Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.