The quantum mechanical (QM) component was characterized using the QCFF/PI approach to define the chromophore in excited states. The MD simulations consisted of 20,000 steps with a timestep of 0.5 fs at a temperature of 300K. We recorded the emission energies of both the isolated chromophore and the chromophore influenced by MMderived charges. The collected data points (2000 in total) were distributed across 20 bins, ranging from the lowest to the highest emission wavelengths. We then calculated the emission wavelength () and their corresponding relative intensities as a fraction of occurrences per bin, normalized against the total number of data points. The emission maximum (max) for the phenolate form in RLuc8, after correcting for the water reference value using Eqn. (3) (also refer to Table for the reference emission wavelength in water), was determined to be 467 nm. This closely matches the experimentally observed value of 480 nm. Our calculated QCFF/PI vertical emission spectrum is depicted in Figure . Additionally, we examined the neutral chromophore while keeping Asp162 unionized.