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MOLECULAR PHARMACOLOGY 51:147-151 (1997).

Evidence for a Role of a Perferryl-Oxygen Complex, FeO³⁺, in the N-Oxygenation of Amines by Cytochrome P450 Enzymes

F. Peter Guengerich, Alfin D. N. Vaz, Gregory N. Raner, Steven J. Pernecky, and Minor J. Coon

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 (F.P.G.), and Department of Biological Chemistry, the University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 (A.D.N.V., G.N.R., S.J.P., M.J.C.)

Most cytochrome P450 (P450)-catalyzed reactions are believed to involve an FeO³⁺ intermediate as the actual oxygenating species. However, studies on the mechanism of steroid aromatization and subsequent model work have provided evidence that a peroxo-iron form (formally FeO₂⁺) can be involved directly in some oxidations. The possible involvement of peroxo-iron was considered in P450-catalyzed N-oxygenations, because there is precedent for the use of H₂O₂ and organic peroxides in such reactions in the literature concerning synthetic and flavin reactions. The approach used was to compare P450 reactions involving the normal NADPH/NADPH-P450 reductase/O₂ system with those supported by the oxygen surrogates H₂O₂ (which can directly form FeO₂⁺ and subsequently FeO³⁺) and iodosylbenzene (which can form FeO³⁺ but not FeO₂⁺). Iodosylbenzene was effective in supporting rabbit P450 1A2-catalyzed N,N-dimethyl-2-aminofluorene N-oxygenation, human P450 3A4-catalyzed quinidine N-oxygenation, rat P450 2B1-catalyzed oxidation of N-benzyl-(1-phenyl) cyclobutylamine to the N-hydroxyamine and nitrone, and rat P450 2B1-catalyzed and rabbit P450 2B4-catalyzed N-oxygenation of N,N-dimethylaniline (also N-demethylation). H₂O₂ also supported most of these reactions. A mutant of P450 2B4 with the substitution of alanine for threonine at position 302 has been shown to have decreased ability to catalyze reactions involving the putative FeO³⁺ but, presumably because of decreased ability to protonate the FeO₂⁺ complex, to have enhanced activity in oxidative deformylation reactions believed to involve FeO₂⁺. This mutant showed both decreased N,N-dimethylaniline N-demethylation and N-oxygenation activity. Although some contribution of an FeO₂⁺ species to these reactions cannot be ruled out, formation of product in the iodosylbenzene-supported systems cannot be readily explained by an obligatory FeO₂⁺ mechanism and the involvement of FeO³⁺ is concluded to be more likely.