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Reduction of tertiary amine N-oxides by liver microsomal cytochrome P-450.

Reduction of tertiary amine N-oxides in rat liver was investigated with imipramine N-oxide, tiaramide N-oxide, and N,N-dimethylaniline N-oxide as substrates in the presence of NADPH. In hepatocytes most of the activities were located in the microsomal fraction; the activities of other subcellular fractions were low or negligible. NADPH-cytochrome c reductase, solubilized by trypsin or subtilisin, showed no N-oxide reductase activity by itself. This evidence clearly distinguishes N-oxide reductase from nitro and azo reductase activities. The characteristics of N-oxide reductase activity associated with cytochrome P-450 are as follows. (a) The activity of N-oxide reductase is inhibited by an atmosphere of carbon monoxide. The concentration of carbon monoxide giving apparent 50% inhibition of imipramine N-oxide reduction is 1.0 µM. The inhibition of N-oxide reductase activity by carbon monoxide is partially reversed by exposure to white light. (b) The activity is also inhibited in the presence of oxygen, with 50% inhibition occurring at an oxygen concentration of 2-3 µM. (c) Kinetic studies show that the Km values for the reduction of imipramine N-oxide, tiaramide N-oxide, and N,N-dimethylaniline N-oxide are on the order of 0.1 mM. (d) N-Oxide reductase activity is inhibited by nitro, nitroso, and azo compounds and hydrazide, while it is stimulated markedly by flavins. (e) The results of stoichiometric studies show that the ratio between reduced pyridine nucleotide consumption and tertiary amine formation is 1:1, and therefore it is presumed that the final products are a tertiary amine and water. The results obtained suggest that 2 electrons are transferred directly from ferrous iron of cytochrome P-450 to the N-oxide in the reaction. The following observations suggest that N-oxide coordinates to heme iron at the sixth position: first, reduction of N-oxides was inhibited by oxygen; second, N-oxides produced spectral changes with reduced cytochrome P-450; finally, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine, which form a hemichrome with reduced cytochrome P-450, markedly inhibited N-oxide reduction. The close relationship observed between cytochrome P-450 content and N-oxide reductase activity in microsomes from phenobarbital-treated rats of either sex and various ages further supports our hypothesis that NADPH-dependent N-oxide reduction is catalyzed by cytochrome P-450 of liver microsomes.