Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions. In this work, we present the simulation of the absorption and emission spectra of the possible natural oxyluciferin forms in water and some of their analogues considering both the solvent/oxyluciferin interactions and the dynamical effects by using MD simulations and QM/MM methods. On the one hand, the absorption band shapes have been rationalized by analyzing the electronic nature of the transitions involved. On the other hand, the simulated and experimental emission spectra have been compared. In this case, an ultrafast excited state proton transfer (ESPT) occurs in oxyluciferin and its analogues, which impairs the detection of the emission from the protonated state by steady-state fluorescence spectroscopy. Transient absorption spectroscopy was used to evidence this ultrafast ESPT and rationalize the comparison between simulated and experimental steady-state emission spectra. Finally, this work shows the suitability of the studied oxyluciferin analogues to mimic the corresponding natural forms in water solution, as an elegant way to block the desired interexchange reactions allowing the study of each oxyluciferin form separately.

Absorption and emission energies calculation covering both implicit and explicit solvation models using oxyluciferin as the case of study.