Hydrogen peroxide-supported oxidation of NG-hydroxy-L-arginine by nitric oxide synthase.

Abstract

The ability of murine macrophage nitric oxide synthase (NOS) to utilize peroxides in place of O2 and NADPH was investigated using hydrogen peroxide (H2O2), tert-butylhydroperoxide, and cumene hydroperoxide with both L-arginine and NG-hydroxy-L-arginine (L-NHA) as substrates. Of the three peroxides examined, only H2O2 was able to support product formation using L-NHA as a substrate. No product formation was observed from L-arginine with any peroxide tested. Therefore, the L-NHA/H2O2 reaction was examined in greater detail. The products of the reaction were 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 Km and Vmax of the reaction, determined under aerobic conditions, were affected by (6R)-tetrahydro-L-biopterin (H4B). Chemiluminescence experiments failed to detect nitric oxide (.NO) as a reaction product. However, spectral spectral experiments with L-NHA and H2O2 under anaerobic conditions demonstrated the appearance of a ferrous heme-.NO 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) complex. Preliminary experiments detected nitrous oxide (N2O) formation by gas chromatography under anaerobic conditions. Stable isotope labeling experiments with [18O]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.