Mechanism and selectivity of the dinuclear iron benzoyl-coenzyme A epoxidase BoxB

Benzoyl-CoA epoxidase is a dinuclear iron enzyme that catalyzes the epoxidation reaction of the aromatic ring of benzoyl-CoA with chemo-, regio- and stereo-selectivity. It has been suggested that this enzyme may also catalyze the deoxygenation reaction of epoxide, suggesting a unique bifunctionality among the diiron enzymes. We report a density functional theory study of this enzyme aimed at elucidating its mechanism and the various selectivities. The epoxidation is suggested to start with the binding of the O₂ molecule to the diferrous center to generate a diferric peroxide complex, followed by concerted O–O bond cleavage and epoxide formation. Two different pathways have been located, leading to (2S,3R)-epoxy and (2R,3S)-epoxy products, with barriers of 17.6 and 20.4 kcal mol^–1, respectively. The barrier difference is 2.8 kcal mol^–1, corresponding to a diastereomeric excess of about 99 : 1. Further isomerization from epoxide to phenol is found to have quite a high barrier, which cannot compete with the product release step. After product release into solution, fast epoxide–oxepin isomerization and racemization can take place easily, leading to a racemic mixture of (2S,3R) and (2R,3S) products. The deoxygenation of epoxide to regenerate benzoyl-CoA by a diferrous form of the enzyme proceeds via a stepwise mechanism. The C2–O bond cleavage happens first, coupled with one electron transfer from one iron center to the substrate, to form a radical intermediate, which is followed by the second C3–O bond cleavage. The first step is rate-limiting with a barrier of only 10.8 kcal mol^–1. Further experimental studies are encouraged to verify our results.