Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes

Cytochrome P450 enzymes play critical roles in many important biological processes, including steroid hormone biosynthesis, drug metabolism, and xenobiotic carcinogenesis. The radical rebound mechanism of P450 enzymes was widely accepted for decades but more recently was challenged by mechanisms involving a cation intermediate. In order to address this question, we developed the α‐ and β‐thujones as novel radical clocks and investigated their oxidation and that of the 7‐deuterated derivative by cytochrome P450 cam , a P450 cam L244A mutant, P450 BM‐3 , human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Quantitative analysis of thujone metabolites indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate of between 0.7–12.5 x 10 10 s −1 for trapping of the carbon radical by the P450 iron‐bound hydroxyl radical equivalent. The involvement of a carbon radical intermediate is also strongly supported by the observation of inversion of the C‐4 methyl group in the reaction. The absence of a solvent isotope effect on product distribution in the P450cam reaction argues against a significant role of the ferric hydroperoxide precursor of the ferryl species in substrate oxidation. The results provide strong support for exclusive carbon hydroxylation by the ferryl species and the involvement of radical intermediates whose detailed properties are substrate‐ and enzyme‐dependent. This work was supported by grant NIH GM25515.