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

In the quest for the identification of the light emitter(s) responsible for the firefly 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 pKa values do not differ much from their reference values, it turns out that the AMP protonation state is analogue-dependent and never doubly-deprotonated.

Excited-State Proton Transfer in Oxyluciferin and Its Analogues.

One of the most characterized bioluminescent reactions involves the firefly luciferase that catalyzes the oxidation of the luciferin producing oxyluciferin in its first excited state. While relaxing to the ground state, oxyluciferin emits visible light with an emission maximum that can vary from green to red. Oxyluciferin exists under six different chemical forms resulting from a keto/enol tautomerization and the deprotonation of the phenol or enol moieties.

Deciphering the pH-dependence of ground- and excited-state equilibria of thienoguanine.

The thienoguanine nucleobase (G) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, G exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of G as a function of pH (2 to 12).

Simulation and Analysis of the Spectroscopic Properties of Oxyluciferin and Its Analogues in Water.

Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions.