3.7 Electrostatic effect of the protein environment on the fluorescence spectrum. After elucidating the electrostatic effect of polar solvents and OEEFs, we now seek to gain insight into how the protein environment affects the fluorescence spectrum of 1 HFOH*. A direct comparison between System I and System III shows that λ F is redshifted from 412 nm to 493 nm by the electrostatic effect of the protein environment. As shown above, either increasing solvent polarity or applying an OEEF in the negative F X direction can lead to a significant redshift of λ F . In the implicit solvent model of chlorobenzene (ε = 5.70) that is commonly used to mimic the protein electrostatic environment, 105-108 the calculated λ F is redshifted to 442 nm (Figure ), yet it is still 48 nm shorter than the 490 nm observed experimentally in the protein. Interestingly, the redshift of the emission wavelength in the protein can be rationalized by the presence of an internal electric field (IEF) of F X = -0.0057 a.u. = -29.3 MV/cm (refer to Figure ). Notably, the IEF within the protein cavity is not uniform, but rather varies across the isoalloxazine ring of the fluorophore. Therefore, the estimated electric field can be viewed as an effective field representing the spatially heterogeneous IEF. The key charged residues that contribute to the IEF along the long axis of the fluorophore (X-axis), in both positive and negative directions, are shown in Figure . Negatively charged residues located on the left side, such as Glu175, Glu200, and Asp314, as well as positively charged residues on the right side, like His45, Arg107, Lys112, Lys286, and Arg85 of chain B, produce a negative F X field. Conversely, the positively charged residues on the left side, including Lys201 and Arg290, and the negatively charged residues on the right side, such as Glu43, Asp111, Asp113, and Glu88 of the chain B, generate a positive F X field. Therefore, mutations at these positions are predicted to lead to considerable changes in the emission wavelength (λ F ). Based on this understanding, a rational design of new variants of bacterial luciferase becomes possible. For example, to obtain fluorescence with a longer wavelength, one would need to introduce a more negative F X field, which could be achieved by engineering either positively charged residues on the right side or negatively charged residues on the left side of the fluorophore through site-directed mutagenesis.