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

• Published in: Biochemistry (Easton) Vol. 39; no. 9; pp. 2332 - 2339
• Main Authors: Boggs, Susan, Huang, Liuxin, Stuehr, Dennis J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.03.2000
• Subjects: Animals; Arginine - analogs & derivatives; Arginine - chemistry; Catalysis; Citrulline - chemistry; Ferrous Compounds - chemistry; Heme - chemistry; Kinetics; Models, Chemical; Nitric Oxide - biosynthesis; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase Type I; Nitrites - chemistry; Oxygen - chemistry; Rats; Spectrophotometry - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9920228

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 H4B and NOHA was provided with 0.5 or 1 electron per heme and then exposed to air at 25 °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 °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.