HYDROGEN PEROXIDER-SUPPORTED OXIDATION OF N-G-HYDROXY-L-ARGININE BY NITRIC-OXIDE SYNTHASET

The ability of murine macrophage nitric oxide synthase (NOS) to utiliz e peroxides in place of O-2 and NADPH was investigated using hydrogen peroxide (H2O2), tert-butylhydroperoxide, and cumene hydroperoxide wit h both L-arginine and N-G-hydroxy-L-arginine (L-NHA) as substrates. Of the three peroxides examined, only H2O2 was able to support product f ormation using L-NHA as a substrate. No product formation was observed from L-arginine with any peroxide tested. Therefore, the L-NHA/H2O2 r eaction was examined in greater detail. The products of the reaction w ere citrulline and nitrite/nitrate (NO2-/NO3-) with a stoichiometry of approximately 0.75:1 (citrulline to NO2-/NO3-). Product formation was greater in the presence of oxygen. Both the K-m and V-max of the reac tion, determined under aerobic conditions, were affected by (6R)-tetra hydro-L-biopterin (H4B). Chemiluminescence experiments failed td detec t nitric oxide ((NO)-N-.) as a reaction product. However, spectral exp eriments with L-NHA and H2O2 under anaerobic conditions demonstrated t he appearance of a ferrous heme-(NO)-N-. complex with a Soret peak at 440 nm and a broad single alpha/beta peak at 578 nm, which is believed to arise from single electron transfer of a ferric-NO- (nitroxyl) com plex. Preliminary experiments detected nitrous oxide (N2O) formation b y gas chromatography under anaerobic conditions. Stable isotope labeli ng experiments with [O-18]H2O2 conclusively established incorporation of label exclusively into the ureido position of citrulline. Based on these results, a mechanism of oxidation of L-NHA by H2O2 is proposed.