OXIDATION OF N-HYDROXYGUANIDINES BY CYTOCHROMES P450 AND NO-SYNTHASES AND FORMATION OF NITRIC OXIDE

Abstract

Microsomal cytochromes P450 and tetrahydrobiopterin (BH₄) free-NOS II catalyze the oxidation of N-hydroxyguanidines by NADPH and O₂ with formation of nitrogen oxides including NO. These reactions are not selective in terms of substrates, as they occur on most N-hydroxyguanidines, and of products, as they not only lead to corresponding ureas but also to cyanamides. These non selective reactions are mainly due to O^√−₂ derived from the oxidase function of those hemeproteins. By contrast, NO synthase (NOS) containing BH₄ catalyze the selective monooxygenation of some N-hydroxyguanidines by NADPH and O₂ with formation of NO and the corresponding ureas in a 1 : 1 molar ratio. Those reactions are not inhibited by superoxide dismutase (SOD) and are performed by the NOS Fe(II)–O₂ complex. The endogenous NOS substrate N^ω-hydroxy-L-arginine (NOHA), and its close analogue homo-NOHA, are selectively oxidized in this manner by NOS whereas nor-NOHA and dinor-NOHA are not. Moreover, some non α-amino acid N-hydroxyguanidines act as NOS substrates in a manner similar to NOHA. This includes a small number of simple N-alkyl N′-hydroxyguanidines with R(alkyl)=propyl, butyl, and pentyl, and some N-aryl N′-hydroxyguanidines that involve a relatively small and preferably electron-rich aryl substituent. The best exogenous substrate of NOS reported so far is N-butyl N′-hydroxyguanidine; this compound is oxidized by NOS II with formation of NO with a catalytic efficiency (kcat/Km) only two times lower than NOHA itself. N-butyl N′-hydroxyguanidine is also a good substrate for NOS I and NOS III. However, some N-aryl N′-hydroxyguanidines, with Ar=p-chlorophenyl and p-methylphenyl, are selective substrates of NOS II. These results show that exogenous N-hydroxyguanidines not bearing an α-amino acid function are efficiently and selectively oxidized by NOS with formation of NO. They open the way toward the research of new NO donors based on selective substrates of each class of NOS.

Key Words :

• Tetrahydrobiopterin
• L-arginine
• N^ω-hydroxy-L-arginine
• Superoxide radical monoxygenases
• N-Butyl N′-hydroxyguanidine