Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence

The first systematic steady-state and time-resolved emission study of firefly oxyluciferin (emitter in firefly bioluminescence) and its analogues in aqueous buffers provided the individual emission spectra of all chemical forms of the emitter and the excited-state equilibrium constants in strongly polar environment with strong hydrogen bonding potential. The results confirmed the earlier hypothesis that excited-state proton transfer from the enol group is favored over proton transfer from the phenol group. In water, the phenol-keto form is the strongest photoacid among the isomers and its conjugate base (phenolate-keto) has the lowest emission energy (634 nm). Furthermore, for the first time we observed green emission (525 nm) from a neutral phenol-keto isomer constrained to the keto form by cyclopropyl substitution. The order of emission energies indicates that in aqueous solution a second deprotonation at the phenol group after the enol group had dissociated (that is, deprotonation of the phenol-enolate) does not occur in the first excited state. The pH-dependent emission spectra and the time-resolved fluorescence parameters revealed that the keto-enol tautomerism reaction, which can occur in a nonpolar environment (toluene) in the presence of a base, is not favored in water.