Ring Current Effects in the Active Site of Medium-Chain Acyl-CoA Dehydrogenase Revealed by NMR Spectroscopy

• Published in: Journal of the American Chemical Society 2005-06, Vol.127 (23), p.8424-8432
• Main Authors: Wu, Jiaquan, Bell, Alasdair F, Jaye, Andrew A, Tonge, Peter J
• Format: Article
• Language: English
• Subjects: Acyl-CoA Dehydrogenase - chemistry; Acyl-CoA Dehydrogenase - metabolism; Animals; Binding Sites; Biological and medical sciences; Carbon Isotopes; Cattle; Enoyl-CoA Hydratase - chemistry; Enoyl-CoA Hydratase - metabolism; Fundamental and applied biological sciences. Psychology; Kinetics; Mechanisms. Catalysis. Electron transfer. Models; Models, Molecular; Molecular biophysics; Nuclear Magnetic Resonance, Biomolecular - methods; Physical chemistry in biology; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Structure-Activity Relationship; Swine; Titrimetry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja050083p

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using 13C NMR and 1H−13C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the 1H chemical shift of the C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchanged upon binding. These changes in chemical shift are unexpected given the results of previous Raman studies which revealed that the C3C2−C1O HD enone fragment is polarized upon binding to MCAD such that the electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al. Biochemistry 2000, 39, 13982−13992). To investigate the apparent discrepancy between the NMR and Raman data for HD-CoA bound to MCAD, 13C NMR spectra have been obtained for HD-CoA bound to enoyl-CoA hydratase, an enzyme system that has also previously been studied using Raman spectroscopy. Significantly, binding to enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by 4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement with the alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu, J.; Feng, Y.; Tonge, P. J. Biochemistry 2001, 40, 1725−33). The large increase in shielding experienced by the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring current field from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present when the enzyme is placed in an external magnetic field, also explains the differences in 13C NMR chemical shifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used to rationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligands are bound to MCAD than when they are free in the protein solution.