Oxidations of N-omega-hydroxyarginine analogues and various N-hydroxyguanidines by NO synthase II: Key role of tetrahydrobiopterin in the reaction mechanism and substrate selectivity

Oxidations of L-arginine 2, homo-L-arginine 1, their N-omega-hydroxy deriva tives 4 and 3 (NOHA and homo-NOHA, respectively), and four N-hydroxyguanidi nes, N-omega-hydroxynor-L-arginine 5 (nor-NOHA), N-omega-hydroxydinor-L-arg inine 6 (dinor-NOHA), N-(4-chlorophenyl)-N'-hydroxyguanidine (8), and N-hyd roxyguanidine (7) itself, by either NOS II or (6R)-5,6,7,8-tetrahydroL-biop terin (BH4)-free NOS II, have been studied in a comparative manner. Recombi nant BH4-free NOS II catalyzes the oxidation of all N-hydroxyguanidines by NADPH and Oz, with formation of NO2- and NO3- at rates between 20 and 80 nm ol min(-1) (mg of protein)(-1). In the case of compound 8, formation of the corresponding urea and cyanamide was also detected besides that of NO2- an d NO3-. These BH4-free NOS II-dependent reactions are inhibited by modulato rs of electron transfer in NOS such as thiocitrulline (TC) or imidazole (Im H), but not by Arg, and are completely suppressed by superoxide dismutase ( SOD), They exhibit characteristics very similar to those previously reporte d for microsomal cytochrome P450-catalyzed oxidation of N-hydroxyguanidines . Both P450 and BH4-free NOS II reactions appear to be mainly performed by O-2(.-) derived from the oxidase function of those heme proteins. In the pr esence of increasing concentrations of BH4, these nonselective oxidations p rogressively disappear while a much more selective monooxygenation takes pl ace only with the N-hydroxyguanidines that are recognized well by NOS II, N OHA, homo-NOHA, and 8. These monooxygenations are much more chemoselective (8 being selectively transformed into the corresponding urea and NO) and ar e inhibited by Arg but not by SOD, as expected for reactions performed by t he NOS Fe-II-O-2 species. Altogether, these results provide a further clear illustration of the key role of BH4 in regulating the monooxygenase/oxidas e ratio in NOS. They also suggest a possible implication of NOSs in the oxi dative metabolism of certain classes of xenobiotics such as N-hydroxyguanid ines, not only via their monooxygenase function but also via their oxidase function.