title: 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.
authors.affiliation: Interdepartmental Program in Medicinal Chemistry, College of Pharmacy University of Michigan, Ann Arbor 48109-1065.
authors.forename: R A
authors.initials: RA
authors.lastname: Pufahl
authors.forename: J S
authors.initials: JS
authors.lastname: Wishnok
authors.forename: M A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D002460:Cell Line; D002956:Citrulline / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D009249:NADP / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D010545:Peroxides / Q000378:metabolism; D020122:tert-Butylhydroperoxide
chemical_list: D001555:Benzene Derivatives; D009566:Nitrates; D009573:Nitrites; D010545:Peroxides; D002956:Citrulline; D009249:NADP; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C007164:cumene hydroperoxide; D010100:Oxygen