Culmodulin (CaM) binding to neuronal nitric oxide synthase (nNOS) allows NADPII-derived electrons to be transferred from the reductase domain to the oxygenase domain resulting in nitric oxide production. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the reaction between nitric oxide and Superoxide fO: ). we employed the adrenochrome assay to examine whether Ov could be detected during catalytic turnover ol" nNOS in the presence of L-arginine. In order to differentiate between the role played by the reductase domain from that of the oxygenase domain in O," production, we compared nNOS against a nNOS Cys331Ala mutant (CYS331 ). which, as isolated was impaired in the ability to transfer NADPHderived electrons to the heme iron, and against the flavoprotein module of nNOS. We report that O2· - production by nNOS and the CYS-331 mutant is CaM-dependent and that OV production can be modulated by substrates and inhibitors of nNOS. O 2· was also produced by the reductase domain of nNOS; however. it did not display the same CaM dependency. We conclude that both the reduclase and oxygenase domains of nNOS produce O2·-, but that the reductase domain is both necessary and sufficient for O2 production.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology