Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach

Abstract

Chemical modifications of spinach leaf nitrate reductase, and its 28 000 M r fragment with phenylglyoxal, 2,3-butanedione and pyridoxal phosphate reduce the catalytic activity of the enzyme. The kinetics of the modification indicate a rapid inactivation followed by a slower rate of inactivation. NADH-nitrate reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase activities of the nitrate reductase complex are inactivated at a faster rate when compared to the loss of FMNH 2-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28 000 M r fragment of nitrate reductase. These data suggest that nitrate reductase contains active sites of arginine and lysine residues that are involved in the NADH binding site of the enzyme.