A combined experimental and theoretical investigation of the deuterium isotope effects on the bacterial luciferase reaction is described. The experimental studies focus on determining if the unusual aldehydic deuterium isotope effect of ~1.5 observed in these reactions is an intrinsic isotope effect resulting from a single rate-limiting step or is a composite of multiple rate-limiting steps. The isotope effect observed is not significantly affected by variation in the aldehyde chain length, changes in the pH over a range of 6-9, use of αC106A and αC106S site-directed mutants, or chloride substitution at the 8-position of the reduced flavin, though the isotope effect is decreased when the 8-methoxy-substituted flavin is used as a substrate. From these observations it is concluded that the aldehydic isotope effect arises from the change in rate of a single kinetic step. A stopped-flow kinetic analysis of the microscopic rate constants for the reactions of 1-[1H]decanal and 1-[2H]decanal in the bacterial luciferase reaction was carried out, and aldehyde hydration isotope effects were determined. From the results it is estimated that the aldehydic deuterium isotope effect is ~1.9 after formation of an intermediate flavin C4a- hydroperoxy hemiacetal. Ab initio calculations were used to examine the transformation of the aldehyde into a carboxylic acid and to predict isotope effects for possible mechanisms. These calculations indicate that the mechanism involving rate-limiting electron transfer from the flavin C4a- hydroxide to an intermediate dioxirane is consistent with the enigmatic aldehydic isotope effect and that the intermediacy of a dioxirane is energetically plausible.
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