Red Light in Chemiluminescence and Yellow-Green Light in Bioluminescence:  Color-Tuning Mechanism of Firefly, Photinus pyralis, Studied by the Symmetry-Adapted Cluster−Configuration Interaction Method

Abstract

The yellow-green luminescence from firefly luciferase has long been understood to be the emission from enol-oxyluciferin. However, a recent experiment showed that an oxyluciferin constrained to the keto form produced a yellow-green emission in luciferase (Branchini, B. R.; Murtiashaw, M. H.; Magyar, R. A.; Portier, N. C.; Ruggiero, M. C.; Stroh, J. G. J. Am. Chem. Soc. 2002, 124, 2112-2113). The present quantum mechanical/molecular mechanical and symmetry-adapted cluster-configuration interaction (SAC-CI) theoretical study supports the keto-form to be the yellow-green bioluminescence state in luciferase. We give the theoretically optimized structure of the excited state of oxyluciferin within luciferase, which gives luminescence calculated by the SAC-CI method that is close to the experimental value. Coulombic interactions with neighboring residues, in particular Arg218 and the phosphate group of AMP, play important roles in the color-tuning mechanism. Transformation to the enol form is energetically unfavorable in the luciferase environment. The twisted intramolecular charge-transfer (TICT) state is meta stable and would be easily relaxed to the co-planer structure. Further analyses were performed to verify the spectral-tuning mechanism based on the protonation state and the resonance structure of oxyluciferin.

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

“…[12,[22][23][24][25][26][27][28] In contrast, bioluminescence of the Maki analogue 5 with different Fluc mutants occurred with almost identical emission wavelength maxima and only very small nonspecific shifts between Fluc mutants ( Figure S2 a), [19] supporting the theory that the phenolic hydroxy group is crucial for the color-tuning mechanism of luciferin. [22][23][24][25][26][27][28] These results suggest that iLH 2 6 would be of use for in vivo multiparametric imaging, whereas the Maki analogue 5 would not be. The other redshifted luciferin analogues (Figure 1) may also not be suitable because of the substitution of the phenolic hydroxy group for an amine.…”

“…[11][12][13] Current measurements and calculations suggest that color modulation is due to perturbing interactions in the microenvironment surrounding the anionic phenolate of excited-state oxyluciferin (2) in the Fluc active site. [22][23][24][25][26][27][28] Additionally, p-p overlap between the benzothiazole and thiazolone heterocycles in 2 also appears to be important. [29][30][31] Maki and co-workers demonstrated the importance of extended p-conjugation in luciferin derivatives which led to the development of 5.…”

“…This highlights the importance of retaining the 6'-hydroxy group for color modulation. [22][23][24][25][26][27][28] In vitro bioluminescence spectra of iLH 2 6 ethyl ester (saponified with PLE (pig liver esterase) in situ immediately prior to use) with purified wild-type (WT) Fluc, the x5 Fluc mutant (a thermostable Fluc with similar properties to WT but with higher quantum yields), [35,36] and the x5 S284T Fluc mutant (a bright red-shifted point mutant of x5) [37,38] showed marked red-shifted peak maxima of 100 nm magnitude compared to the l max of each enzyme with 1 ( Figure 2, Table 1). This effect is remarkable considering that these mutants were originally engineered for different emission …”

Theoretical Study of the Relationships between Excited State Geometry Changes and Emission Energies of Oxyluciferin

“…[1][2][3][4] Interestingly, while keto-1 exhibits red fluorescence in DMSO or in aqueous solution, 2,3,[5][6][7][8][9] its bioluminescence spectra can be shifted to the green by interactions with the luciferases. [10][11][12][13][14][15][16] Several mechanisms have been proposed to explain this phenomenon.…”

“…[10][11][12][13][14][15][16] Several mechanisms have been proposed to explain this phenomenon. 1,2,4,6,11,12,14,[17][18][19][20][21][22][23][24][25] To the present, the detailed mechanism of the bioluminescence and its shifts in color remain unclear due to the complexity of the microenvironment in vivo.…”