such as ethylene, carbon monoxide, dinitrogen and others did not alter the enzyme activity. From the two partial activities of nitrate reductase, only the terminal nitrate reductase was impaired by acetylene while the dehydrogenase activity was rather stimulated. Functional dehydrogenase activity was required for inactivation when NADH was the reductant. Dithionite, dithionite + MV or dithionite + FMN were also able to sustain acetylene inactivation, whether or not nitrate reductase was previously depleted of its dehydrogenase activity. However, ascorbate or ascorbate + DCIP did not cooperate with acetylene for inactivating nitrate reductase. Nitrate and the competitive inhibitors with respect to nitrate of nitrate reductase, namely azide, cyanate and carbamyl phosphate, protected nitrate reductase from acetylene inactivation. Cyanide-inactivated nitrate reductase was still sensitive to acetylene, since, once the cyanide-inactivated enzyme was placed under acetylene, no ferricyanide reactivation could be attained. These results suggest that reduced nitrate reductase might bind acetylene at the nitrate active site, where molybdenum is supposed to be implicated, thus impairing the reduction of nitrate.