Neuronal nitric oxide synthase (nNOS) contains FMN, FAD and heme bound to distinct structural domains in stoichiometric amounts. Urea has been employed as a perturbant to correlate the binding of flavins with structural changes in nNOS. Changes in the fluorescence of flavins are thought to arise from changes in the binding interactions of the flavins with the protein. The rate of, as well as the extent of, total flavin release varied as a function of the [urea] (0-8 M). However, analysis of ultrafiltrates showed that FMN was released at lower [urea] than FAD. Thus, at 2 M urea, 65% of FMN had been released, while all of the FAD remained bound. At 4 M urea, 100% of both FMN and FAD were released. These data indicate that the structural interactions involved in the flavin domains of nNOS are analogous to those of NADPH-cytochrome P450 reductase, another flavoprotein containing FMN and FAD in stoichiometric quantities. However, important distinctions exist between the two proteins. For example, in the case of reductase, changes in trp/tyr fluorescence qualitatively paralleled changes in unbound [FAD]. In other words, trp/tyr fluorescence values were constant from 0-1 M urea and increased continuously between 2-8 M urea. In contrast, increases in trp/tyr fluorescence were observed through the entire range of [urea] employed (0-8 M). Further studies are in progress to examine the influence of urea on catalytic activity of nNOS and on the structure of the heme and flavin domains.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology