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Ipso-Substitution by Cytochrome P450 with Conversion of p-Hydroxybenzene Derivatives to Hydroquinone: Evidence for Hydroperoxo-Iron As the Active Oxygen Species

https://doi.org/10.1006/abbi.2001.2665Get rights and content

Abstract

Evidence for multiple functional active oxidants in cytochrome P450-catalyzed reactions was previously obtained in this laboratory with mutants in which proton delivery was perturbed by replacement of the highly conserved threonine residue in the active site by alanine, thus apparently interfering with the conversion of the peroxo-iron to the hydroperoxo-iron and the latter to the oxenoid-iron species. These enzymes have now been employed to examine the reaction in which cytochrome P450 in liver microsomes is known to effect ipso-substitution, the elimination of p-substituents in phenols to yield hydroquinone. As shown with purified NH2-truncated cytochromes in a reconstituted enzyme system, the reaction exhibits an absolute requirement for cytochrome P450 and NADPH–cytochrome P450 reductase. Under optimal conditions truncated cytochrome P450 2E1 is active with 10 of the p-substituted phenols examined. Of particular interest, the corresponding cytochrome with threonine-303 replaced by alanine is from 1.5- to 50-fold higher in activity with the p-chloro, -bromo, -nitro, -cyano, -hydroxymethyl, -formyl, and -acetyl derivatives, and the reaction with the p-benzoyl, -methyl, and -t-butyl compounds is catalyzed by the mutant enzyme only. The results implicate the hydroperoxo-iron species as an electrophilic active oxidant in cytochrome P450-catalyzed aromatic ipso-substitution.

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    This investigation was supported by Grant DK-10339 from the National Institutes of Health. Preliminary reports of this study have been presented at the 12th International Symposium on Microsomes and Drug Oxidations (Abstract No. 410; Montpellier, France, 1998), the 11th International Conference on Cytochrome P450 (Abstract No. P-36; Sendai, Japan, 2000), and the annual meeting of Experimental Biology (FASEB J.14, A1335, 2000).

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    To whom correspondence and reprint requests should be addressed: Fax: (734) 763-4581. E-mail: [email protected].

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