Redox modulation of acyl -CoA dehydrogenases (ACDs) and their ligands in several ACD -analog systems

Human beings and all other mammals store excess energy as fat in adipose tissue for later use. A family of non-membrane-bound mitochondrial enzymes, known as acyl-CoA dehydrogenases (ACDs), catalyze the first step of beta-oxidation, the primary means through which adipose energy stores are accessed. As such, deficiencies in this family of enzymes can lead to serious metabolic disorders, even death. A thorough understanding of enzyme and ligand (i.e. substrate) activation in the ACD catalyzed oxidation of fatty acyl-CoAs will allow ACDs to be more accurately utilized as model systems of more complex, membrane-bound enzymes.;It is well established that acyl-CoA dehydrogenases are activated by substrate/product binding to accept electrons from their substrates. For example, the midpoint potential of the flavin cofactor in medium chain acyl-CoA dehydrogenase (MCAD) has been shown to shift positively by over 120 mV upon natural substrate/product (octanoyl-/octenoyl-CoA) binding. The source of this activation energy, however, has not yet been identified.;My research utilized spectrally active alternative substrates and products to elucidate the ligand's role in activation. The thermodynamic effects of substrate/product binding on the ligands themselves were directly studied for the first time by combining spectroelectrochemical techniques and ligands with spectrally distinct redox states. These experiments demonstrated that the substrate/product couple is indeed activated for electron transfer, likely via polarization. However, the extent of this activation is significantly smaller than anticipated, leading to the conclusion that other factors such as active site desolvation, pKa changes, and charge changes also play significant roles in ACD activation.;Non-reactive substrate and product analogs were also studied. These allowed for work with both mammalian MCAD and bacterial short chain acyl-CoA dehydrogenase (bSCAD). The spectral properties of these ligands allowed for the study of enzyme-induced polarization in conjunction with the study of ligand-induced enzyme activation. Results of these studies again suggest that enzyme activation is due to more than simple ligand polarization.