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 polypep tide subunit is approximately 100 kDa in size and contains three separ ate domains, one each for a flavin, a heme-iron, and a molybdopterin c ofactor. The heme-iron domain of nitrate reductase has homology with t he simple redox protein, cytochrome b5, whose crystal structure was us ed to predict a three-dimensional structure for the heme domain. Two h istidine residues have been identified that appear to coordinate the i ron of the heme moiety, while other residues may be important in the f olding or the function of the heme pocket. Site-directed mutagenesis w as employed to obtain mutants that encode nitrate reductase derivative s 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 nonfunctio nal enzyme whereas replacement of the other histidine resulted in a st able and functional enzyme with a lower affinity for heme. Certain ami no acid substitutions appeared to cause a rapid turnover of the heme d omain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacemen ts within the heme domain led to a dramatic change in regulation of ni trate reductase synthesis, with significant expression of the enzyme e ven in the absence of nitrate induction.