A density functional study of the factors that influence the regioselectivity of toluene hydroxylation by cytochrome P450 enzymes

Density functional theory calculations have been performed to elucidate the factors that influence the regioselectivity of toluene hydroxylation by a model of the active species of cytochrome P450 enzymes, so-called Compound I (Cpd I). Cpd I can hydroxylate the benzylic C-H and generate benzyl alcohol, or it can activate the phenyl group and produce p-cresol and p-methylcyclohexanone products. The reactions take place via two-state reactivity on competing doublet and quartet spin state surfaces. In the gas phase, the benzyl alcohol is preferred over p-cresol by more than three orders of magnitude. Environmental perturbations, namely, NH⋯S hydrogen bonding to the thiolate ligand and bulk polarity of the protein, lower this preference to roughly 10:1 in favour of benzyl alcohol. Substitution of methyl hydrogen atoms by deuterium atoms raises the barriers that lead to benzyl alcohol without affecting those leading to p-cresol. Therefore combining toluene deuteration with the effects of NH⋯S hydrogen bonding and bulk polarity lowers the free energy difference between the two processes to only 0.4 kcal mol^-1, and the two processes become competitive. These results as well as the calculated kinetic isotope effects are in good general agreement with experimental data.