1 LFOH * . As noted by List et al., the electrostatic effect imposed by the protein can be determined by comparing the results obtained with the bare fluorophore in System III and the results obtained with the fluorophore embedded in the protein (System I). After examining the importance of geometry confinement exerted by the protein environment, we now turn to investigating the electronic emission properties of HFOH employing the bare fluorophore with the planar structure observed in the protein (System III). Notably, the fluorophore in these two cases adopts the same geometry, as obtained using the QM/MM method (refer to Figure ), to eliminate the interference of conformational effect. Similarly, the electrostatic effect of polar solvents can be obtained by comparing the results for the planar geometry of the bare chromophore in the gas phase and in different solvents. The fluorescence spectral properties of HFOH were calculated using the simplified model molecule LFOH, with TD-CAM-B3LYP/def2-TZVP method consistent with above QM/MM calculations. The first excited state (S 1 ) is primarily (96.5%) represented by a one-electron promotion from the highest occupied molecular orbital (HOMO, π) to the lowest unoccupied molecular orbital (LUMO, π*). The calculated vertical emission energy from the S 1 to S 0 state is 3.01 eV, corresponding to a fluorescence wavelength of 412 nm (Figure ). Notably, it is about 80 nm shorter than the emission maximum of ca. 490 nm observed in the protein environment. With the planar conformation, the calculated oscillator strength (f) is 0.106, much stronger than the value of 0.001 estimated for the distorted conformation. Analysis of the frontier molecular orbitals shows the HOMO is delocalized over the isoalloxazine ring, while the LUMO is mainly concentrated on the pyrimidine ring (insets of Figure ). Accordingly, the right pyrimidine ring becomes more electron-rich, while the rest of the molecule becomes electron-deficient upon S 0 to S 1 promotion, which is verified by the hole and electron map (Figure ). The calculated dipole moment of the S 0 state (μ 0 ) is 6.20 D, pointing approximately in the negative X direction (Figure ). For the S 1 state, the dipole moment (μ 1 ) is much larger with a magnitude of 14.33 D, pointing nearly in the same direction (Figure ). The large difference dipole moment (∆μ = μ 1μ 0 = 8.30 D) indicates that the S 1 state has a significant charge transfer (CT) character, which can be further confirmed from the hole and electron map shown in Figure .