Identification of the Luciferase-Bound Flavin-4A-Hydroxide as the Primary Emitter in the Bacterial Bioluminescence Reaction

The luciferase light-emitting reaction was carried out at 1°C by mixing purified luciferase-FMN-4a-hydroperoxide with long chain aldehyde (decanal). Simultaneous kinetic measurements of bioluminescence and absorbance showed that light emission decayed more rapidly than oxidized FMN appeared, indicative of a transient intermediate species subsequent to light emission. The same species was found in reaction mixtures examined immediately after light emission was completed. Both its absorption spectrum (λmax, 360nm) and its fluorescence emission (λmax, 490nm) are consistent with the hypothesis that the chromophore is the luciferase-bound flavin-4a-hydroxide, the ground state of the primary emitter in the reaction. It has a relatively short lifetime (7 min at 9°C) and decays to the stable product, FMN, by losing water. The activation energy for this step was determined to be 83 kJ mol-1