The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

The cytochrome P450 enzyme, CYP199A4 from Rhodopseudomonas palustris strain HaA2, is able to oxidize parasubstituted benzoic acids. This enzyme was used to compare aromatic versus aliphatic C−H bond oxidation, common reactions catalyzed by the P450 superfamily of heme monooxygenases. CYP199A4 was able to bind 4-phenylbenzoic acid and 4cyclohexylbenzoic acid, and the crystal structures demonstrated that both substrates are bound within the active site in a similar fashion. Despite this, while 4-cyclohexylbenzoic acid was efficiently hydroxylated, no detectable enzyme catalyzed oxidation of the aromatic 4-phenylbenzoic acid was observed. The selectivity of 4-cyclohexylbenzoic acid oxidation favored C−H bond abstraction at one of the β-sites in an enantioselective fashion (66%, 95:5 er), over C−H bond abstraction at the benzylic position (33%). In addition, unlike the oxidation of smaller alkyl-substituted benzoic acids (4-ethyl-and 4-isopropyl-), little or no desaturation of the cyclohexyl ring to give an alkene was detected (∼1%). Molecular dynamics simulations suggested that the cyclohexyl ring of 4cyclohexylbenzoic acid was able to achieve a suitable orientation to enable efficient C−H bond abstraction and oxidation by the enzyme at the expected positions. In contrast, when the distance and angle of attack were considered, the alignment of the phenyl ring of 4-phenylbenzoic acid rarely attained a productive geometry for aromatic oxidation to occur. Overall, these results illustrate the chemoselectivity that may arise due to the different geometrical requirements for efficient aromatic oxidation versus aliphatic C− H bond hydroxylation by cytochrome P450 enzymes.

DFT calculations explain the counterintuitive phenomenon that silanes reduce electron-rich (alkyl) phosphine oxides faster than electron-poor (aryl) phosphine oxides.

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4‐cyclohexylbenzoic acid but not the aromatic oxidation of 4‐phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4‐benzyl‐, 4‐phenoxy‐ and 4‐benzoyl‐benzoic acid and methoxy‐substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4‐benzylbenzoic acid. The methoxy‐substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4‐(3′‐methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4‐phenylbenzoic acid. 4‐Phenoxy‐ and 4‐benzoyl‐benzoic acid bound with the ether or ketone oxygen atom hydrogen‐bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4‐phenylbenzoic acid; however the F182L mutant was able to catalyse 4‐phenylbenzoic acid oxidation generating 2′‐hydroxy‐, 3′‐hydroxy‐ and 4′‐hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild‐type enzyme, the phenyl ring of 4‐phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.