Analysis of Neuronal NO Synthase under Single-Turnover Conditions: Conversion ofNω-Hydroxyarginine to Nitric Oxide and Citrulline

Abstract

Nitric oxide synthases (NOSs) are proposed to generate NO and citrulline from l-arginine in two steps: initial N-hydroxylation to generate N^ω-hydroxyarginine (NOHA) followed by a three-electron oxidation of the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation of O₂. Studies done under multiple-turnover conditions suggest that 0.5 mol of NADPH is consumed to convert 1 mol of NOHA to products, implying that one electron from NADPH may be sufficient. To test this, we studied NOHA oxidation under single-turnover conditions using neuronal NOS (nNOS), whose heme iron reduction requires bound calmodulin. The heme iron in calmodulin-bound nNOS was reduced with excess NADPH under anaerobic conditions, calmodulin was then dissociated from nNOS to prevent subsequent heme iron reduction, NOHA was added, and the reaction initiated by exposure to air. Spectra obtained at each step were consistent with buildup of NOHA-bound ferrous nNOS prior to air exposure. Reactions containing graded amounts of nNOS produced l-citrulline in linear relation (1.2 ± 0.1 mol of citrulline per mole of nNOS). Nitrite and nitrate also accumulated as NO-derived products. Control reactions that contained l-arginine instead of NOHA, no enzyme, or ferric nNOS did not generate products. Thus, supplying a single electron from NADPH to the heme iron permits nNOS to catalyze one full round of citrulline and NO synthesis from NOHA upon exposure to O₂. These data provide a molecular explanation for the NADPH requirement in the second step of the biosynthetic reaction, implicate ferrous−dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.