CALCULATED AND EXPERIMENTAL ABSOLUTE STEREOCHEMISTRY OF THE STYRENE AND BETA-METHYLSTYRENE EPOXIDES FORMED BY CYTOCHROME-P450(CAM)

Cytochrome P450cam oxidizes styrene to styrene oxide and a trace of phenylacetaldehyde; cis-beta-methylstyrene to cis-beta-methylstyrene oxide, cis-3-phenyl-2-propen-1-ol, and a trace of 1-phenyl-2-propanone; and trans-beta-methylstyrene to trans-beta-methylstyrene oxide, trans-3-phenyl-2-propen-1-ol, and a trace of 1-phenyl-2-propanone. Aromatic ring hydroxylation is also observed as a very minor process with each of the three substrates. Benzaldehyde is formed as a major product in all the reactions, but its formation is due to oxidation of the olefins by the H2O2 produced by uncoupled turnover of the enzyme. Stoichiometry studies indicate that oxidation of the styrenes is highly uncoupled relative to the oxidation of camphor. Analysis of the absolute stereochemistry of the three epoxides by chiral gas-liquid chromatography shows that the stereoisomers are formed in the following ratios: styrene oxide, S:R 83:17; cis-beta-methylstyrene oxide, 1S,2R: 1R,2S 89:11; trans-beta-methylstyrene oxide, 1S,2S:1R,2R 75:25. Calculation of the minimum energy conformations of the three olefins, docking of the preferred conformations in the active site of cytochrome P450cam with use of AMBER to calculate the minimum energy orientations, and molecular dynamic simulations independently predict the following epoxide stereoisomer ratios: styrene oxide, S:R 65:35; cis-beta-methylstyrene oxide, IS,2R:1R,2S 84:16; trans-beta-methylstyrene oxide, IS,2S:1R,2R 75:25. The excellent agreement between theory and experiment supports the validity of the computational methodology and provides insight into the factors that control the stereoselectivity and outcome of cytochrome P450-catalyzed oxidations.