In the bioluminescence mechanism, the excited state of the amide anion is an intermediate, as outlined in the proposed reaction pathway depicted in Scheme 1. The emission peak corresponding to this intermediate species is reported for RLuc8-D120A mutant. Our PDLD titration calculation for this mutant revealed that the ionization states of the amino acids closely resemble those in the RLuc8. However, notable exceptions include the neutral state of His142 and the ionized state of Asp162 (refer to Table in the Supporting Information for full ionization configurations). This suggests that preceding the transition of the amide ion to a neutral emitter via a PT process (see Scheme 1), Asp162 remains in its ionized form. The un-ionized state of His142 implies a more intricate network of PT processes potentially involving nearby residues or the bulk solvent, as the emitter transitions from the amide ion to the neutral or phenolate form.