Further investigation is necessary to explore the significance of these intriguing findings within the protein environment. Additionally, considering that the experiment used a higher concentration of potassium phosphate buffer solution as mentioned above, further experimental studies focusing on the impact of buffer solution concentration effect on the emission spectra could provide insights into the biomolecular state of the emitter in RLuc8-D162A. Our study also extended to the engineered design AncFT-D160A enzyme (PDB ID: 7QXQ), known for its similarities to RLuc8-D162A in structure and active-site chemistry, yet displaying a strikingly different emission wavelength. Specifically, AncFT-D160A demonstrated a blue-shifted emission maximum at 390 nm, in stark contrast to RLuc8-D162A emission at 520 nm. The underlying cause of this shift is likely due to be the absence of an adjacent Aspartate residue, which would typically deprotonate the hydroxyl group of the R2 6-(p-hydroxyphenyl) substituent, thereby maintaining the chromophore in a neutral state. Our calculations for the neutral chromophore's emission spectra in AncFT-D160A yield a value of 375 nm, remarkably consistent with experimental observations. This finding highlights the pivotal role of hydrogen bond proton acceptor residues in determining the enzyme's emission characteristics.