Bioluminescence is a widespread light-emitting phenomenon of living organisms, ranging from bacterial, fungal, insect, and marine creatures. Due to its simple operation, short detection time, and high sensitivity, bioluminescence has been extensively used in many fields, such as biological analysis, bioimaging, detection of environmental toxicants, and many others. However, its application is somewhat limited by the fact that the photon energy is weakened by the scattering and absorption of biological tissue. To further improve its spectroscopic properties and expand its applications, an in-depth understanding of its spectral tuning mechanism is highly desired. Bioluminescent bacteria are the most widely distributed bioluminescent species in the world. The bioluminescence found in bacteria is a result of the activity of the luciferase enzyme, known as bacterial luciferase. Bacterial luciferase is a unique flavin-dependent monooxygenase, utilizing reduced flavin mononucleotide (FMNH 2 ), O 2 , and a long-chain aliphatic aldehyde to produce visible light. Previous NMR studies have demonstrated that the luciferase-bound FMNH 2 molecule adopts an anionic form (FMNH -, I-1). FMNH -can activate a dioxygen molecule to form 4a-hydroperoxy-5-hydro-FMN (HFOOH, I-2) with the assistance of a proton supplier. HFOOH further reacts with the aliphatic aldehyde, yielding the 4a-peroxyhemiacetal-5-hydro-FMN species (HFOOCH(OH)R, I-3). Decomposition of I-3 generates the 4a-hydroxy-5-hydro-FMN intermediate in its first singlet excited state HFOH * (I-4), which is responsible for light emission. I-4 returns to its ground state (HFOH, I-5) after radiative decay. Finally, HFOH can rapidly convert to the oxidized flavin mononucleotide (FMN) species via a dehydration reaction, which can be further transformed to FMNH -to initiate another luminescence process. HFOH exhibits a fluorescence peak around 490 nm when bound in the active site of the luciferase protein. However, it is unstable and readily converts to FMN with the loss of one water molecule in solution, as shown in Scheme 1. Consequently, N(5)-alkylated 4a-hydroxy flavins are commonly used as experimental models to mimic HFOH. Interestingly, the N5alkylated analogs display weak fluorescence in solution, yet their fluorescence is greatly enhanced when bound to the enzymatic binding pocket. Additionally, the fluorescence quantum yield was found to be markedly enhanced in a frozen state of solvents than at room temperature. A joint computational and experimental study indicated that an orthogonal out-ofplane distortion of the terminal pyrimidine ring leads to the excited state deactivation via a conical intersection (CI) mediated internal conversion. The geometry confinement imposed by the frozen solvent or the protein cavity restraints the ring distortion and blocks the CI-mediated internal conversion, readily explaining the fluorescence enhancement. Scheme 1. The proposed bioluminescence process of bacterial luciferase. R′ denotes CH 2 (CH 2 OH) PO 4 H 2 .