Theoretical Insight into the Fluorescence Spectral Tuning Mechanism: A Case Study of Flavin-dependent Bacterial Luciferase

Bioluminescence of bacteria is widely applied in biological imaging, environmental toxicants detection, and many other situations. Understanding the spectral tuning mechanism not only helps explain the diversity of colors observed in nature, but also provides principles for bioengineering new color variants for practical applications. In this study, time-dependent density functional theory (TD-DFT) and quantum mechanics and molecular mechanics (QM/MM) calculations have been employed to understand the fluo-rescence spectral tuning mechanism of bacterial luciferase, with a focus on the electrostatic effect. The spectrum can be tuned by both the homogeneous dielectric environment and oriented external electric fields (OEEFs). Increasing solvent polarity leads to a redshift of the fluorescence emission maximum, λF, accompanied by an increase in density. In contrast, applying an OEEF along the long axis of the isoalloxazine ring leads to a significant red- or blue-shift in λF, depending on the direction of the OEEF, but with negligible changes in its intensity. The effect of polar solvents is directionless, and the red-shifts can be attributed to the larg-er dipole moment of the S1 state compared to the S0 state. However, the effect of OEEFs directly correlates with the difference dipole moment between the S1 and S0 states, which is directional and determined by the charge redistribution upon excitation. Moreover, the electrostatic effect of bacterial luciferase is in line with the presence of an internal electric field (IEF) pointing in the negative X direction with a magnitude of ca. 30 MV/cm. Finally, key residues that contribute to this IEF and strategies for modu-lating the spectrum through site-directed point mutations are discussed.