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

M Miyajima, I Sagami, S Daff, C T Migita, T Shimizu

Research output: Contribution to journalArticlepeer-review

Abstract

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 Ca2+/calmodulin (CaM) and NADPH under anaerobic conditions, Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding: inhibitor, completely inhibited azo reduction. The omission of Ca2+/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 Ca2+/CaM. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)752-758
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume275
Issue number3
Publication statusPublished - 7 Sep 2000

Keywords

  • nitric oxide synthase
  • electron transfer
  • site-directed mutagenesis
  • azo reduction
  • methyl red
  • FMN
  • NADPH-cytochrome P450 reductase
  • SUPEROXIDE GENERATION
  • ELECTRON-TRANSFER
  • DISTAL SITE
  • L-ARGININE
  • OXYGEN
  • TETRAHYDROBIOPTERIN
  • CYTOCHROME-P450
  • MECHANISM
  • DOMAIN
  • ACTIVATION

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