Radical rebound mechanism in cytochrome P-450-catalyzed hydroxylation of the multifaceted radical clocks α- and β-thujone

Abstract

α-Thujone (1α) and β-thujone (1β) were used to investigate the mechanism of hydrocarbon hydroxylation by cytochromes P-450 cam (CYP101) and P-450BM3 (CYP102). The thujones are hydroxylated by these enzymes at various positions, but oxidation at C-4 gives rise to both rearranged and unrearranged hydroxylation products. Rearranged products result from the formation of a radical intermediate that can undergo either inversion of stereochemistry or ring opening of the adjacent cyclopropane ring. Both of these rearrangements, as well as a C-4 desaturation reaction, are observed. The ring opening clock gives oxygen rebound rates that range from 0.2 × 1010 to 2.8 × 1010 s-1 for the different substrate and enzyme combinations. The C-4 inversion reaction provides independent confirmation of a radical intermediate. The phenol product expected if a C-4 cationic rather than radical intermediate is formed is not detected. The results are consistent with a two-state process and provide support for a radical rebound but not a hydroperoxide insertion mechanism for cytochrome P-450 hydroxylation.