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 prop erties to those of NADPH-cytochrome P450 reductase (P450R), contains bindin g sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to el ucidate the mechanism of activation of the flavin domain by calcium/calmodu lin (Ca2+/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 t he corresponding FAD-FMNH. of P450R. In the absence or presence of Ca2+/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 tech nique and a rapid scan spectrophotometry. The reduction of the oxidized enz yme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-ele ctron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca2+/C aM. 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 Ca2+/CaM. The F1H2, which is the fully reduced form of the air-stable semiquinone, can donate o ne electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca2+/CaM complex is discussed on the basis of that provide d by P450R. (C) 1999 Elsevier Science B.V. All rights reserved.