Peroxynitrite reduction of calmodulin stimulation of neuronal nitric oxide synthase.
The Ca(2+)-dependent binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) stimulates the catalytic oxidation of L-arginine to nitric oxide. The CaM-dependent increase in catalytic activity is associated with an increase in the flow of electrons from the flavoprotein to the heme domain. In the presence of suboptimal arginine concentrations, uncoupled turnover of nNOS produces both nitric oxide and superoxide, reactive species which combine to form peroxynitrite. We demonstrate here that peroxynitrite and other oxidants produced by nNOS oxidize the methionine residues of CaM and show that the ability of CaM to stimulate nNOS is impaired by this oxidative modification. Of the nine Met residues, those at the C-terminus (Met-144, -145, -124, -109) are most sensitive to oxidation. Correlation of the Met oxidation pattern with ability to stimulate nNOS suggests that oxidation of Met-36 is particularly important for the stimulation of nNOS. Incubation of nNOS with suboptimal concentrations of arginine results in sulfoxidation of the CaM methionine residues. Although nitration of the tyrosine residues in CaM could also occur, this does not occur to a significant extent in the present system. The results suggest that peroxynitrite may exert a feedback effect on its own formation by oxidizing CaM and thereby decreasing its ability to stimulate the turnover of nNOS.
Hühmer
A F
AF
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045, USA.
Gerber
N C
NC
de Montellano
P R
PR
Schöneich
C
C
D001667:Binding, Competitive; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000187:drug effects; D015536:Down-Regulation; D005246:Feedback / Q000187:drug effects; D007700:Kinetics; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000187:drug effects*; D013481:Superoxides / Q000032:analysis
D002147:Calmodulin; D003574:Cytochrome c Group; D009566:Nitrates; D013481:Superoxides; C039642:peroxynitric acid; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase