Our findings reveal that in RLuc8, the chromophore (coelenteramide) predominantly exists in its phenolate form when the nearby Asp162 is protonated. This conclusion is supported by our QCFF/PI calculations, which matches closely the experimental values of the emission maxima. Furthermore, we studied the proton transfer (PT) process that leads to the formation of the primary emitter in the RLuc8's bioluminescent reaction, utilizing both PDLD/S-LRA and QCFF/PI methods. Our calculations demonstrate that incorporating the deprotonation reaction energy between the neutral emitter and Asp162 in both the excited and ground states into the emission energy calculations accurately replicates the emission maximum of the phenolate ion. This indicates strongly that Asp162 serves as a potential proton acceptor, making the phenolate ion the dominant emitting species in RLuc8.