Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain.
The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.
Nishino
Yoshitaka
Y
Graduate School of Life Science, Himeji Institute of Technology, University of Hyogo, Kouto 3-2-1, Kamigori, Hyogo 678-1297, Japan.
Yamamoto
Keita
K
Kimura
Shigenobu
S
Kikuchi
Akihiro
A
Shiro
Yoshitsugu
Y
Iyanagi
Takashi
T
D003198:Computer Simulation; D004579:Electron Transport; D005415:Flavins / Q000737:chemistry*; D008956:Models, Chemical; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D017434:Protein Structure, Tertiary
D005415:Flavins; D010088:Oxidoreductases; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III