We modeled the pyrazine ion with an explicit Na + positioned 2. pyrazine ion (as depicted in Scheme 2). Examining the X-ray crystal structure of RLuc8-D162A reveals that the nearest proton accepting residue Asp158 is located 4.9 Å from the oxygen of phenolic group of the emitter. This donor-acceptor gap is considerably lengthy for efficient proton transfer, hinting at an unfavorable process. The deprotonation of the phenolic group could potentially occur to the bulk solvent, or through a Grotthusstype water-mediated proton transfer involving the phenolic group of the emitter and Asp158. To assess the proposed hypothesis, we investigated the emission maximum of the pyrazine ion with Asp158 in both its protonated and unprotonated states.