Spinach nitrate reductase. Kinetic studies of NADH-diaphorase

Abstract

NADH-cytochrome c reductase (diaphorase), a functional moiety of the spinach NADH-nitrate reductase complex, uses one molecule of NADH to reduce two of ferricytochrome c. Initial velocity experiments in the absence of products produce patterns of parallel lines when plotting 1/ v vs. 1/(substrate) at different fixed concentrations of the other substrate. The results agree with a Ping Pong mechanism in which the first product (NAD +) is released, with formation of a reduced free form of the enzyme, prior to binding of the first molecule of the second substrate (ferricytochrome c). Product inhibition experiments indicate the production of an abortive complex between NAD + and the oxidized enzyme form that binds NADH. The data are consistent with a Hexa Uni Ping Pong mechanism in which an abortive complex is formed between NAD + and the enzyme form binding NADH.