Spectroscopic Studies of the Light-Color Modulation Mechanism of Firefly (Beetle) Bioluminescence

To reveal the light-color modulation mechanism of firefly (beetle) bioluminescence, we investigated the spectroscopic properties of the phenolate anion 1-O− generated from 5,5-dimethyloxyluciferin (1-OH) using various base/solvent combinations. Phenolate anion 1-O− is a model compound for the keto form of wild-type oxyluciferin phenolate anion (OL−), which is postulated to be the emitter of the bioluminescence. The fluorescence maxima of 1-O− were found to depend on the base/solvent combination used, and they varied in the range 541−640 nm, which covers the almost whole range of the bioluminescence emission maximum. In a polar solvent, where 1(1-O−)* and the countercation (the conjugate acid of a base) make a solvent-separated ion pair or a free ion couple, the emission maxima of 1-O− were found to be modulated by the solvent polarity. In a less polar solvent, where 1(1-O−)* and the countercation are formed as a contact ion pair, the strength of the covalent character of the O8′···H bond between 1(1-O−)* and the countercation is operative. The effect of the base/solvent combination on the emission properties of 1(1-O−)* was also verified using fluorescence lifetime measurements and density functional theory calculations on 1-O− and its ion-pair models. On the basis of these results, we propose the following light-color modulation mechanism: (1) the light emitter is the excited singlet state of OL− [1(OL−)*], and (2) light emission from 1(OL−)* is modulated by the polarity of the active-site environment of a luciferase and the degree of covalent character of the O8′···H bond between 1(OL−)* and a protonated basic moiety in the active site. Mechanisms for variation of the bioluminescence colors and their applications are discussed.