However, if the emitter remains protonated, it emits a photon of purple light (405 nm). Additionally, earlier studies by McCapra et al. and Shimomura et al. have also explored the possibility of the amide anion formed as a result of chemiluminescence as the potential emitter. Consequently, coelenteramide is believed to exist in various bimolecular states, distinguished by their protonation states (either neutral or anionic) and the specific site of deprotonation (either phenolate or amide ion). In the presence of alkali metals, coelenteramide can also adopt a pyrazine form, where the alkali metal acts as a counter ion to the phenolate emitter, facilitating the formation of the pyrazine structure through resonance (vide infra). Since RLuc utilizes the same substrate as the aequorin and obelin proteins, produces identical products, and emits light with similar spectral characteristics, the reaction mechanisms are likely analogous. However, understanding the detailed catalytic mechanisms and spectral properties of RLuc has proven to be a significant challenge, largely due to the scarce availability of structural data for substrate-and product-bound enzyme complexes. To address this issue, mutagenesis experiments were conducted to create mutants that are more conducive to crystallization. In one of the earlier investigations, Loening et al. determined the structure of a stabilized form of RLuc, which contains eight amino acid substitutions (RLuc8), both with and without the coelenteramide product bound. While the coelenteramide was found in one of these structures (PDB ID: 2PSJ), its location on the outer edge of the active site was interpreted as a nonproductive binding mode. Recently, Damborsky et al. co-crystallized coelenteramide bound RLuc8 complex with a resolution of 1.9 Å (PDB ID: 6YN2) in the presence of excess of the substrate coelenterazine, providing biological relevant structural insights. In another recent study conducted by Marek et al., a crystal structure of the RLuc8-coelenteramide complex was obtained with a high-resolution of 1.5 Å (PDB ID: 7OMR). The authors of this work carried out a detailed mechanistic study for this complex, taking into account multiple co-crystal structures of the stabilized AncFT surrogate enzyme and RLuc8 luciferases, given their structural similarities. This study was backed by mutagenesis, experimental approaches, and molecular dynamics studies. Furthermore, they assigned the bioluminescence states of the coelenteramide emitter in RLuc8 and its mutants, which is the main focus of our work.