Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical eel filtration, consisted of a mixture of monomers and dimers, H4B-free NOS catalyzed the oxidation of N-G-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O-2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O-2 reactions were determined to be citrulline and N-delta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (. NO) in the NADPH/O-2 reaction. The initial inorganic product of the NADPH/O-2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH, NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degrees C, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min(-1) mg(-1) and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min(-1) mg(-1) with a K-M,K-app for NHA of 129 +/- 9 mu M. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min(-1) mg(-1) and the NADPH-dependent reaction to produce . NO and citrulline at 171 +/- 20 nmol min(-1) mg(-1) with a K-M,K-app for NHA in the NADPH reaction of 36.9 +/- 0.3 mu M.