Our consensus PDLD/S-LRA pKa calculated values for the emitter and D162 at pH 7 in RLuc8 are 7.6 and 4.9, respectively, in the GS, and -0.8 and 5.1 in the ES. Utilizing Eqn. 12, this translates to reaction free energies (∆𝐺) of +3.7 kcal/mol in the GS and -8.1 kcalmol -1 in the ES, signifying that the PT reaction is energetically more favored in the ES. It should be noted that our analysis focuses solely on thermodynamic aspects, and the depicted barrier in Figure serves an illustrative purpose only. Nevertheless, the PT process in the ES is typically associated with an energy barrier of a few kcal/mol, as observed in other bioluminescent proteins. To estimate the emission spectra during the PT reaction, we incorporated the free energy change (∆∆𝐺) into our calculated QCFF/PI emission maximum for the neutral emitter. This yielded an emission maximum of 455 nm, which is in reasonable agreement with the observed value of 480 nm, and closely matches with our calculated emission maximum of 467 nm predicted for the phenolate ion, thus providing evidence of the excited-state PT process in RLuc8 and that the emitter exclusively is in phenolate form.