NO accounts completely for the oxygenated nitrogen species generated by enzymic L-arginine oxygenation

Abstract

We have assessed the stoichiometry of the nitric oxide (NO) synthase reaction by using a novel e.p.r. technique. NO generated by crude and partially purified NO synthase from endothelial cells and Escherichia coli-lipopolysaccharide-activated macrophages was trapped by a ferrous diethyldithiocarbamate complex dispersed in yeast. The paramagnetic ferrous mononitrosyl dithiocarbamate complex formed exhibited a characteristic e.p.r. signal at g perpendicular = 2.035 and g parallel = 2.02 with a triplet hyperfine structure (hfs) at g perpendicular. NO, 3-morpholinosydnonimine and S-nitroso-L-cysteine, but not nitrite or hydroxylamine, generated a similar e.p.r. signal. NO generated by NO synthase and by SIN-1 accumulated at a constant rate for 1 h, as measured by continuous e.p.r. registration at 37 degrees C. The formation of e.p.r.-detectable NO by NO synthases was inhibited by NG-nitro-L-arginine. Incubation with [15N]NG-L-arginine caused an e.p.r. signal with doublet hfs, indicating that the nitrosyl nitrogen derived exclusively from the guanidino nitrogen. The amount of NO generated by NO synthase as measured by e.p.r. technique was compared with formation of L-[3H]citrulline from L-[3H]arginine. NO and L-citrulline were detected at a 1:1 ratio with both NO synthase preparations. GSH and thiol depletion did not significantly affect NO synthase activity, excluding S-nitrosothiols as intermediates in the NO synthase reaction. We conclude that NO fully accounts for the immediate oxygenated nitrogen species derived from the enzymic oxygenation of L-arginine.