Structural Modulation of 2-Enoyl-CoA Bound to Reduced Acyl-CoA Dehydrogenases: A Resonance Raman Study of a Catalytic Intermediate1

Abstract

A catalytic intermediate, the so-called “purple complex,” of acyl-CoA dehydrogenase is produced on its reaction with the substrate, acyl-CoA. The purple complex is a charge-transfer complex between the reduced enzyme and the product, enoyl-CoA. Resonance Raman spectra of the purple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydrogenases] were measured with excitation at 632.8 nm within charge-transfer absorption bands. The 1,577 cm⁻¹ band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1)=O stretching of crotonyl-CoA, judging from the isotopic frequency shifts upon ¹³C or ¹⁸O substitution of butyryl-CoA. The 1,627 cm⁻¹ band of the C(1)=O moiety of crotonyl-CoA in solution shifted downward by 50 cm⁻¹ on complexation with reduced SCAD. This large frequency shift indicates a substantial interaction between C(1)=O and the enzyme, and is further evidence for an appreciable contribution of a polarized form of the C(1)=O moiety in the enzyme-bound enoyl-CoA. This frequency shift can be explained by the hydrogen bond of C(1)=O. The 1,577 cm⁻¹ band of the MCAD purple complex remained constant, regardless of the acyl carbon-chain length (from C4 to C16 of the substrate, acyl-CoA); the alkyl chain scarcely affected the interaction of the C(1)=O moiety in the active site. The frequency of the 3-methylcrotonyl-CoA carbonyl C(1)=O moiety shifted from 1,626 cm⁻¹ in solution downward by 45 cm⁻¹ when the CoA thioester bound to reduced IVD, but by 28 cm⁻¹ when the thioester bound to reduced SCAD or MCAD. This indicates that the hydrogen bond at C(1)=O of 3-methylcrotonyl-CoA in SCAD or MCAD is weaker than in the case of IVD; the steric repulsion of the 3-methyl group probably changes the orientation of the −C(3)H=C(2)H−C(1)=O moiety and thus affects the hydrogen bonding. Tyr-375, which is conserved in straight chain acyl-CoA dehydrogenases, may be responsible for the steric repulsion and thus play a role in the substrate specificity