Localized electron polarization in a substrate analog binding to the active site of enoyl‐CoA hydratase: Raman spectroscopic and conformational analyses of rotamers of hexadienoyl thiolesters

The Raman bands associated with different rotamers of hexadienoyl ethyl thiolester, CH₃CHCHCHCHC(O)SCH₂CH₃, have been partially assigned using recent conformational and vibrational spectroscopic analyses of ethyl thiocrotonate CH₃CHCHC(O)SCH₂CH₃.¹ In particular, rotational isomers involving the CHC(O) and SCH₂ axes have been characterized. The assignment of the vibrational modes of different conformers of hexadienoyl ethyl thiolester was further facilitated by variable‐temperature (+20°C to −90°C) Raman studies on the neat thiolester. High‐quality Raman spectra of hexadienoyl‐coenzyme A bound to the enzyme enoyl‐CoA hydratase were obtained using 647.1‐nm excitation and Raman difference spectroscopy. The findings provided by analysis of the ethyl thiolester model compound enable us to conclude that conformational selection occurs for the CoA analog upon binding, resulting in the presence of a single‐rotamer population in the hexadienoyl moiety on the enzyme. The hexadienoyl‐CoA was labeled with ¹⁸O in the CO group and, separately, with ¹³C at the C2 position. A comparison of Raman data for the free and bound ligands, isotopically labeled and unlabeled, indicates that strong π‐electron polarization occurs in only a part of the hexadienoyl chain, viz, in the CCCO fragment, upon binding. The polarization gives rise to important contributions from canonical (resonance) forms of the type C⁺CCO⁻. In contrast, the C4C5 linkage (where the carbon atom numbering is C6C5C4C3C2C1O) seems little perturbed in the bound ligand. The causes and mechanistic advantage of the observed localized polarization are discussed. © 1995 John Wiley & Sons, Inc.