BRCHIVES OF RIOCHEMISTRY AND BIOPHYSICS 161, 441-447 (1974) Nitrate Reductase from Spinacea oleracea Reversible Inactivation by NAD(P)H and by Thiols’ ENRIQUE PALACIAN, FERNANDO DE LA ROSA, FRANCISCO CASTILLO, AND CARLOS GOMEZ-IVIOREKO Departamento de Bioquimica, Facultad de Ciencias y CSIC, Universidad de Sevilla, Spain Received September 26, 1973 The enzymatic complex nitrate reductase from Spinacea oleracea is inactivated by NADH or NADPH and by simple thiols. The inactivation affects FNHs-nitrate re- ductase but not NADH-diaphorase. Reactivation can be achieved by addition of ferricyanide. The extent of inactivation by dithioerythritol is increased by NAD+, but not by NADP+. Nitrate protects against inactivation by NADH or NADPH, and abolishes the effect of NAD+ on the inactivation by dithioerythritol. The NAD(P)H- inactivation of nitrate reductase requires that the diaphorase moiety of the complex be functional. However, there is no proportionality between NADH-diaphorase or NADH-nitrate reductase activities and the susceptibility of the enzymatic prepara- tion to NADH or NADPH. It seems likely that the nitrate reductase complex con- tains a specific regulatory site, different from the catalytic site, the reduction of which is accompanied by the production of an inactive form of the complex. Nitrat’e reductase (EC 1.6.6.1) from spinach catalyzes the reduction of nitrate to nitrite using NADH as electron donor (1). This enzyme complex, with a molecular weight around 500,000 (1, 2), has two different moieties : an FAD-dependent NADH-diaphorase, and an FNHz-nitrat’e reductase2 that contains molybdenum and catalyzes the reduction of nitrate using reduced flavine nucleotides as elect’ron donors (l-3). The two moieties of the com- plex are affected in different ways by certain treatments and inhibitors: NADH-diapho- rase is inact’ivated by heating at 45°C and by the sulfhydryl-group reagent, p-chloromer- curibenzoate, while FNHz-nitrate reductase is inactivated by cyanide and azide (1, 2). In the presence of KADH, the inactivation by cyanide takes place at a much lower con- centration (2). 1 This work was supported by a grant from Philips Research Laboratories to Prof. M. Losada. 2 Abbreviations : FNHz, reduced FAD or FMN ; DTE, 2,3-dihydroxy-1,4-dithiolbutane (dithio- erythritol). 441 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. In unicellular algae, inactivation of n- trate reductase by reduction and reactiva- tion by oxidation seems to be a mechanism of regulation that operates in response to different nutritional and physiological condi- tions (4-6). The nitrate reductases obtained from Chlorella fusca, C. vulgaris, and Chlamydomonas reinhardii are reversibly inactivated by NAD(P)H, reactivation being achieved by treatment with ferricyanide (5, 7-9). Therefore, it was of interest to study this process in a nitrate reductase from a higher plant, like spinach. This paper reports the reversible inactiva- tion of nitrate reductase from spinach by NAD (P)H and by simple thiols, and how the inactivation by NAD(P)H requires that the first moiety of the complex, namely NADH- diaphorase, be functional. MATERIALS AND METHODS Preparation of nitrate reductase. Nitrate reduc- tase was partially purified from spinach leaves (Spinacea oleracea L.) by a procedure described previously (lo), consisting of the following steps: (1) preparation of the cell-free extract, (2) passage