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

Abstract

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.

Study on the Effects of Intermolecular Interactions on Firefly Multicolor Bioluminescence

“…[20] Calculations by Nakatani et al [21] discarded the resonance-based structure as an explanation, as was corroborated by our own time-dependent DFT (TD-DFT) calculations. [22] Also, several TD-DFT calculations [22][23][24] demonstrated that interaction of a cation with the benzothiazole moiety of OxyLH 2 results in a blueshift, thus refuting the hypothesis formulated by Hirano et al [18] Theoretical [25] and experimental [18] studies also discarded the hypothesis formulated by Nakatsu et al, [17] which stated that the rigidity of the active site modulates the color of light emitted. More recently, we began to pursue clarification of this topic, having reviewed computational studies on multicolor bioluminescence.…”

“…[13] Several hypotheses have been advanced over the years to try to explain this phenomenon. Keto-enol tautomerism, [14] rotation about a CÀC bond, [15] resonance-based structure, [16] rigidity of the active site, [17] and interaction of the emitter with a cation [18] have all been proposed, but were rejected because they cannot explain the multicolor variation. Branchini et al demonstrated that the keto form of OxyLH 2 suffices for multicolor bioluminescence, [19] and theoretical calculations demonFirefly luciferase exhibits a color-tuning mechanism based on pH-induced changes in the structure of the active site.…”

Photochemical properties of phytochrome and firefly luciferase chromophores: A theoretical study

“…This finding together with the acurracy of the calculated absorption and emission maxima indicate that the well-known trend that hybrid functionals give more accurate excitation energies than pure functionals does not apply for bilin The characteristic glow of a firefly is created by a bioluminescent reaction inside the active pocket of the enzyme firefly luciferase. In this reaction, the enzyme catalyzes a conversion of the ground-state heterocyclic acid D-luciferin into the [35][36][37] and spectroscopic [38,39] studies have proposed that the phenolate-keto-OxyLH − form is the light emitter, Naumov and coworkers [40] have recently reported experimental data favoring an enolate species (enolate-OxyLH − or OxyL 2− ). In Paper III, the most probable form as the light emitter was predicted based on the intrinsic tendency of the light emitter to prefer a particular form of OxyLH 2 in aqueous solution at pH 7.…”

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…Recent studies favor the dependence of color on the polarization of the emitter oxyluciferin and the degree of covalent interaction between oxyluciferin and luciferase. 46,47 A small shift of kmax is generally attributed to the local polarity changes in the emitter sites. 48,49 The polarizability of the introduced residue is positively correlated to red shift.…”