Choline oxidase catalyzes the flavin-linked oxidation of choline to glycine betaine, with betaine aldehyde as intermediate and oxygen as electron acceptor, Here, the effects of oxygen concentration and temperature on the kinetic isotope effects with deuterated choline have been investigated. The D(Kcat/Km) and (D)k(cat) values with 1,2-[H-2(4)]-choline were pH-independent at saturating oxygen concentrations, whereas they decreased at high pH to limiting values that depended on oxygen concentration at <= 0.97 mM oxygen. The k(cat)/K-m and k(cat) pH profiles had similar patterns reaching plateaus at high pH. Both the limiting k(cat)/K-m at high pH and the pK(a) values were perturbed to lower values with choline and <= 0.25 mM oxygen. These data suggest that oxygen availability modulates whether the reduced enzyme-betaine aldehyde complex partitions forward to catalysis rather then reverting to the oxidized enzyme-choline alkoxide species. At saturating oxygen concentrations, the D(kcat/Km) was 10.6 +/- 0.6 and temperature independent, and the isotope effect on the preexponential factors (A(H)(')/A(D)(')) was 14 +/- 3, ruling out a classical over-the-barrier behavior for hydride transfer. Similar enthalpies of activation (Delta H-double dagger) with values of 18 +/-2 and 18 +/- 5 kJ mol(-1) were determined with choline and 1,2(H-2(4)]-choline. These data suggest that the hydride transfer reaction in which choline is oxidized by choline oxidase occurs quantum mechanically Within a 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 donor and acceptor of the hydride.