Glu376, the base involved in substrate alphaH+ abstraction at the active center of medium-chain acyl-CoA dehydrogenase (MCAD), has been mutated to Gln and Gly. The mutants are active; however, their rates of dehydrogenation are lowered by approximately 5 orders of magnitude. Binding of the substrate octanoyl-CoA to Glu376Gln-MCAD involves (at least) two steps. The ensuing dehydrogenation reaction that corresponds to reduction of the flavin cofactor also occurs in two phases. These are interpreted to consist of a first, reversible step, followed by a slower, practically irreversible one. For Glu376Gln-MCAD, the log of the rates of dehydrogenation increases linearly with pH (slope = 1) in the pH range of 6-10, suggesting HO- as a reactant. The rates of the same reactions in D2O have the same pD profile and reflect a solvent kinetic isotope effect (SKIE) of approximately 8.5. Glu376Gln+Glu99Gly-MCAD (studied to assess the role of Glu99 also present at the bottom of the active center cavity) has activities and activity profiles similar to those of Glu376Gln-MCAD. This excludes Glu99 as the active center base for Glu376Gln-MCAD catalysis. Proton inventories for the two phases of the dehydrogenation reaction were investigated at 4 and 25 degrees C. The inventories at 25 degrees C reflect a SKIE of approximately 4.5; the profiles are "bowl-shaped", in which a transition-state contribution predominates. The profiles for the 4 degrees C reaction are very unusual. That for the first phase can be analyzed on a two-step model with one step (80% rate-limiting) having a conformational reorganization with an isotope effect of 90-100, from small isotope effects at many protein sites, and the other step (20% rate-limiting) having an inverse isotope effect of ca. 2, characteristic of the reaction of hydroxide ion as a base. For the second phase, only a contribution from many protein sites with a KIE of approximately 4.5 is observed. The results are compatible with a very rigid active site framework that must undergo rearrangements for dehydrogenation to take place, and specifically to allow access of HO-, the reactant that must neutralize the H+ abstracted from the alphaC-H substrate. The large isotope effects are attributed to the changes in state of several H-bonds that occur during the process.