The hydride transfer reaction catalyzed by choline oxidase under irreversible regime, i.e., at saturating oxygen, was shown in a recent study to occur quantum mechanically within a highly preorganized active site, with the reactive configuration for hydride tunneling being minimally affected by environmental vibrations of the reaction coordinate other than those affecting the distance between the alpha-carbon of the choline alkoxide substrate and the N(5) atom of the enzyme-bound flavin cofactor [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 17954-17961]. In this study, we have determined the effects of pH and temperature on the substrate kinetic isotope effects with 1,2-[2H4]choline as substrate for choline oxidase at 0.2 mM oxygen to gain insights on the mechanism of hydride transfer under reversible catalytic regime. The data presented indicated that the kinetic complexity arising from the net flux through the reverse of the hydride transfer step changed with temperature, with the hydride transfer reaction becoming more reversible with increasing temperatures. After this kinetic complexity was accounted for, analyses of the kcat/Km and D(kcat/Km) values determined at 0.2 mM according to the Eyring and Arrhenius formalisms suggested that the quantum mechanical nature of the hydride transfer reaction is, not surprisingly, maintained during enzymatic catalysis under reversible regime. A comparison of the thermodynamic and kinetic parameters of the hydride transfer reaction under reversible and irreversible catalytic regimes showed that the enthalpies of activation (DeltaH++) were significantly larger in the reversible catalytic regime. This reflects the presence of an enthalpically unfavorable internal equilibrium of the enzyme-substrate Michaelis complex occurring prior to, and independently from, CH bond cleavage. Such an internal equilibrium is required to preorganize the enzyme-substrate complex for efficient quantum mechanical tunneling of the hydride ion from the substrate alpha-carbon to the flavin N(5) atom.