Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains.
Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.
Guan
Zhi-Wen
ZW
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, 678-1279 Japan.
Kamatani
Daiki
D
Kimura
Shigenobu
S
Iyanagi
Takashi
T
D002147:Calmodulin / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D009249:NADP; D005486:Flavin Mononucleotide; C496320:NOS1 protein, human; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II