1992 Volume 111 Issue 6 Pages 699-706
Resonance Raman (RR) spectra of the complex of pig kidney medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the dehydrogenase were obtained. RR spectra were also measured for the complexes prepared by using isotopically labeled compounds, i.e., [3-13C]-, [1, 3-13C]-, and [2, 4-13C2] acetoacetyl-CoA; [1-13C] octanoyl-CoA; the dehydrogenase reconstituted with [4a-13C]- and [4, 10a-13C2] FAD. Both bands of oxidized flavin and acetoacetyl-CoA were resonance-enhanced in the 632.8nm excited spectra of the acetoacetyl-CoA complex; this confirms that the broad long-wavelength absorption band is a charge-transfer absorption band between oxidized flavin and acetoacetyl-CoA. The 1, 622cm-1 band was assigned to the C(3)=O stretching mode coupling with the C(2)-H bending mode of the enolate form of acetoacetyl-CoA and the bands at 1, 483 and 1, 119cm-1 were assigned to bands associated with the C(2)=C(1)-O- moiety. Both bands of fully reduced flavin and the substrate were resonance-enhanced in the 632.8nm excited spectra of the purple complex. As the enzyme is already reduced, the substrate must be oxidized to octenoyl-CoA; the complex is a charge-transfer complex between the reduced enzyme and octenoyl-CoA. The low frequency value of the 1, 577cm-1 band, which is associated with the C(2)-C(1)=O moiety of the octenoyl-CoA, suggests that the enzyme-bound octenoyl-CoA has an appreciable contribution of C(2)=C(1)-O-. The large contribution of ionic resonance structure, C(2)=C(1)-O-, for the ligands in both complexes, can be explained by the existence of an electrophilic group near the O atom of the C(1)=O group of the ligands and the electrostatic interaction between the group and the O atom of the ligand. This electrostatic interaction probably lowers the pKa value of a substrate at the C(2)-H in an early step of reductive half-reaction, facilitating the abstraction of the α-proton
