Exploring the Possibility of Excited State Keto‐Enolate Transformation of the Oxyluciferin‐Luciferase Complex with QM/MM Free Energy Simulations

Abstract

Firefly bioluminescence has been widely studied due to its high quantum yield and its broad range of color modulation, but debates on the origin of its color modulation and the exact chemical form of the emitter are still ongoing. Herein, the ketoenolate transformation of the oxyluciferin-luciferase complex is investigated using quantum mechanics/molecular mechanics (QM/MM) free energy simulations. We find that the free energy profile of this transformation largely depends on the protonation state of the phenoxy oxygen in oxyluciferin. Although the keto-enolate transformation can be facilitated by protonation of this oxygen atom, the overall transformation is still unlikely and only the keto form will predominantly exist along our hypothesized reaction path. Nevertheless, we show with additional calculations with a modestly modified protein electric field that the energetics of the transformation can be heavily modulated with the protein electrostatics. This suggests that carefully modeling the protein-ligand interaction is key to understanding the reaction mechanism of the firefly bioluminescence and that it is not yet appropriate to completely rule out the possibility of having enolate participation.

Spectrochemistry of Firefly Bioluminescence

“…24 These localized charges not only induce changes in the local electric field of the cavity but are also known to potentially cause conformational changes that can have additional consequences on the local electric field. The local electric field is also heavily affected by the dynamics of the reaction, most notably, the oxyluciferin tautomerization 130 and the decomposition of the dioxetanone. 148 More advanced calculations of the electronic transitions that have utilized QM/MM methods (applying electronic embedding to classical representations) to achieve a better description of the local electric field have concluded that the emission energy of oxyluciferin is red-shifted when the electric field of the luciferase cavity is taken into account.…”

“…Another approach used to take into account the non-uniformity of the cavity is to mimic the directionality of the cavity’s polarity by applying a uniform external electric field. ,− This approach reflects better the fact that the substrate is not free to move in the cavity. In the case of oxyluciferin, the degree of movement in the cavity is limited due to a relatively narrow cavity (for a flat molecule), as can be seen with all classical molecular dynamics simulations perfomed on this system. ,, We remind the reader that a charge q at position P induces at a point M of space at the distance r PM an electric field in the direction PM proportional to q / r PM 2 .…”

“…An estimate of the mean local electric field inside the cavity (calculated by removing the oxyluciferin) has been reported at slightly less than 300 mV/Å, with projections along the x , y , and z axes of −134.7, +148.1, −180.2 mV/Å (Chart ), respectively. This value is higher than the commonly accepted value for oxyluciferin from Stark studies (150–200 mV/Å) because only the electric field along a single axis is considered at a time. ,− …”

“…These localized charges not only induce changes in the local electric field of the cavity but are also known to potentially cause conformational changes that can have additional consequences on the local electric field. The local electric field is also heavily affected by the dynamics of the reaction, most notably, the oxyluciferin tautomerization and the decomposition of the dioxetanone . More advanced calculations of the electronic transitions that have utilized QM/MM methods (applying electronic embedding to classical representations) to achieve a better description of the local electric field have concluded that the emission energy of oxyluciferin is red-shifted when the electric field of the luciferase cavity is taken into account. ,,,,,− Comparison of the calculated spectra in water while taking into account the solvent effects within the polarizable continuum model (PCM) or with explicit water molecules in a QM/MM scheme with the experimental spectra showed the importance of accounting explicitly for the solvent. − Neglecting the hydrogen bonds while considering the effect of the protein environment on the absorption and emission spectra of the light emitter is a big approximation.…”