Interflavin one-electron transfer in the inducible nitric oxide synthase reductase domain and NADPH-cytochrome P450 reductase.
In this study, we have analyzed interflavin electron transfer reactions from FAD to FMN in both the full-length inducible nitric oxide synthase (iNOS) and its reductase domain. Comparison is made with the interflavin electron transfer in NADPH-cytochrome P450 reductase (CPR). For the analysis of interflavin electron transfer and the flavin intermediates observed during catalysis we have used menadione (MD), which can accept an electron from both the FAD and FMN sites of the enzyme. A characteristic absorption peak at 630 and 520 nm can identify each FAD and FMN semiquinone species, which is derived from CPR and iNOS, respectively. The charge transfer complexes of FAD with NADP+ or NADPH were monitored at 750 nm. In the presence of MD, the air-stable neutral (blue) semiquinone form (FAD-FMNH*) was observed as a major intermediate during the catalytic cycle in both the iNOS reductase domain and full-length enzyme, and its formation occurred without any lag phase indicating rapid interflavin electron transfer following the reduction of FAD by NADPH. These data also strongly suggest that the low level reactivity of a neutral (blue) FMN semiquinone radical with electron acceptors enables one-electron transfer in the catalytic cycle of both the FAD-FMN pairs in CPR and iNOS. On the basis of these data, we propose a common model for the catalytic cycle of both CaM-bound iNOS reductase domain and CPR.
Yamamoto
Keita
K
Graduate School of Life Science, Himeji Institute of Technology, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Hyogo 678-1297, Japan.
Kimura
Shigenobu
S
Shiro
Yoshitsugu
Y
Iyanagi
Takashi
T
D001483:Base Sequence; D002384:Catalysis; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D011809:Quinones / Q000737:chemistry; D013053:Spectrophotometry
D005415:Flavins; D011809:Quinones; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D009251:NADPH-Ferrihemoprotein Reductase