Distinguishing between keto-enol and acid-base forms of firefly oxyluciferin through calculation of excited-state equilibrium constants

Abstract

While recent years have seen much progress in the elucidation of the mechanisms underlying the bioluminescence of fireflies, there is to date no consensus on the precise contributions to the light emission from the different possible forms of the chemiexcited oxyluciferin (OxyLH2) cofactor. Here, this problem is investigated by the calculation of excited-state equilibrium constants in aqueous solution for keto-enol and acid-base reactions connecting six neutral, mono-anionic and di-anionic forms of OxyLH2.Particularly, rather than relying on the standard Förster equation and the associated assumption that entropic effects are negligible, these equilibrium constants are for the first time calculated in terms of excited-state free energies of a Born-Haber cycle.Performing quantum chemical calculations with density functional theory methods and using a hybrid cluster-continuum approach to describe solvent effects, a suitable protocol for the modeling is first defined from benchmark calculations on phenol. Applying this protocol to the various OxyLH2 species and verifying that available experimental data (absorption shifts and ground-state equilibrium constants) are accurately reproduced, it is then found that the phenolate-keto-OxyLH -mono-anion is intrinsically the preferred form of OxyLH2 in the excited state, which suggests a potential key role for this species in the bioluminescence of fireflies. Keywords Graphical Table of ContentsAqueous keto-enol and acid-base excited-state equilibrium constants between six neutral, mono-anionic and di-anionic forms of oxyluciferin, the cofactor responsible for the bioluminescence of firefly luciferase, are for the first time calculated from free energies of a Born-Haber cycle, rather than using the Förster equation. Thereby, it is found that the phenolate-keto-OxyLH -mono-anion is the preferred excited-state form of oxyluciferin in aqueous solution, attributing a potential key role to this species in the bioluminescence of fireflies.

How accurate are TD‐DFT excited‐state geometries compared to DFT ground‐state geometries?

“…Initial work along those lines includes an insightful study focused on exciton properties [ 110 ] and assessments of how accurately TD‐DFT reproduces experimental free‐energy barriers and equilibrium constants of excited‐state intramolecular proton transfer reactions. [ 111,112 ]…”

The Fluorescent Properties of pH‐Independent Cypridina Oxyluciferin Derivatives

“…In the same way, the excited state dissociation constant (pKa*) can be obtained using the same equations when considering molecules in their excited state. This method provided a better description of pKa and pKa* of firefly oxyluciferin and coelenteramide …”

“…This method provided a better description of pKa and pKa* of firefly oxyluciferin and coelenteramide. [52,[56][57][58][59] The Gibbs energy in the gas phase was calculated by vibrational frequency analysis. The zero point effects were accounted in the calculations.…”

Vibronic Structures in Absorption and Fluorescence Spectra of Firefly Oxyluciferin in Aqueous Solutions

“…On the other hand, the hydrogen bonding interactions may have to be considered in the case of phenolate‐keto and keto. In these days, the absorption energies for oxyluciferin and its conjugate bases with eleven water molecules using PCM method were reported . Even if eleven water molecules were explicitly included in the model system, it is not easy to obtain the theoretical absorption energies which are nearly equal to the experimental ones.…”