Formation of Nδ-Cyanoornithine from NG-Hydroxy-l-arginine and Hydrogen Peroxide by Neuronal Nitric Oxide Synthase: Implications for Mechanism

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Abstract
Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-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 Nδ-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, O2, and NHA (Vmax,app = 49 ± 2 nmol min-1 mg-1 for the reactions with 10 μM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [Km,app(H2O2) = 10 ± 1 mM for the reactions with 10 μM 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.

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