Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH-and NAD ؉ -dependent carboxylation reaction that forms -keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. Xanthobacter strain Py2 is one of several bacteria capable of growth with short chain aliphatic alkenes as carbon and energy sources (1). The first step in alkene metabolism involves an oxidative insertion of an oxygen atom into the olefin bond in a reaction that is catalyzed by an inducible (2), multiprotein (3) alkene monooxygenase as shown for the substrate propylene and product epoxypropane in Equation 1.Epoxides formed in this manner are further metabolized via a novel ring opening and carboxylation reaction that requires CO 2 as a cosubstrate and forms a -keto acid as product as shown in Equation 2 (4, 5).Aliphatic epoxides such as epoxypropane have toxic, mutagenic, and potential carcinogenic properties (6), and their metabolism in bacteria and mammalian systems has been the focus of considerable research in recent years. Epoxide carboxylation as described for Xanthobacter Py2 represents the most recently discovered biological epoxide transformation reaction, the others involving conjugation to glutathione, hydration to dihydrodiols (7), or isomerization to an aldehyde (8). Initial studies of the epoxide-carboxylating enzyme, designated an epoxide carboxylase, indicate that it has cofactor requirements, molecular properties, and a catalytic mechanism as unique as the epoxide carboxylation reaction itself.With respect to cofactor requirements, in vitro epoxide carboxylation requires a source of reductant (DTT, 1 other dithiols, or NADPH) and an oxidant (NAD ϩ ) (4, 9). These cofactor requirements are unprecedented for all other carboxylases that have been characterized. The requirement of oxidant and reductant is intriguing since there is no net redox chemistry involved in epoxide carboxylation. In the course of epoxide carboxylation, there is an apparent transhydrogenation reaction wherein the reductant becomes oxidized and NAD ϩ becomes reduced, although this has not to date been unequivocally demonstrated.With respect to molecular properties, epoxide carboxylase appears to function as a multiprotein complex (10, 11). Fractionation of Xanthobacter cell extracts by anion-exchange chromatography resolved epoxide carboxylase into three fractions, designated fractions I, II, and III based on their order of elution, that could be recombined with restoration of activity (11). The active component of one of these fractions was purified to homogeneity on the basi...