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 domain of corn leaf NADH:nitrate reductase (EC 1.6.6.1) were modified by site-directed mutagenesis. At least two amino acid replacement mutants were generated for each of the 5 cysteines of this domain. Characteristics of the amino acid replacement mutants correlated well with the structural location of the cysteine residues in the preliminary three-dimensional 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 b5 reductase, as well as, most other members of this flavoenzyme family. No substitution for the invariant cysteine yielded highly active enzyme, although these mutants had normal visible spectra. When the invariant cysteine was mutated to serine, the cytochrome b reductase domain 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 invariant cysteine plays an important role in catalysis and may be essential for high catalytic efficiency of nitrate reductases.
Dwivedi
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UN
Phytotechnology Research Center, Michigan Technological University, Houghton 49931-1295.
Shiraishi
N
N
Campbell
W H
WH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000032:analysis*; D003579:Cytochrome Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004274:DNA, Recombinant; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D003313:Zea mays / Q000201:enzymology
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D003579:Cytochrome Reductases; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D003545:Cysteine