Molecular enigma of multicolor bioluminescence of firefly luciferase

Abstract

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL(-) [(1)(OL(-))*], and light emission from (1)(OL(-))* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.

Fig. 1

Two-step oxidation of luciferin during firefly luciferase reaction to produce light, oxyluciferin, CO₂, and AMP. Oxyluciferin emits light through keto or enol tautomer

Fig. 2

TICT excited state of oxyluciferin with φ = 90° (right). The structure of dimethyl oxyluciferin (left)

Fig. 3

Structure of a stable analogue of luciferin adenylate intermediate structure; 5-O-[N-(dehydroluciferyl) sulfamoyl] adenosine (DLSA)

Scheme 1

Charge delocalization of the anionic oxyluciferin

Scheme 2

Chemiexcitation process of the anionic dioxetanone intermediate of oxyluciferin

Fig. 4

Bioluminescence emission spectra produced by the native form Photinus pyralis (1) and mutant (2, Arg³⁵⁶; 3, Lys³⁵⁶; 4, Glu³⁵⁶; 5, Gln³⁵⁶) luciferases by a luciferase-catalyzed reaction at pH 7.8. Reprinted from [94] with permission from American Chemical Society (ACS)

Fig. 5

Superposition of flexible loop (residues 350–365) models from native (green) and mutant (Lys³⁵⁶, red; Arg³⁵⁶, orange; Glu³⁵⁶, yellow; Gln³⁵⁶, blue) luciferases. The insertion of new residues forms a longer flexible loop in comparison to native luciferase. Reprinted from [94] with permission from American Chemical Society (ACS)

Fig. 6

Possible trans-C2–C2′ bond chemical forms of the firefly emitter oxyluciferin, OxyLH₂

Fig. 7

Assignment of the absorption and emission spectra of the firefly emitter oxyluciferin, based on experimental and theoretical spectroscopic data (*Unstable species; **Difficult to be determined experimentally because of the small concentration in mixture with other species). The figures in the middle show the color change of oxyluciferin (from left to right) in DMSO without added base, in water with and without base, and in DMSO in presence of base. Reprinted from [101] with permission from American Chemical Society (ACS)

Fig. 8

Road map for understanding the bioluminescence color variation as a function of a solvent-polarity parameter. Reprinted from [68] with permission from American Chemical Society (ACS)

Fig. 9

The active site model of firefly luciferase. The amide carbonyls of Ser316 and Gln340 play an important role as basic moieties similar to non-polar solvents. Reprinted from [68] with permission from American Chemical Society (ACS)

Fig. 10

Graphic HOMO and LUMO model of oxyluciferin. According to molecular modeling, transition from S₁ to S₀ leads to an internal negative charge transfer from the thiazolone ring near the AMP to the benzothiazole ring. Reprinted from [105] with permission from American Chemical Society (ACS)