In the RLuc8-D162A variant, when Asp158 is unprotonated, our calculated emission maximum for the pyrazine ion is 552 nm. Conversely, when Asp158 is protonated, the emission maximum shifts to 555 nm. These predicted values, while slightly red-shifted by 32 and 35 nm respectively, align closely with the experimental value of 520 nm. This agreement suggests the presence of the pyrazine ion in this variant. Furthermore, our findings imply that D158 may serve as a proton acceptor from the chromophore, facilitated by a water-assisted Grotthuss-type mechanism as previously mentioned. It is important to note, however, that while our emission maximum is in good agreement with the observed value, the experimental study did also report a smaller peak at 400 nm, which was absent in our simulated spectra (see Fig. ). This discrepancy may be attributed to additional structural changes in the emitter within the protein environment, possibly involving another π to π* transitions. Prior experimental studies conducted in the absence of protein environment have demonstrated that the presence of a counter ion can toggle the biomolecular state of the emitter, resulting in two distinct peaks. The higher peak corresponds to the pyrazine anion, while the lower peak is linked to the amide anion.