The regiospecificity and stereoselectivity of alkane hydroxylation and alkene epoxidation by the particulate methane monooxygenase from Methylococcus capsulatus (Bath) was evaluated over a range of substrates. Oxidation products were identified by conventional GC analysis, and the stereoselectivity of oxidation was determined by a combination of chiral GC and HPLC methods, as well as 1H NMR analysis of the corresponding (R)-2-acetoxy-2-phenylethanoate ester derivatives in the case of alkanol products. Alkane hydroxylation was found to proceed favoring attack at the C-2 position in all cases, and the stereoselectivity for n-butane and n-pentane was characterized by an enantiomeric excess of 46% and 80%, respectively, with preference for the (R)-alcohol noted for both substrates. Epoxides were formed with smaller stereoselectivities. Together, the regio- and stereoselectivity results suggest that an equilibrium of competing substrate binding modes exists. A simple substrate-binding model that incorporates preferential C-2 oxidation with the observed stereoselectivity of alkane hydroxylation is proposed, and hypotheses for the general mechanism are suggested and discussed.

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