論文

査読有り 国際誌
2007年9月1日

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain.

Archives of biochemistry and biophysics
  • Yoshitaka Nishino
  • ,
  • Keita Yamamoto
  • ,
  • Shigenobu Kimura
  • ,
  • Akihiro Kikuchi
  • ,
  • Yoshitsugu Shiro
  • ,
  • Takashi Iyanagi

465
1
開始ページ
254
終了ページ
65
記述言語
英語
掲載種別
研究論文(学術雑誌)

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.

リンク情報
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/17610838

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