Thiol Modification and Site-Directed Mutagenesis of the Flavin Domain of Spinach NADH:Nitrate Reductase

Abstract

Incubation of eitherChlorellanitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such asN-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°C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both theChlorellaand spinach enzymes followed the orderp-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid >N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate orp-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 μmol NADH consumed/min/nmol FAD and an increase in theKmfor NADH from 12 to 35 μMwhen compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 μmol NADH consumed/min/nmol FAD with aKmfor NADH of 14 μM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2couple in the C54S (E0′ = −197 mV), C62S (E0′ = −226 mV), and C240S (E0′ = −219 mV) mutants compared to the wild-type domain (E0′ = −268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.