IDENTIFICATION OF AN ESSENTIAL CYSTEINE OF NITRATE REDUCTASE VIA MUTAGENESIS OF ITS RECOMBINANT CYTOCHROME-B REDUCTASE DOMAIN

Five cysteine residues in the recombinant cytochrome b reductase domai n of corn leaf NADH:nitrate reductase (EC 1.6.6.1) were modified by si te-directed mutagenesis. At least two amino acid replacement mutants w ere generated for each of the 5 cysteines of this domain. Characterist ics of the amino acid replacement mutants correlated well with the str uctural location of the cysteine residues in the preliminary three-dim ensional model of the cytochrome b reductase domain: somewhat exposed cysteines could be replaced by hydrophilic amino acid residues, while more buried cysteines by hydrophobic residues. An exception was found for the invariant cysteine near the C terminus, which is found in all nitrate reductases and also in the closely related NADH: cytochrome b( 5) reductase, as well as, most other members of this flavoenzyme famil y. No substitution for the invariant cysteine yielded highly active en zyme, although these mutants had normal visible spectra. When the in v ariant cysteine was mutated to serine, the cytochrome b reductase doma in was resistant to inhibition by pchloromercuribenzoate, an inhibitor of nitrate reductases. Kinetic analysis suggested that the catalytic efficiency of the mutant was markedly reduced. We concluded, the invar iant cysteine plays an important role in catalysis and may be essentia l for high catalytic efficiency of nitrate reductases.