Theoretical Study of Metal Ion–Induced Modulation of Firefly Bioluminescence Spectra

Firefly bioluminescence (BL), owing to its high sensitivity and low background, has emerged as a powerful tool for imaging and biosensing. Experimentally, the presence of heavy metal ions induces a red shift in the emission spectrum, yet the microscopic origin of this modulation remains unclear. In this study, we present a theoretical investigation of firefly BL spectra in the presence of Ag ⁺ , Zn ²⁺ , Cd ²⁺ , and Hg ²⁺ using molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. The results demonstrate that the spectral tuning does not follow simple periodic trends (e.g., ionic charge and radius) of the metal ions but is governed by a specific structural remodeling of the active site. We identify that metal ions act as allosteric triggers that disrupt the native salt-bridge network through specific coordination geometries. This perturbation remodels the enzyme microenvironment, inducing both steric and electrostatic changes that collectively regulate the emitter. Structurally, the altered spatial constraints force the light emitter oxyluciferin ( oLu ) to undergo a planar-to-curved geometric transition, which indirectly modulates its electronic structure. Electrostatically, the remodeling repositions residue Lys529 into close proximity with the oLu , dramatically enhancing the internal electric field (IEF). By drawing an analogy to the modulation induced by uniform external electric fields (EEF), we demonstrate that this enhanced IEF regulates the oLu 's electronic structure via an electrochromic effect. These findings elucidate the structural origins of the differential sensitivity to various heavy metals and establish a theoretical foundation for "electrostatic engineering" in the rational design of biosensors.