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 NH(2)-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.