We next explored the pyrazine anion as a potential emitter, as identified for RLuc8-D162A. Previous research by Shimomura and colleagues demonstrated that in various solvents and with alkali metals, the phenolate ion can transition to a pyrazine ion, a resonance structure of phenolate. However, this experiment was observed in the absence of protein environment. In the recent study of RLuc8-D162A, the experiment was conducted in 100 mM potassium phosphate buffer, providing a high concentration of potassium ions and therefore the resonance structure of phenolate emitter might form due to the presence of this alkali metal ion. The potassium ion thus acts as a counter ion near the pyrazine ring, creating a polarized N -...K + bond. This interaction subsequently leads to the phenolate oxygen transitioning to a keto form, a process driven by charge delocalization (refer to Scheme 2).