Azo reduction of methyl red by neuronal nitric oxide synthase: The important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reduct ase domain. The reductase domain has NADPH, FAD, and FMN binding sites, Wil d-type nNOS reduced the azo bond of methyl red with a turnover number of ap proximately 130 min(-1) in the presence of Ca2+/calmodulin (CaM) and NADPH under anaerobic conditions, Diphenyleneiodonium chloride (DPI), a flavin/NA DPH binding: inhibitor, completely inhibited azo reduction. The omission of Ca2+/CaM from the reaction system decreased the activity to 5%, The rate o f the azo reduction with an FMN-deficient mutant was also 5% that of the wi ld type. NADPH oxidation rates for the wild-type and mutant enzymes were we ll coupled with azo reduction. Thus, we suggest that electrons delivered fr om the FMN of the nNOS enzyme reduce the azo bond of methyl red and that th is reductase activity is controlled by Ca2+/CaM. (C) 2000 Academic Press.