STEPS IN THE POPULATION OF THE EMITTER IN THE BACTERIAL LUCIFERASE REACTION

Abstract— The reaction of luciferase-bound flavin hydroperoxide with both I-¹H and 1–²H decanal has been examined at 2°C in both low (0.01 M ) and high (0.35 M ) phosphate buffer, pH 7, where the kinetics and deuterium isotope effects are quite different. Upon reaction in both buffers there are rapid (<2 ms) increases in absorption at both 380 and W nm, followed by decay over the subsequent seconds and minutes. The changes at 380 nm exhibit a primary isotope effect and are rapid compared to bioluminescence, indicating that the scission of the aldehyde C — H bond occurs prior to the step responsible for populating the electronically excited state. However, the final absorbance change at 600 nm decays in parallel to bioluminescence under the several different conditions studied, suggesting the involvement of a long-wavelength absorbing flavin species in the production of, the excited state. Evidence is also presented indicating that under certain conditions there may be two (sequential) steps, each of which exhibits a primary isotope effect involving the same H atom.