Moreover, our calculations also provide theoretical evidence supporting the bioluminescence mechanism in the RLuc8-D120A mutant, where the light emitter is in its amide ion form. The emission maximum of this form aligns well with experimental findings. Further investigation into the bioluminescence of the RLuc8-D162A mutant reveals that the presence of a counter ion (Na + ) is essential for the formation of the pyrazine anion, with its emission maximum closely matching observed value. However, our method did not replicate the second, lower emission peak reported for RLuc8-D162A.