Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N ‐hydroxyguanidines by hydrogen peroxide

Nitric oxide (NO) is a potent intra‐ and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l ‐arginine catalysed by hemeproteins called NO‐synthases with intermediate formation of N ω ‐hydroxy‐ l ‐arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N ‐(4‐chlorophenyl)‐ N ′‐hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4‐chlorophenylcyanamide was observed from incubations of N ‐(4‐chlorophenyl)‐ N ′‐hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, N δ ‐cyano‐L‐ornithine, was too unstable under the conditions used and l ‐citrulline was the only product identified. A NO‐specific conversion of 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide to 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl and formation of MP8–Fe–NO complexes were observed by EPR spectroscopy and low‐temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N ‐hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo .