Tautomer-Selective Fluorescence Spectroscopy of Oxyluciferin Anions

Bioluminescence in fireflies and related insects arises as emission from the fluorophore oxyluciferin, yet the color of the emission in these insects can range from red to green. The chromophore's microenvironment or multiple tautomeric forms may be responsible for the color tuning; however, these effects are difficult to separate in condensed phases. To investigate the role of oxyluciferin tautomerization in the color tuning mechanism, gas-phase spectroscopy eliminates solvent effects and allows us to study the fluorescence from individual tautomers. Using a home-built mass-spectrometry setup with a cylindrical ion trap cooled with liquid nitrogen, we measure fluorescence from the enol-locked form of oxyluciferin in the gas phase and characterize the photophysics of both keto and enol forms. At 100 K, the enol-locked form has an emission maximum of 564 ± 1 nm, coinciding with a previously reported assignment in oxyluciferin. We measure the absorption spectrum and find a maximum at 560.5 ± 0.5 nm, which implies a Stokes shift of 110 cm^-1. The absorption spectrum is compared to Franck-Condon simulated spectra that identify one dominant vibrational mode in the transition. Additionally, we ultimately separated the emission by the enol and keto forms present in the trap by selectively exciting each form. We demonstrate that fluorescence measured close to the 0-0 transition limits the reheating of the ions, thereby providing the coldest ions and therefore the narrowest emission spectra. These experimental data are also crucial benchmarks for computational studies, offering actual emission spectra in the gas phase for both tautomeric forms. Thus, our findings serve as essential reference points for excited-state calculations aimed at understanding the color tuning mechanism of bioluminescence computationally.