Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis.
The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.
Okamoto
P M
PM
Department of Biochemistry, Ohio State University, Columbus 43210.
Marzluf
G A
GA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D015139:Blotting, Southern; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D014170:Transformation, Genetic
D000596:Amino Acids; D006418:Heme; D006639:Histidine; D009565:Nitrate Reductases; D050901:Nitrate Reductase