Theoretical Insight\ninto the Fluorescence Spectral\nTuning Mechanism: A Case Study of Flavin-Dependent Bacterial Luciferase

Bioluminescence of bacteria is widely applied in biological\nimaging,\nenvironmental toxicant detection, and many other situations. Understanding\nthe spectral tuning mechanism not only helps explain the diversity\nof colors observed in nature but also provides principles for bioengineering\nnew color variants for practical applications. In this study, time-dependent\ndensity functional theory (TD-DFT) and quantum mechanics and molecular\nmechanics (QM/MM) calculations have been employed to understand the\nfluorescence spectral tuning mechanism of bacterial luciferase with\na focus on the electrostatic effect. The spectrum can be tuned by\nboth a homogeneous dielectric environment and oriented external electric\nfields (OEEFs). Increasing the solvent polarity leads to a redshift\nof the fluorescence emission maximum, λ<sub>F</sub>, accompanied\nby a substantial increase in density. In contrast, applying an OEEF\nalong the long axis of the isoalloxazine ring (<i>X</i>-axis)\nleads to a significant red- or blue-shift in λ<sub>F</sub>,\ndepending on the direction of the OEEF, yet with much smaller changes\nin intensity. The effect of polar solvents is directionless, and the\nred-shifts can be attributed to the larger dipole moment of the S<sub>1</sub> state compared with that of the S<sub>0</sub> state. However,\nthe effect of OEEFs directly correlates with the difference dipole\nmoment between the S<sub>1</sub> and S<sub>0</sub> states, which is\ndirectional and is determined by the charge redistribution upon deexcitation.\nMoreover, the electrostatic effect of bacterial luciferase is in line\nwith the presence of an internal electric field (IEF) pointing in\nthe negative <i>X</i> direction. Finally, the key residues\nthat contribute to this IEF and strategies for modulating the spectrum\nthrough site-directed point mutations are discussed.