Abstract
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.
Highlights
Erated foreach of the 5 cysteines of this domain
Proteolytic degrelated well with the structural locationof the cysteine radation of the native, intact nitrate reductase showed that residues in the preliminary three-dimensional modeolf largefragments of nitratereductase could be cleaved and the cytochrome b reductase domain: somewhat exposed physically separated with retention of the characteristic partial cysteines could be replaced by hydrophilic amino acid residues, while more buried cysteines by hydrophobic residues.An exception was found forthe invariant cysteine near the C terminus, whichis found in all nitrate reductasesand in the closely related NADH: activities of the cofactor-binding domains (1, 6).Subsequently, individual domains and a combination of two domains were overexpressed in Escherichiacoli using fragments of cDNA clones (7-10)
Kinetic analysis suggested that the catalytic efficiency b reductase domain of corn leaf NADH:nitrate reductase exof the mutant was markedly reduceW d,e concluded,the pressed in E. coli, which we have previously called the FAD or invariant cysteine plays an important role in catalysis flavin domain (4,7)
Summary
Impact on the Cytochrome b Reductase Domain of Amino Acid Substitutions for Cysteine Residues-Each of the 5 cyssodium phosphate, pH 7.3, 1mM EDTA. The en- teine residues was replaced in the recombinant cytochrome b reductase domain of corn leaf NADH:nitratereductase (Fig. 1). The abbreviation used is: pCMB, p-chloromercuribenzoic acid. When a singleserine was substitutedfor a cysteine residue, the five resulting mutant cytochrome b reductase domains were.
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