Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain.
The neuronal NO synthase (nNOS) flavin domain, which has similar redox properties to those of NADPH-cytochrome P450 reductase (P450R), contains binding sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium/calmodulin (Ca(2+)/CaM). In this study, we used the recombinant nNOS flavin domains, which include or delete the calmodulin (CaM)-binding site. The air-stable semiquinone of the nNOS flavin domains showed similar redox properties to the corresponding FAD-FMNH(&z.ccirf;) of P450R. In the absence or presence of Ca(2+)/CaM, the rates of reduction of an FAD-FMN pair by NADPH have been investigated at different wavelengths, 457, 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry. The reduction of the oxidized enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH. The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca(2+)/CaM. The F(1)H(2), which is the fully reduced form of the air-stable semiquinone, can donate one electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca(2+)/CaM complex is discussed on the basis of that provided by P450R.
Matsuda
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H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, Japan.
Iyanagi
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T
D001665:Binding Sites; D002147:Calmodulin / Q000494:pharmacology*; D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005415:Flavins / Q000737:chemistry*; D009249:NADP / Q000737:chemistry; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D010957:Plasmids; D013056:Spectrophotometry, Ultraviolet
D002147:Calmodulin; D003574:Cytochrome c Group; D005415:Flavins; D009569:Nitric Oxide; D009249:NADP; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I