Theoretical insights into the effect of pH values on oxidation processes in the emission of firefly luciferin in aqueous solution

To elucidate the emission process of firefly d-luciferin oxidation across the pH range of 7-9, we identified the emission process by comparison of the potential and free-energy profiles for the formation of the firefly substrate and emitter, including intermediate molecules such as d-luciferyl adenylate, 4-membered dioxetanone, and their deprotonated chemical species. From these relative free energies, it is observed that the oxidation pathway changes from D-luciferin -> deprotonated D-luciferyl adenylate -> deprotonated 4-membered dioxetanone -> oxyluciferin to deprotonated D-luciferin -> deprotonated D-luciferyl adenylate -> deprotonated 4-membered dioxetanone oxyluciferin with increasing pH value. This indicates that deprotonation on 6'OH occurs during the formation of dioxetanone at pH7-8, whereas luciferin in the reactant has a 6'OH-deprotonated form at pH9.