The Influence of Chaotropic Reagents on Neuronal Nitric Oxide Synthase and Its Flavoprotein Module. Urea and Guanidine Hydrochloride Stimulate NADPH–Cytochrome c Reductase Activity of Both Proteins

Abstract

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P.M. Horowitz, and B.S.S. Masters (1995)Arch. Biochem. Biophys.316, 267–274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2murea. Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH–cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3–4murea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/CaCl2and 0.5mGnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4murea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.