This peak might be associated with another biomolecular state of the emitter that our approach did not capture, suggesting a need for further investigation. More sophisticated theoretical approaches will be required to fully understand and assess the significance of these intriguing results. Additionally, our analysis of the AncFT-D160A engineered enzyme, which is structurally analogous to RLu8-D162A yet exhibiting a distinctly blueshifted emission maximum, shows it lacks aspartate residues on the rim of catalytic pocket that can accept protons. This inhibits the deprotonation of the hydroxyl group on the R2 6-(p-hydroxyphenyl) substituent of the emitter, thereby maintaining the emitter in a neutral state. Our predicted emission spectra for this neutral form align well with experimental results, and this might explain the differences in observed emission spectra.