1. Geometry confinement by the protein environment. As alluded to above, the luciferase protein affects the emission properties of the chromophore either by modifying its geometry or through electrostatic interactions. To understand how the geometry of the light-emitter (HFOH) is influenced by the protein, we optimized its first excited state (S 1 ) geometry both in the protein environment (System I) and in the gas phase (System II), as shown in Figure and 1B, respectively. The geometry embedded in the protein was calculated using the QM/MM method at the TD-CAM-B3LYP/6-31+G**//MM level, while the gas-phase geometry was optimized with the TD-DFT method at the TD-CAM-B3LYP/6-31+G** level. Notably, a simplified model of HFOH, denoted as LFOH, was used in the QM calculations, where the ribose phosphate group was replaced by a methyl group, to ensure computational accuracy and efficiency (refer to Scheme 2). In the presence of the protein environment (System I), the isoalloxazine ring adopts a more planar conformation, with a C 9 -N 10 -C 10a -N 1 dihedral angle of 147.6⁰ (Figure ). The vertical energy difference between the S 1 and S 0 states (E F ) estimated at the TD-CAM-B3LYP/def2-TZVP//MM level is 2.52 eV, corresponding to an emission maximum (λ F ) of 493 nm, which is close to the experimentally determined value of ca. 490 nm. In contrast, the optimized geometry in the gas phase (System II) shows an out-of-plane distortion of the pyrimidine ring, with a C 9 -N 10 -C 10a -N 1 dihedral angle of 72.9⁰ (Figure ). The E F in the gas phase (System II) estimated with this distorted geometry at the TD-CAM-B3LYP/def2-TZVP level is rather small with a value of only 0.81 eV. The distorted geometry is similar to the intersection structure obtained with N(5)-methylated LFOH using ab initio multiconfigurational quantum chemistry, in which the dihedral angle is ca. 85⁰. Furthermore, the oscillator strength (f) under vacuum was calculated to be 0.001, a rather small value indicating negligible fluorescence. In contrast, the value in the protein was calculated to be 0.133, more than 100-fold enhancement compared to the case under vacuum. Therefore, our results support the assertion that the protein environment restraints the isoalloxazine ring to its planar fluorescent state, which blocks the CI-mediated internal conversion pathway and thus increases the fluorescence quantum yield. Additionally, the results obtained with TD-DFT method are generally consistent with those obtained using the more computationally expensive stateof-the-art multireference method, demonstrating the informative nature of the TD-DFT approach.