Formation of N-delta-cyanoornithine from N-G-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: Implications for mechanism

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of N-G-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N-delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O-2, and NHA (V-max,V-app = 49 +/- 2 nmol min(-1) mg(-1) for the reactions with 10 mu M added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K-m,K-app(H2O2) = 10 +/- 1 mM for the reactions with 10 mu M added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.