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-phenylmethylcy clopropa and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that diff erent structural rearrangements occur for the two types of intermediates. T he P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from rabbit), the expressed truncated isozymes P45 0 Delta 2B4 and P450 Delta 2E1 (ethanol-inducible, from rabbit), and mutant s of the latter two in which an active site threonine was replaced with ala nine, Delta 2B4 T302A, and Delta 2E1 T303A. Cationic rearrangement products were found from both probes. Oxidations of trans, trans-2-methoxy-3-phenyl methylcyclopropane gave small amounts of radical-derived rearrangement prod ucts indicating that hydroxylation occurs via insertion reactions with tran sition state lifetimes in the 80-200 fs range. A mechanistic description of cytochrome P450-catalyzed hydroxylations that is in accord with the presen t and previous radical probe results is presented. This description incorpo rates the recent demonstrations that two electrophilic oxidants are produce d in the natural course of P450 oxidation reactions and that both electroph ilic oxidant forms can effect hydroxylation reactions. Following production of a peroxo-iron species, protonation gives a hydroperoxo-iron species. Pr otonation of the hydroperoxo-iron species gives an iron-ore species and wat er. Hydroxylations by both the hydroperoxo-iron and iron-ore species occur by insertion reactions. The hydroperoxo-iron species inserts the elements o f OH+ producing protonated alcohol products that can react in solvolysis-ty pe reactions to give cationic rearrangement products. The iron-ore species reacts by insertion of an oxygen atom.