Acyl-CoA dehydrogenases are flavoproteins that catalyze the conversion of a fatty acyl thioester substrate to the corresponding alpha,beta-enoyl-CoA product. It has been well established that a glutamate residue in the active site [e.g., E367 in short-chain acyl-CoA dehydrogenase (SCAD) of Megasphaera elsdenii] is responsible for the initial alpha-proton abstraction. Early studies have also shown that this class of enzymes is capable of catalyzing gamma-H abstraction to afford the allylic isomerization between alpha,beta- and beta,gamma-enone thioesters and/or inactivation by 2- or 3-acetylenic acyl-CoA derivatives. Although a dual role has been proposed for the glutamate residue in both alpha- and gamma-deprotonation, the existence of a second active-site basic group to mediate the observed reactions occurring at gamma-C remains a feasible mechanism. In an attempt to discern between these two possibilities, we have prepared a few oxirane-containing acyl-CoA derivatives aimed at trapping active-site bases in the vicinity of the alpha- and/or gamma-C. It was found that 2,3-epoxybutyryl-CoA is a new class-selective irreversible inactivator against SCAD; however, the inability of other oxirane-containing probes to react with these enzymes prompted us to tackle this mechanistic problem by directly studying the role of Glu-367 in SCAD-catalyzed 1,3-isomerization. The effect of E367Q mutation on the proficiency of SCAD to mediate the gamma-H exchange of crotonoyl-CoA was examined. The capabilities of the wild-type SCAD and its E367Q mutant to catalyze the gamma-H abstraction during the inactivation by 2-butynoyl-CoA was also compared. The fact that the mutant protein fails to promote gamma-H exchange/abstraction provides strong evidence supporting a one-base mechanism of this enzyme-catalyzed allylic isomerization. Since the catalysis of acyl-CoA dehydrogenases is closely related, such a one-base mechanism is expected to be conserved within this family of enzymes.