ANALYSIS OF NEURONAL NO SYNTHASE UNDER SINGLE-TURNOVER CONDITIONS - CONVERSION OF N-OMEGA-HYDROXYARGININE TO NITRIC-OXIDE AND CITRULLINE

Nitric oxide synthases (NOSs) are proposed to generate NO and citrulli ne from L-arginine in two steps: initial N-hydroxylation to generate N -omega-hydroxyarginine (NOHA) followed by a three-electron oxidation o f the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation O-2. Studies done under mu ltiple-turnover conditions suggest that 0.5 mol of NADPH is consumed t o convert 1 mol of NOHA to products, implying that one electron from N ADPH 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, calmod ulin was then dissociated from nNOS to prevent subsequent heme iron re duction, NOHA was added, and the reaction initiated by exposure to air . Spectro obtained at each step were consistent with buildup of NOHA-b ound ferrous nNOS prior to air exposure. Reactions containing graded a mounts of nNOS produced L-citrulline in linear relation (1.2 +/- 0.1 m ol of citrulline per mole of nNOS). Nitrite and nitrate also accumulat ed 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 N OHA upon exposure to O-2. These data provide a molecular explanation f or the NADPH requirement in the second step of the biosynthetic reacti on, implicate ferrous-dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.