In our study, we utilized the high-resolution X-ray structure of RLuc8 (PDB ID: 7OMR), which was complexed with the coelenteramide chromophore, to perform a QCFF/PI MD simulation on the first excited singlet state surface. It is important to note that in the X-ray crystal structure of RLuc8, the residue at position 162 is an alanine, resulting from a mutation of the native aspartate (referred to as RLuc8-D162A). Given that the emission spectra for the phenolate forms of the emitter have been experimentally reported in RLuc8, we reverted this alanine to its original aspartate (Asp) using a rotamer library within the Chimera software suite. Prior to analyzing the emission spectra of the studied systems, we first examined rigorously the ionization states of the protein residues.