Medium-chain acyl-coenzyme A dehydrogenase bound to a product analogue, hexadienoyl-coenzyme A: Effects on reduction potential, pK(a), and polarization

2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) fro m pig kidney. HD-CoA is a thermodynamically stabilized product analogue tha t binds tightly to oxidized MCAD (K-dox = 3.5 +/- 0.1 muM, pH 7.6) and elic its a redox potential shift that is 78% of that observed with the natural s ubstrate/ product couple [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990 ) Biochemistry 29, 3709-3715]. The midpoint potential of the MCAD HD-CoA co mplex exhibits a pH dependence that is consistent with the redox-linked ion ization of two key glutamic acids as well as the flavin adenine dinucleotid e (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a, ox) approximate to 9.3) and Glu99-COOH (pK(a,ox) approximate to 7.4) in the oxidized MCAD HD-CoA complex indicate that while binding of the Cg analogu e makes Glu376 a stronger catalytic base (pK(a,ox) approximate to 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximate to 7. 5, free MCAD) [Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605 - 14612]. This find ing is in agreement with the apparent pK of 9.2 determined for Glu376 in th e human MCAD .4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorp e, C. (1998) Biochemistry 37, 8437-8445]. The pK(a)s estimated for Glu376 a nd Glu99 in the reduced pig kidney MCAD HD-CoA complex, 9.8 and 8.6, respec tively, suggest that both of these residues remain protonated in the charge -transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK, and redox potent ial shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman differen ce spectroscopy. Binding to either the oxidized or reduced enzyme results i n localized pi -electron polarization of the hexadienoyl C(1)=O and C(2)=C( 3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by bind ing. These results suggest that, upon binding to MCAD, HD-CoA is selectivel y polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.