Raman and FT-IR study on hydrogen-bond in the active site of acyl-CoA dehydrogenase

Abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the oxidation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. The C(1)=O of the acyl-CoA is hydrogen-bonded to the ribityl-2'-OH of FAD and the main chain amide N-H of Glu376 in MCAD, and the hydrogen bonds are important in the catalysis. The details of the hydrogen bonds were investigated through the use of artificial FADs by Raman and FT-IR spectroscopies. 8-NH₂-FAD-MCAD binds a substrate octanoyl-CoA, but cannot oxidize it, thus the complex is a valuable sample for exploration of the details of enzyme-substrate complex. FT-IR difference spectra between non-labeled and [1-¹³C]octanoyl-CoA were measured free in solution and bound to 8-NH₂-FAD-MCAD. The 1668-cm⁻¹ band of C(1)=O stretch of octanoyl-CoA free in solution shifted to 1626 cm⁻¹ in the bound form. This 42-cm⁻¹ downward shift reflects an appreciable contribution of a polarized form of the C(1)=O moiety in the enzyme-bound acyl-CoA, and can be explained by the hydrogen bonds of C(1)=O in the active site. The similar downward shifts have been observed by resonance Raman method in the C(1)=O stretch of octenoyl-CoA in MCAD purple complex (reduced MCAD-product complex). We estimated the hydrogen bond enthalpy change (ca. 15 kcal/mol in both cases) for the transfer of the substrate and product from aqueous solution to the active site in oxidized and reduced MCAD, respectively, based on the shift in frequency. [Jpn J Physiol 54 Suppl:S64 (2004)]