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 reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.
Miyajima
M
M
Institute for Chemical Reaction Science, Sendai, 980-8577, Japan.
Sagami
I
I
Daff
S
S
Taiko Migita
C
C
Shimizu
T
T
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D016296:Mutagenesis; D009249:NADP / Q000037:antagonists & inhibitors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013053:Spectrophotometry; D013113:Spin Labels
D001391:Azo Compounds; D002147:Calmodulin; D009419:Nerve Tissue Proteins; D009861:Onium Compounds; D013113:Spin Labels; D009249:NADP; C008492:methyl red; C007517:diphenyleneiodonium; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D010100:Oxygen; D002118:Calcium