Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

Abstract

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K(m) values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K(m) values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.Unlike that of the wild-type, the nitrate reductase from nar1a did not bind to blue dextran-Sepharose. The nar1a enzyme did bind to Affi Gel Blue, but recoveries were low. The NADH and NADPH nitrate reductase activities of nar1a were not separated by affinity chromatography. The nitrate reductase in nar1a is a different enzyme than the wild-type NADH nitrate reductase and appears to be a NAD(P)H-bispecific enzyme.