Cytochrome P450-catalyzed hydroxylation of mechanistic probes that distinguish between radicals and cations. Evidence for cationic but not for radical intermediates

Oxidation of the mechanistic probes trans, trans-2-methoxy-3-phenylmethylcyclopropa and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that different structural rearrangements occur for the two types of intermediates. The P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from rabbit), the expressed truncated isozymes P450 Delta 2B4 and P450 Delta 2E1 (ethanol-inducible, from rabbit), and mutants of the latter two in which an active site threonine was replaced with alanine, Delta 2B4 T302A, and Delta 2E1 T303A. Cationic rearrangement products were found from both probes. Oxidations of trans, trans-2-methoxy-3-phenylmethylcyclopropane gave small amounts of radical-derived rearrangement products indicating that hydroxylation occurs via insertion reactions with transition state lifetimes in the 80-200 fs range. A mechanistic description of cytochrome P450-catalyzed hydroxylations that is in accord with the present and previous radical probe results is presented. This description incorporates the recent demonstrations that two electrophilic oxidants are produced in the natural course of P450 oxidation reactions and that both electrophilic oxidant forms can effect hydroxylation reactions. Following production of a peroxo-iron species, protonation gives a hydroperoxo-iron species. Protonation of the hydroperoxo-iron species gives an iron-ore species and water. Hydroxylations by both the hydroperoxo-iron and iron-ore species occur by insertion reactions. The hydroperoxo-iron species inserts the elements of OH+ producing protonated alcohol products that can react in solvolysis-type reactions to give cationic rearrangement products. The iron-ore species reacts by insertion of an oxygen atom.