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Chem Res Toxicol. 2001 Feb;14(2):202-10.

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.

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Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris V, 45 Rue des Saints-Pères, 75270 Paris Cedex 06, France.


Oxidations of L-arginine 2, homo-L-arginine 1, their N(omega)-hydroxy derivatives 4 and 3 (NOHA and homo-NOHA, respectively), and four N-hydroxyguanidines, N(omega)-hydroxynor-L-arginine 5 (nor-NOHA), N(omega)-hydroxydinor-L-arginine 6 (dinor-NOHA), N-(4-chlorophenyl)-N'-hydroxyguanidine (8), and N-hydroxyguanidine (7) itself, by either NOS II or (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4)-free NOS II, have been studied in a comparative manner. Recombinant BH4-free NOS II catalyzes the oxidation of all N-hydroxyguanidines by NADPH and O2, with formation of NO2(-) and NO3(-) at rates between 20 and 80 nmol 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(-) and NO3(-). These BH4-free NOS II-dependent reactions are inhibited by modulators of electron transfer in NOS such as thiocitrulline (TC) or imidazole (ImH), but not by Arg, and are completely suppressed by superoxide dismutase (SOD). They exhibit characteristics very similar to those previously reported for microsomal cytochrome P450-catalyzed oxidation of N-hydroxyguanidines. Both P450 and BH4-free NOS II reactions appear to be mainly performed by O2(.-) derived from the oxidase function of those heme proteins. In the presence of increasing concentrations of BH4, these nonselective oxidations progressively disappear while a much more selective monooxygenation takes place only with the N-hydroxyguanidines that are recognized well by NOS II, NOHA, homo-NOHA, and 8. These monooxygenations are much more chemoselective (8 being selectively transformed into the corresponding urea and NO) and are inhibited by Arg but not by SOD, as expected for reactions performed by the NOS Fe(II)-O2 species. Altogether, these results provide a further clear illustration of the key role of BH4 in regulating the monooxygenase/oxidase ratio in NOS. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics such as N-hydroxyguanidines, not only via their monooxygenase function but also via their oxidase function.

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