This finding is in good accordance with the reported emission maximum of neutral coelenteramide (400 nm) observed in other bioluminescent proteins such as Ca 2+ regulated photoprotein. However, this value is significantly different from the observed emission in RLuc8. This discrepancy, along with the good agreement between the experimental and calculated emission maximum of the phenolate anion, emphasizes the importance of Asp162 protonation state. It strongly suggests that in RLuc8, the coelenteramide emitter exists in the phenolate form during its photochemical and photophysical processes.