Formation and reactions of the heme-dioxygen intermediate in the first andsecond 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 studi ed conversion of N-hydroxy-L-arginine (NOHA) or L-arginine (Arg) to citrull ine and NO under single-turnover conditions using the oxygenase domain of n euronal nitric oxide synthase (nNOSoxy) and rapid scanning stopped-flow spe ctroscopy. When anaerobic nNOSoxy saturated with H4B 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 th at NOHA conversion had 1:1 stoichiometry with respect to electrons added. I dentical experiments with Arg produced substoichiometric amounts of NOHA or citrulline even when up to 3 electrons were provided per heme. Transient s pectral intermediates were investigated at 10 degrees C. For NOHA, four spe cies were observed in the following sequence: starting ferrous nNOSoxy, a t ransient ferrous-dioxygen complex, a transient ferric-NO complex, and ferri c nNOSoxy. For Arg, transient intermediates other than the ferrous-dioxygen species were not apparent during the reaction. Our results provide a kinet ic 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 comp lex.