Excited-State Proton Transfer of Firefly Dehydroluciferin

Steady-state and time-resolved emission techniques were used to study the protolytic processes in the excited state of dehydroluciferin, a nonbioluminescent product of the firefly enzyme luciferase. We found that the ESPT rate coefficient is only 1.1 × 1010 s–1, whereas those of d-luciferin and oxyluciferin are 3.7 × 1010 and 2.1 × 1010 s–1, respectively. We measured the ESPT rate in water–methanol mixtures, and we found that the rate decreases nonlinearly as the methanol content in the mixture increases. The deprotonated form of dehydroluciferin has a bimodal decay with short- and long-time decay components, as was previously found for both d-luciferin and oxyluciferin. In weakly acidic aqueous solutions, the deprotonated form’s emission is efficiently quenched. We attribute this observation to the ground-state protonation of the thiazole nitrogen, whose pK a value is ∼3.