pH-dependent absorption spectrum of oxyluciferin analogues in the active site of firefly luciferase

International audience; In the quest for the identication of the light emitter(s) responsible for the rey bioluminescence, the study of oxyluciferin analogues with controlled chemical and electronic structures is of particular importance. In this article, we report the results of our experimental and computational investigation of the pH-dependent absorption spectra characterizing three analogues bound into the luciferase cavity, together with adenosine-monophosphate (AMP). While the analogue microscopic pK a values don't dier much from their reference values, it turns out that AMP protonation state is analogue-dependent and never doubly-deprotonated. Careful analysis of the interactions evidences the main role of E344 glutamic acid, as well as the exibility of the cavity which can accommodate any oxyluciferin analogue. The consideration of the absorption spectra suggests that the oxyluciferin enolate form has to be excluded from the list of the bioluminescence reaction products.