Formation and reactions of the heme-dioxygen intermediate in the first and second steps of nitric oxide synthesis as studied by stopped-flow spectroscopy under single-turnover conditions.
To better understand the mechanism of nitric oxide (NO) synthesis, we studied conversion of N-hydroxy-L-arginine (NOHA) or L-arginine (Arg) to citrulline and NO under single-turnover conditions using the oxygenase domain of neuronal nitric oxide synthase (nNOSoxy) and rapid scanning stopped-flow spectroscopy. When anaerobic nNOSoxy saturated with H(4)B and NOHA was provided with 0.5 or 1 electron per heme and then exposed to air at 25 degrees C, it formed 0.5 or 1 mol of citrulline/mol of heme, respectively, indicating that NOHA conversion had 1:1 stoichiometry with respect to electrons added. Identical experiments with Arg produced substoichiometric amounts of NOHA or citrulline even when up to 3 electrons were provided per heme. Transient spectral intermediates were investigated at 10 degrees C. For NOHA, four species were observed in the following sequence: starting ferrous nNOSoxy, a transient ferrous-dioxygen complex, a transient ferric-NO complex, and ferric nNOSoxy. For Arg, transient intermediates other than the ferrous-dioxygen species were not apparent during the reaction. Our results provide a kinetic framework for formation and reactions of the ferrous-dioxygen complex in each step of NO synthesis and establish that (1) the ferrous-dioxy enzyme reacts quantitatively with NOHA but not with Arg and (2) its reaction with NOHA forms 1 NO/heme, which immediately binds to form a ferric heme-NO complex.
Boggs
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Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Huang
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L
Stuehr
D J
DJ
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D002384:Catalysis; D002956:Citrulline / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000737:chemistry; D052248:Nitric Oxide Synthase Type I; D009573:Nitrites / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*; D051381:Rats; D013053:Spectrophotometry / Q000379:methods
D005296:Ferrous Compounds; D009573:Nitrites; D002956:Citrulline; D009569:Nitric Oxide; D006418:Heme; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C034236:ferrous oxide; D010100:Oxygen