| Acyl-coenzyme A dehydrogenases (ACDs) are responsible for catalyzing the 2,3-dehydrogenation of acyl-CoA thioesters to trans-2-enoyl-CoA thioester products. During substrate oxidation, two electrons are transferred from the substrate to a flavin adenine dinucleotide (FAD) cofactor. This electron-transfer reaction is highly regulated by non-covalent interactions between the substrate, the flavin, and the protein environment in the enzyme active-site. Objectives of this work were: (i) to determine how the electronic structures of substrate/product and substrate/product analogues are affected by ACD binding, and (ii) to identify and characterize specific apoprotein-flavin interactions that stabilize formation of reduced flavin.;To achieve these objectives, the thermodynamic properties of two ACD systems were investigated. Medium-chain acyl-CoA dehydrogenase (MCAD) was complexed with a product analog, hexadienoyl-CoA, and the two-electron reduction potential (Eox/hq) of the complex was measured as a function of pH. The Eox/hq versus pH profile exhibited a pH dependence consistent with the redox-linked ionization of two key glutamic acids as well as the FAD cofactor. An increase in the p Ka of Glu376 is proposed to allow HD-CoA to become polarized in the active-site of MCAD. The results of Raman studies on the MCAD·HD-CoA complex confirmed this hypothesis, and moreover, indicated that electronic rearrangement occurs such that partial positive charge selectively develops on a region of the analogue that is only ca. 3.3 A from the flavin ring.;A second ACD, short-chain acyl-CoA dehydrogenase (SCAD) from Megasphaera elsdenii, was used to test the effects of aromatic stacking and hydrogen bonding interactions on the stabilization of the reduced FAD. Amino acid substitutions were systematically introduced at three positions (Tyr366, Phe160, and Thr129) and the redox properties of the resulting mutant enzymes were characterized using spectroelectrochemical techniques. The results suggest that favorable pi-sigma interactions between the aromatic residues and the isoalloxazine ring of the flavin are important for shifting Eox/hq in the positive direction. In addition, a hydrogen bond formed between the threonine hydroxyl group and N(1) on the flavin was found to be crucial for stabilizing the development of negative charge in the N(1)-C(2)=O region. |
