This dissertation investigates aspects of two interesting flavoproteins, the mammalian medium chain acyl-CoA dehydrogenase (MCAD) and the yeast sulfhydryl oxidase ERV2p (essential for respiration and vegetative growth) respectively. MCAD is a medically-important enzyme involved in fatty acid catabolism and the present study identifies a class of novel inhibitors that might be developed into therapeutics for metabolic syndromes such diabetes. ERV2p is a compact yeast ER luminal protein with modest sulfhydryl oxidase activity. A comparative study with its enigmatic homolog, the mammalian augmenter of liver regeneration (ALR), a growth factor with a diverse range of physiological roles, and the mechanistically best-understood quiescin sulfhydryl oxidase (QSOX) is presented with a perspective concerning oxidative protein folding.

MCAD is rapidly inhibited by R-(-)-3,4-decadienoyl-CoA, with subsequent slow reactivation of the enzyme following the conversion of the inhibitor to the conjugated 2,4-diene species. α-Proton abstraction yields an enolate to flavin charge-transfer intermediate prior to adduct formation. Both the rate and the half-life of the covalent, yet kinetically reversible, inhibition are structurally modulated, with a broad range of variability, as shown by the alteration of the acyl chain length and truncation of the CoA moiety. The crystal structure of the reduced, inactivated enzyme shows a single covalent bond linking the inhibitor and the flavin prosthetic group. These reduced enzyme structures suggest that active site H-bond patterns and the orientation of the catalytic base are important determinants of adduct stability. Unlike the R-isomer, the S-(-)-3,4-decadienoyl-CoA is readily isomerized to the conjugated diene. Modeling studies suggest that the S-enantiomer is precluded from adduct formation, and instead can be facilely reprotonated by the catalytic base. Several allenic inhibitors are potent inhibitors of fatty acid oxidation in respiring mitochondria. These studies suggest that allenic inhibitors may serve as potential drug leads for the reversible attenuation of fatty acid oxidation flux in vivo.

This dissertation also examines the enzymology of ERV2p and compares its the redox behavior with ALR and the multi-domain QSOX. Dithionite and photochemical reductions of ERV2p show full reduction of the flavin cofactor after the addition of 4-electrons with a mid-point potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in ERV2p, consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn²⁺, full reduction of ERV2p requires 6-electrons. Zn ²⁺ also strongly inhibits ERV2p when assayed using tris(2-carboxyethyl)phosphine as the reducing substrate of the oxidase. In contrast to QSOX, ERV2p shows a comparatively low turnover with both a range of small thiol substrates, and with reduced protein substrates. Rapid reaction studies confirm that reduction of the flavin center of ERV2p is rate-limiting during turnover with molecular oxygen. Finally, a comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.