What Exactly Is the Light Emitter of a Firefly?

Firefly bioluminescence attracts people by its glaring beauty and fascinating applications, but what is the light emitter of a firefly? The answer to this question has been explored since before the 1960s. The unanimously accepted answer is that excited-state oxyluciferin is the light emitter. The complexity of this question arises from the existence of six chemical forms (keto, enol, keto-1, enol-1, enol-1′, and enol-2) of oxyluciferin. After decades of experimental and theoretical efforts, a consistent conclusion was almost reached in 2011: excited-state keto-1 is the only light emitter in fireflies. However, the debate is raised again by the latest in vitro experimental results. This study will solve this contradiction via hybrid quantum mechanics and molecular mechanics (QM/MM) calculations combined with molecular dynamics (MD). The calculations were performed in the real protein for the six chemical forms of oxyluciferin and their corresponding analogues employed in the latest experiments. By considering the real environment, the pH value, and a possible equilibrium of the chemical forms of oxyluciferin in vivo, the calculated results indicate that the main emitter is still the excited-state keto-1 form.