THIOL MODIFICATION AND SITE-DIRECTED MUTAGENESIS OF THE FLAVIN DOMAINOF SPINACH NADH-NITRATE REDUCTASE

Incubation of either Chlorella nitrate reductase or the recombinant fl avin domain of spinach nitrate reductase with reagents specific for mo dification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of in activation for both the Chlorella and spinach enzymes followed the ord er p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-malei midylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the s pinach flavin domain, inactivation by methyl methanethiosulfonate or p -chloromercuribenzoate was found to be concentration independent sugge sting the absence of nonspecific modifications. Initial rate studies o f the methyl methanethiosulfonate-modified flavin domain indicated a r eduction in NADH:ferricyanide activity (V-max) from 85 to 44 mu mol NA DH consumed/min/nmol FAD and an increase in the K-m for NADH from 12 t o 35 mu M when compared to the native enzyme, confirming a role for cy steine residue(s) in maintaining diaphorase activity. Site-directed mu tagenesis of the four individual cysteines (residues 17, 54, 62, and 2 40) in the recombinant spinach flavin domain resulted in mutant protei ns with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of seri ne for cysteine 240 decreased diaphorase activity with maximal NADH:fe rricyanide activity for the C240S mutant corresponding to 51 mu mol NA DH consumed/min/nmol FAD with a K-m for NADH of 14 mu M. Mutation of C 240 to Ala or Gly resulted in greater loss of activity. The thermal st ability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest inst ability. In contrast to the effects on diaphorase activity, square wav e voltammetric studies indicated changes in the oxidation-reduction mi dpoint potential for the FAD/FADH(2) couple in the C54S (E(0') = -197 mV), C62S (E(0)' = -226 mV), and C240S (E(0)' = -219 mV) mutants compa red to the wild-type domain (E(0)' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase fl avin domain, only C240 plays a role in maintaining diaphorase activity , while C54 has the greatest influence on flavin redox potential and t hat no correlation between changes in catalytic activity and flavin re dox potential was observed. (C) 1996 Academic Press, Inc.