Abstract

Previous investigations have shown that the antiprotozoal drug pentamidine is N-hydroxylated by rabbit and rat liver microsomal fractions. Indirect evidence for the participation of the cytochrome P-450 enzyme system was obtained. In this study, rabbit liver cytochrome P-450 2C3 is shown by reconstitution experiments with highly purified variants of P-450 2C3 isolated from rabbit liver and purified variants of P-450 2C3 expressed by recombinant Escherichia coli to be a microsomal pentamidine N-hydroxylase. The two variants, P-450 2C3 (6 beta H) and P-450 2C3 (6 beta L), are equally efficient for the formation of the monoamidoxime derivative of pentamidine. N-hydroxypentamidine is further oxidized to the respective amide by reconstituted rabbit liver P-450 enzyme systems involving the oxidase and peroxidase activities of this enzyme. Formation of nitric oxide [(NO); endothelium-derived relaxing factor] during this oxidation is shown by the detection of the cytochrome P-420-Fe(II)-NO complex by visible difference spectroscopy. The possibility for the N-hydroxylation of pentamidine to the corresponding amidoximes and subsequent oxidative conversion to the respective amide derivatives is comparable with the physiological transformation of arginine to citrulline via N-hydroxyarginine with liberation of NO (endothelium-derived relaxing factor). The N-hydroxylated derivatives of pentamidine are easily retroreduced by microsomal fractions from rabbit liver. NADH is preferred to NADPH as cofactor for this reduction, and the reaction is strongly suppressed by the addition of N-methylylhydroxylamine. The N-hydroxylation of pentamidine and the retroreduction are also catalyzed by human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)