Bioluminescence Emission from the Reaction of Luciferase-Flavin Mononucleotide Radical with Opt

The blue neutral luciferase flavin radical has been shown not to be in a catalytically significant equilibrium with species leading to emission of light [Kurfurst, M., Ghisla, S., Presswood, R., & Hastings, J. W. (1982) Eur. J. Biochem. 123, 355-361]. It is shown here that this radical can nevertheless react with 02¯● to form a species that is competent in light emission. From its properties, the species formed is deduced to be luciferase-FMNH 4a-hydroperoxide, a key intermediate in the normal luciferase reaction. Although it is concluded that this intermediate can undergo a reversible homolytic dissociation to yield free superoxide and the corresponding luciferase radical, the slowness of these steps precludes a catalytic significance for these pathways in the normal bioluminescent reaction

Bioluminescence emission of bacterial luciferase with 1-deaza-FMN : evidence for the noninvolvement of N(1)-protonated flavin species as emitters

The reaction of reduced 1-d-FMN with oxygen and decanal results in bioluminescence with kinetic and spectral properties similar to those of the reaction with FMNH2, even though the spectral (absorbance, fluorescence) and chemical properties of the oxidized forms differ greatly. This emission, which is about 10 15% as efficient as with FMNH2, is postulated to involve the intermediacy of the corresponding 4a-hydroperoxide, the fluorescence of which occurred transiently. The N(1) protonated species had been proposed as the emitter in the reaction with FMNH2, but the 1-deaza analog cannot be protonated at the corresponding position, thus excluding this possibility

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

Isolation and characterization of the transient, luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction

Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reaction can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2°C and decays spontaneously to give H2O and luciferase-bound FMN with an activation enthalpy of about 120 kJ/mol. It has an absorption spectrum (λmax = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (λmax = 485 nm) that matches the bioluminescence emission closely