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

Theoretical Study of Metal Ion–Induced Modulation of Firefly Bioluminescence Spectra | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Theoretical Study of Metal Ion–Induced Modulation of Firefly Bioluminescence Spectra Jinyu Wang, Deping Hu, Yajun Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8629136/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract 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. firefly bioluminescence heavy metal ions red-shift mechanism electric field effect QM/MM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Firefly bioluminescence (BL) is an enzyme-catalyzed light-emitting reaction that has been widely used for in vivo imaging [ 1 , 2 ] and cellular monitoring [ 3 ] owing to its high quantum yield and signal-to-noise ratio [ 4 ]. As illustrated in Scheme 1 , in this system, D-luciferin undergoes adenylation followed by an oxygen reaction catalyzed by luciferase, ultimately generating oxyluciferin in its first singlet excited (S₁) state, which emits visible light upon relaxation to the ground (S 0 ) state [ 5 , 6 ]. Remarkably, despite sharing a common substrate and reaction pathway, firefly BL exhibits pronounced color variability, spanning from yellow-green to red, depending on the species as well as environmental factors such as pH, temperature, and the presence of heavy metal ions [ 7 – 10 ]. Among these factors, the mechanisms driving spectral tuning by species, pH, and temperature are relatively well-understood [ 8 , 11 , 12 ]. In contrast, the molecular mechanisms for heavy metal ion-induced spectral shifts remain elusive. To address this issue, it is essential to revisit the fundamental determinants governing firefly BL color. The red shift in firefly BL is generally attributed to two critical factors: the structural form of the light emitter oxyluciferin [ 13 – 16 ] and the regulatory effects of the luciferase microenvironment [ 4 , 17 – 23 ]. The former involves keto–enol tautomerism and modifications of oxyluciferin [ 15 , 16 ], such as the introduction of various substituents or other structural alterations, while the latter encompasses a range of enzyme-associated local environmental factors, such as pocket compactness, hydrogen-bonding networks, polarity of the active site, and the electrostatic effects [ 12 , 20 – 25 ]. Regarding the structural form of the light emitter, in the native firefly BL system employing the unmodified oxyluciferin, the anionic keto form ( oLu ), as shown in Fig. 1 , has been confirmed to be the primary light emitter [ 26 , 27 ]. Based on this structure, numerous red-shifted analogs have been developed to improve in vivo imaging [ 28 ]. Accordingly, the observed firefly BL color in native firefly system is closely associated with fluorescence wavelength ( λ F ) of oLu [ 29 ]. On the basis of these considerations, it is hypothesized that the heavy metal ion-induced red shift in native firefly BL primarily originates from the luciferase microenvironment. Given that the enzyme and oLu are intrinsically coupled—where microenvironmental changes inevitably force conformational adjustments in oLu —the red shift is attributed to the complex interplay between the remodeled microenvironment and the conformational modulation of oLu . However, detecting the subtle conformational changes of oLu is experimentally challenging; thus, previous research has primarily targeted the protein architecture [ 30 – 32 ]. Focusing specifically on the role of critical amino acid residues, Viviani et al. conducted systematic studies that provided crucial insights into the mechanisms underlying the heavy metal ion-induced red shift [ 30 ]. Their studies have shown that an internal salt bridge (see Fig. 2 ) between residues Glu311 (E311) and Arg337 (R337)—located near the benzothiazole moiety of oLu —is crucial for maintaining green emission, as this closed conformation stabilizes a low-polarity active site [ 31 ]. Additional residues, such as His310 (H310) and Glu354 (E354), which can form an external salt-bridge (see Fig. 2 ), have also been shown to influence emission color, with mutations leading to red-shifted spectra [ 32 ]. Mutational analyses further identified H310, E311, and E354 as potential metal-binding sites, leading to the proposal that metal ion binding disrupts key salt-bridge interactions and increases the polarity of the substrate-binding pocket, thereby favoring red-shifted emission [ 30 , 32 ]. Nevertheless, a detailed theoretical understanding connecting these structural perturbations to the spectral red shift is still absent. To disentangle this complexity and identify the fundamental drivers of the red shift, we focused on the Brazilian species Amydetes vivianii ( A. vivianii )as an ideal model system. Unlike many other luciferases, this enzyme exhibits a selective response to heavy metal ions (such as Cd 2+ and Hg 2+ ) while being pH-insensitive, making it a promising candidate for detecting metal ions quantitatively in both cellular and environmental contexts [ 33 , 34 ]. This distinctive behavior allows us to isolate pure metal ion-induced structural perturbations from confounding protonation effects. In this study, we systematically investigate the fluorescence properties of oLu in A. vivianii luciferase 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 (Section 3.1 ). Given that the spectral shift arises from the coupled effects of the oLu 's conformation and the luciferase microenvironment, we adopted a decoupled analytical strategy to systematically identify their specific contributions: First, to isolate the contribution of oLu ’s geometry, we investigated its intrinsic structural distortion induced by metal ion binding and the steric constraints of the enzyme pocket (Section 3.2 ). Second, to clarify the role of the enzymatic microenvironment in the observed red shift—including both steric and electrostatic effects—we systematically investigate microenvironmental changes induced by metal ions, encompassing salt-bridge rearrangements, metal-ion coordination modes, displacements of key active-site residues, and the internal electric fields (IEF) generated by the enzyme. ( Section 3.3 ). Finally, to explicitly quantify the direct electrostatic influence, we examine how IEF regulates spectra, drawing an analogy to the modulation induced by uniform external electric fields (EEF) ( Section 3.4 ) [ 25 ]. This work provides the first theoretical clarification of the mechanisms governing metal ion-regulated BL, offering a structural rationale for metal-ion specificity and establishing a general framework for electrostatic engineering in enzyme color tuning, protein electrostatics, and the rational design of bioluminescent biosensors. 2. Computational Methods Setup of Systems. Five systems were constructed based on a homology model of A. vivianii firefly luciferase, as the available experimental structure lacks the substrate and adopts a non-luminescent open or semi-open conformation [ 35 ]. The model was built using Photinus pyralis ( P. pyralis ) luciferase (PDB ID: 4G36) as the template, which shares 79% sequence identity and preserves a similar salt-bridge architecture near the active site [ 11 ]. The validated model was used to construct a complete wild-type luciferase complex containing oLu and AMP ( System I , Fig. 2 a). Using this structure as a reference, four additional systems were generated by introducing Ag⁺, Zn 2+ , Cd 2+ , or Hg 2+ ions between the internal (E311–R337) and external (H310–E354) salt bridges, yielding metal ion-bound complexes ( Systems II–V , Fig. 2 b). All systems were described using the Amber ff19SB force field for proteins and metal ions [ 36 ], with the GAFF force field applied for oLu [ 37 ]. For Systems II–V , the metal ion-binding clusters formed by the metal ions and coordinating residues were parameterized using the “MCPB.py” tool [ 38 ]. MD Simulation of Systems. After proper setups, each system was equilibrated in several steps: (1) energy minimization was carried out using a combination of steepest descent and conjugate-gradient methods; (2) the system was then gradually heated from 0 K to 300 K over a 400 ps under an NVT ensemble, with a weak constraint applied to the protein backbone atoms; (3) the density was equilibrated for 1 ns at 300 K and 1.0 atm using an isothermal-isobaric Langevin thermostat [ 39 ] and Berendsen barostat [ 40 ] with a collision frequency of 2 ps − 1 and a pressure relaxation time of 1 ps; (4) a further 3 ns equilibration was subsequently carried out in the NPT ensemble to ensure stable pressure and temperature; (5) a 100 ns MD simulation was conducted. Nonbonded interactions were treated with the Particle Mesh Ewald method with a cutoff of 12 Å [ 41 ]. The covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm, enabling a 2-fs integration step. All simulations were performed with the AMBER21 software [ 42 ]. QM/MM Calculations. The equilibrated geometry from the MD trajectory was used as the initial input structure for the QM/MM calculations. For five systems, oLu was put into the QM region, while other atoms were put into the MM region. For the geometry optimizations in S 1 states of five systems, the long-range-corrected functional CAM-B3LYP [ 43 ] was employed for a better description of the charge-transfer transitions of oLu . Consequently, the TD CAM-B3LYP/6–31 + G** method, which has been shown to perform well in predicting the fluorescent properties of oLu , was employed for the QM region [ 44 ]. Atoms within 15 Å of oLu in the MM region were allowed to relax during the optimization process. Fluorescence-related single-point calculations were performed at the TD CAM-B3LYP/ma-def2-TZVP level for the QM region. All QM/MM calculations were carried out using the ChemShell software [ 45 , 46 ], which integrates ORCA 5.0 [ 47 ] for the QM region and DL_POLY [ 48 ] for the MM region. The TD-DFT method [ 49 ] was employed for the QM region of QM/MM calculations, while the Amber ff19SB force field was used to describe the MM protein environment. To include the polarization effect of the MM region on the QM region, the electronic embedding scheme was employed in the QM/MM calculations [ 50 ]. EEF and IEF Calculations. For both EEF and IEF, the molecular coordinate axes were defined relative to the oLu scaffold (Fig. 1 ) as follows: the positive X-axis along C2′→N3, the positive Z-axis along C2′→S1′, and the positive Y-axis along perpendicular to the XZ plane and pointing inward. For both EEF and IEF, the positive field direction was defined along the positive axes. Uniform EEFs ranging from − 0.08 to + 0.08 a.u. were applied independently along each axis to isolated oLu ( Systems i–v ), which were extracted from the QM region of the optimized S₁-state QM/MM structures ( Systems I–V ). EEF calculations were performed using Gaussian 09 at the TD CAM-B3LYP/ma-def2-TZVP level, while IEFs generated by the enzyme environment were computed with a homemade Python script. Further methodological details are provided in the Supporting Information (SI). 3. Results and Discussion 3.1 Fluorescence Properties and Electronic Structure of oLu in Five Systems. The fluorescence properties of oLu in five systems were investigated at the level of TD-CAM-B3LYP/ma-def2-TZVP//MM, as summarized in Table 1 . Across all five systems, the oscillator strengths ( f ) for the S₁ → S 0 transition are approximately 0.6, suggesting that oLu maintains a strong fluorescence emission in each system. In System I , the metal ion-free system, the calculated λ F of oLu is 523 nm, which shows reasonable agreement with the experimental value of 543 ± 5 nm [ 32 , 34 , 51 ], supporting the reliability of both the constructed model and the chosen computational methods. In Systems II–V , the binding of metal ions consistently induces a red shift in the λ F of oLu . The magnitude of this shift follows the order: Hg 2+ > Cd 2+ > Ag⁺ > Zn 2+ , with the largest shift occurring in System V (Hg 2+ bound) and the smallest in System III (Zn 2+ bound), which agrees qualitatively with the experimental observations [ 51 ]. Notably, this observed sequence deviates significantly from the trend predicted by classical electrostatics. Given that all four ions share a common d 10 electronic configuration, their polarizing power is governed strictly by ionic potential (charge-to-radius ratio). Consequently, ions with higher ionic potential possess stronger polarizing power and are theoretically expected to induce larger spectral shifts via electrostatic polarization. Based on this, the theoretically expected order should be: Zn 2+ > Cd 2+ > Hg 2 ⁺ > Ag + [ 52 ]. This striking discrepancy establishes that the metal ion does not act merely as a point source of electrostatic polarization. Instead, its primary role is to act as a structural trigger to modulate the electronic structure, as will be revealed through frontier molecular orbital analysis below. Table 1 Calculated fluorescence wavelengths ( λ F, nm) and oscillator strengths ( f ) of oLu in System I and II-V at TD CAM-B3LYP/ma-def2-TZVP//MM level, along with their corresponding experimental emission wavelength ( Exp . nm). System Metal ion λ F f Exp . I no 523 0.620 543 ± 5 [ 35 , 51 ] II Ag + 531 0.580 - III Zn 2+ 529 0.583 556 ~ 563 [ 51 ] IV Cd 2+ 533 0.591 568 ~ 584 [ 51 ] V Hg 2+ 539 0.532 571 ~ 584 [ 51 ] Frontier molecular orbital analysis in Table S1 and Fig. 3 shows that the fluorescence emission in Systems I and II-V arises from the de-excitation process, where the electron relaxes from the lowest unoccupied molecular orbital (LUMO) to the highest occupied molecular orbital (HOMO). The LUMO is delocalized over the entire molecule, whereas the HOMO is primarily localized on the benzothiazole moiety (see Fig. 1 ), indicating a charge-transfer character, consistent with the charge analysis results (see Table S2 ). Compared with System I , the binding of metal ions in Systems II–V stabilizes both HOMO and LUMO energies, with a greater stabilization of the LUMO. This differential stabilization narrows the HOMO–LUMO gap, which directly accounts for the observed red shift at the electronic-structure level. These changes in the electronic structure can be traced back to metal ion-induced alterations in the molecular geometry of oLu and enzyme microenvironment of oLu . 3.2 Geometric Distortion and Intrinsic Electronic Modulation of the Emitter oLu. The molecular geometry of oLu plays an important role in determining its λ F by modulating its electronic structure. Figure 4 shows the comparison of S 1 -state geometries of oLu across five systems. In System I , oLu adopts a mostly planar conformation. However, upon metal ions binding to the luciferase, oLu adopts slightly bent conformations with the S1' and N3' atoms (see Fig. 1 ) as the central axis in Systems II-V . Detailed geometric parameters are listed in Table S3 . An analysis of these parameters reveals a clear structural trend: while the benzothiazole moiety remains rigid across all systems, the thiazole ring exhibits a critical distortion characterized by both geometric bending and bond elongation. Specifically, the dihedral angle C7′–S1′–N3′–S1 (see Fig. 1 ) deviate significantly from the System I (e.g., shifts from − 177° to ~ 171°), confirming the planar-to-curved transition. Accompanying this bending, the carbonyl C = O bond (R7) consistently elongates in all metal ion-bound systems. Although the magnitude of this elongation is subtle (< 0.02 Å), the trend is uniform, indicating a weakening of the C = O double-bond character. The observed geometric alterations in oLu lead to corresponding changes in its intrinsic electronic structure. To isolate this effect, we analyzed the extracted oLu molecules from the five systems in the gas phase ( Systems i–v ). Frontier molecular orbital analysis ( Fig S3 ) reveals that the HOMO-LUMO gap of oLu in Systems ii–v is smaller than that in System i . Crucially, the orbital distribution explains the mechanistic link: since the HOMO is localized on the benzothiazole moiety, whereas the LUMO is delocalized over the entire molecule, extending significantly to the carbonyl group of the thiazole ring, the elongation of the C = O bond (R7) on thiazole ring directly stabilizes LUMO, lowering its energy level. Consequently, this geometric relaxation narrows the HOMO-LUMO gap, resulting in a systematic red shift of λ F , from 499 nm for System i to 504, 503, 507, and 508 nm for Systems ii–v , respectively. Remarkably, the red-shift trend in these isolated systems (Zn 2+ < Ag + < Cd 2 ⁺ < Hg 2+ ) is qualitatively consistent with that observed in the full protein complex ( Systems I–V ). However, the λ F in isolation (~ 505 nm) is notably shorter than those in the protein (~ 530 nm). This indicates that the geometric distortion contributes to the red shift, while the enzymatic microenvironment further amplifies the shift magnitude. This amplification effect is rooted in the specific structural remodeling of the microenvironment described below. 3.3 Remodeling of the Enzyme Microenvironment. 3.3.1 Metal Ion Coordination and Salt-Bridge Network Disruption The remodeling of the microenvironment initiates with the disruption of the native salt-bridge network, which is caused by the coordination of metal ions. Experimental studies have suggested that both internal and external salt bridges play a role in determining the BL color of firefly luciferase (see Fig. 2 ) [ 32 , 33 ]. Figure 5 presents the changes in salt bridge distances across the five systems over the 100 ns MD simulation (the root mean square deviation and radius of gyration analyses indicate that the protein backbone and oLu remained stable, confirming structural reliability; see Fig S4–S8 ), specific distances are shown in Fig S9 . In System I (metal ion-free), both the external and internal salt bridges are generally maintained throughout the simulation. The external salt bridge is formed between the NE2 atom of H310 and the OE1 or OE2 atom of E354, while the internal salt bridge involves the NH1 atom of R337 and the OE1 or OE2 atom of E311 (see Fig. 2 a). However, in System II (Ag + -bound), the external salt bridge is completely disrupted. In Systems III–V (bound to Zn 2+ , Cd 2+ , and Hg 2+ , respectively), the internal salt bridge is largely dismantled, while the external one is weakened. These results suggest that upon binding to luciferase, the metal ions effectively take over the key residues (H310, E311, E354) through specific coordination with their functional atoms (H310NE2, E311OE1/OE2, E354OE1/OE2), thereby preventing them from forming their original salt bridges. Coordination analysis clarifies the specific atomistic contacts between each metal ion and the potential coordinating atoms (H310NE2, E311OE1/OE2, and E354OE1/OE2), as detailed in Fig S10 and S11 . Based on these data, Fig. 6 summarizes the coordination patterns, revealing distinct geometries for each metal ion: Ag + primarily interacts with E311OE1 and E354OE2, forming a bidentate complex (Fig. 6 a). Zn 2+ and Cd 2+ adopt a pentacoordinate geometry, involving all five potential coordinating atoms (Fig. 6 b and 6 c). Hg 2+ predominantly adopts a tetracoordinate geometry (Fig. 6 d). Collectively, these specific coordination patterns provide the mechanistic explanation for the salt-bridge disruptions observed in Fig. 5 . Importantly, the distinct geometries adopted by each metal ion highlight their ion-specific effects, which drive the rearrangement of key residues and the remodeling of the local molecular interaction network. 3.3.2 Repositioning of Key Residues and Intermolecular Interactions. Figure 7 shows that the key molecular interactions of oLu in System I . Aligning with the stable network described in Section 3.3.1 , the inner and outer salt bridges, formed by R337–E311 and H310–E354, are located on the phenolate side of oLu , creating a relatively closed environment for oLu . The Ser314–Glu315–Gly316 peptide segment is positioned above oLu . On the left side of oLu , a water molecule bridges the O10' atom and the side chain of Ser314, while on the right side, another water molecule connects the O6 atom to the side chain of Glu316, establishing two separate hydrogen-bond networks. Additionally, the positively charged residues Lys529 and Arg218 are positioned near the right-upper and left-lower sides of oLu , respectively, contributing electrostatic attraction to oLu . Collectively, these salt bridges, hydrogen bonds, and electrostatic interactions create a well-defined local environment that maintains the stable conformation of oLu . The key molecular interactions of oLu in metal ion-bound Systems II–V are illustrated in Fig. 8 . As detailed in Section 3.3.1 , the metal ions bind to the luciferase through specific coordination modes. This binding acts as a structural force that pulls the coordinating residues closer to the ions, thereby remodeling the local microenvironment and inducing further adjustments in the overall protein conformation (see Fig S12 ). Despite these structural perturbations, certain interactions remain conserved: the Ser314–Gly316 segment remains positioned above oLu , with the Ser314–R337 hydrogen bond preserved, and O10' of oLu continues to form a hydrogen bond with a water molecule. However, the most critical change involves the positively charged residue Lys529. While Arg218 remains near the lower-left side, Lys529 is repositioned significantly closer to the active center, forming a new hydrogen bond with the O6 atom of oLu . This closer approach exerts a significantly stronger electrostatic attraction on the carbonyl oxygen. Crucially, this specific interaction provides the physical basis for the polarization of the carbonyl oxygen and the concomitant elongation of the C = O bond (R7) identified in the geometric analysis (Section 3.2 ). Concurrently, the electrostatic potential of the active-site pocket also changes ( Fig S13 ), reflecting an adjustment in the electrostatic environment. To quantify the effect of the electrostatic environment change, we evaluated the total IEF exerted on oLu by the enzyme. The calculated field strengths for the five systems are 19.519, 43.366, 42.665, 45.176, and 49.232 MV/cm, respectively. This substantial enhancement of the IEF is likely attributed to the closer proximity of the positively charged Lys529. Consequently, these results suggest that the electric field may directly contribute to the red shift of the λ F . 3.4 Regulation by IEF: Comparison with Uniform EEFs. To further elucidate the regulatory mechanism of the enzyme-generated IEF on λ F , a comparative strategy was employed through the application of uniform EEFs along specific directions. By analyzing oLu ’s response to EEFs in different directions and comparing it with the effect of IEF, the contributions from different directional components of IEF can be assessed, providing a clearer understanding of how the electrostatic effects of enzyme microenvironment modulate its λ F . The effects of EEF on λ F of Systems i–v are shown in Fig. 9 and Fig S14 . For all five systems, λ F is not sensitive to the electric field applied along the Y-axis ( Fig S14 ), but it shows sensitivity to the electric fields applied along the X-axis and Z-axis (Fig. 9 a and b ). When a positive uniform electric field is applied along the negative X-axis (-X) or the positive Z-axis (+ Z), a red shift in λ F is observed. Conversely, applying a negative electric field in these directions induces a blue shift, and the displacement increases with the field strength. Crucially, as shown in Figs. 9 a and b , the response trends and magnitudes of the λ F to the EEF are generally consistent across the five systems. Therefore, using System i as a reference, a quantitative relationship between the λ F and the strength of EEF was fitted as follows: y = -249162.8 x ² − 4608.46 x + 499.07 y = 5504.29 z + 500.27 where y represents the λ F (in nm), and x and z denote the electric field strengths (in a.u.) applied along the X and Z axes, respectively. The directions of the IEF acting on oLu in Systems I-V are shown in Figs. 7 and 8 . Crucially, all systems were found to exhibit IEF components along the -X or + Z directions—directions previously shown by EEF studies in Fig. 9 to effectively induce red shifts in emission. This consistency indicates that that the modulation of oLu 's λ F by the enzyme-generated IEF follows the same physical mechanism as that induced by EEF. On this basis, and given that the purpose of this analysis is to establish the physical relevance and order of magnitude of the electrostatic effect—namely, whether the protein-generated electrostatic field can meaningfully influence oLu through its components along the − X and/or + Z directions identified above— rather than to achieve system-by-system quantitative prediction, System I ( and its corresponding isolated System i ) was selected as a representative case for quantitative illustration. The IEF components along the X and Z axes are − 16.362 and 6.699 MV/cm, respectively (1 au ≈ 5140 MV/cm). Based on the derived relationship between the λ F and EEF, these components are predicted to contribute red shifts of approximately 12 and 8 nm. After accounting for this IEF, the predicted λ F for System I is approximately 519 nm, showing close agreement with the directly computed QM/MM value of 523 nm. This indicates that the IEF inherent to the protein microenvironment, operating through its specific directional components, acts as a key physical factor for the spectral tuning. Figure 10 presents the residue-specific electric field contributions projected along the X (Fig. 10 a) and Z (Fig. 10 b) axes. As established by our EEF calibration, a negative field along the X-axis (-X) and a positive field along the Z-axis (+ Z) are the effective drivers for the red shift. The data reveals a striking contrast in residue behavior. For the majority of active-site residues, metal ion binding induces only minor or inconsistent fluctuations in their field contributions. However, residue Lys529 (K529) exhibits a dramatic and distinct deviation. Along the highly sensitive X-axis (see Fig. 10 a), the contribution of K529 plunges from approximately − 16 MV/cm in the metal ion-free system to ~ -40 MV/cm in all metal ion-bound systems. This corresponds to a net field enhancement of ~ 25 MV/cm specifically favoring the red shift. Although the Z-axis projection (Fig. 10 b) indicates that K529 makes a minor contribution opposing the red shift (negative values), it is completely overwhelmed by the dominant red-shifting effect along the X-axis. This quantitative surge in electric field strength directly corroborates our structural analysis in Section 3.3.3 . It confirms that the metal ion-induced structural reorganization—specifically bringing K529 into close proximity with oLu —is the physical origin of the enhanced IEF. Conclusion In this study, we performed a comprehensive theoretical investigation combining MD simulations, QM/MM calculations, and electric field analysis to decode the molecular mechanism underlying the heavy metal ion-induced red shift in A. vivianii firefly BL. Our results demonstrate that the magnitude of the red shift does not simply correlate with the intrinsic physical properties (e.g., ionic radius or charge) of the metal ions but is governed by the specific remodeling of the enzyme microenvironment. We identified that metal ions act as allosteric triggers that disrupt the native salt-bridge network (H310, E311, and E354) through specific coordination geometries. This structural perturbation reshapes the active site, inducing coupled steric and electrostatic changes that collectively regulate oLu . Structurally, the altered spatial constraints force the oLu to undergo a planar-to-curved geometric transition, specifically elongating the carbonyl C = O bond. While this geometric distortion contributes to the red shift, our analysis shows it is insufficient to account for the full magnitude of the shift. Electrostatically, the environmental remodeling repositions residue Lys529 into close proximity with the emitter. This rearrangement acts as an electrostatic amplifier, boosting the IEF exerted on oLu along the sensitive axis (from ~ 16 to ~ 40 MV/cm). By drawing an analogy to the modulation induced by uniform EEF, we confirm that this enhanced IEF regulates the oLu 's electronic structure via an electrochromic effect. Therefore, the observed red shift arises from the combined effects of the environment-enforced geometric distortion and the direct electrostatic modulation. In summary, the heavy metal ion-induced red shift arises from a precise " oLu -Luciferase" reorganization, where the metal ion coordinates the positioning of key electrostatic residues. These findings clarify the structural origins of the observed metal-ion specificity and provide a theoretical blueprint for rational biosensor design. Future efforts should focus on "electrostatic engineering," where binding pockets are designed so that the target analyte drives charged residues (like Lys529) toward the emitter, allowing for fine-tuned control over the bioluminescent color. Declarations Supporting Information The Supporting Information contains computational details, additional tables and figures, molecular dynamics analyses, electronic structure results, and Cartesian coordinates supporting the conclusions of this work. Competing Interests: The authors have no competing interests to declare that are relevant to the content of this article. Author Contribution **Jinyu Wang** : Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization, Data curation. **Deping Hu** : Writing – review & editing, Methodology, Software **Ya-Jun Liu** : Writing – review & editing, Supervision, Project administration, Formal analysis, Resources, Conceptualization. Acknowledgments: This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 22373010 and 22403008), the Beijing Natural Science Foundation (Grant No. 2244074) and the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2025A1515012183). Computing resources were provided by the Interdisciplinary Intelligence Supercomputer Center of Beijing Normal University at Zhuhai. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Zhao, J. Y., Lin, S. X., & Huang, Y. (2013). Mechanism-based design of a photoactivatable firefly luciferase. Journal of the American Chemical Society , 135 (19), 7410–7413. https://doi.org/10.1021/ja4013535 Feng, P., Zhang, H., Deng, Q., & Liu, Y. (2016). Real-time bioluminescence imaging of nitroreductase in mouse model. Analytical Chemistry , 88 (11), 5610–5614. https://doi.org/10.1021/acs.analchem.6b01160 Ikeda, Y., Saitoh, T., Niwa, K., & Amano, R. (2018). An allylated firefly luciferin analogue with luciferase specific response in living cells. Chemical Communications , 54 (14), 1774–1777. https://doi.org/10.1039/c7cc09720d Ando, Y., Niwa, K., Yamada, N., Enomoto, T., Irie, T., Kubota, H., et al. (2007). Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. Nature Photonics , 2 (1), 44–47. https://doi.org/10.1038/nphoton.2007.251 Liu, Y. J. (2022). Understanding the complete bioluminescence cycle from a multiscale computational perspective: A review. Journal of Photochemistry and Photobiology C: Photochemistry Reviews , 52 , 100537. https://doi.org/10.1016/j.jphotochemrev.2022.100537 McElroy, W. D., Seliger, H. H., White, E. H., & Copley, M. (1969). Mechanism of bioluminescence, chemi-luminescence and enzyme function in the oxidation of firefly luciferin. Photochemistry and Photobiology , 10 (3), 153–170. https://doi.org/10.1111/j.1751-1097.1969.tb05676.x Wood, K. V. (1995). The chemical mechanism and evolutionary development of beetle bioluminescence. Photochemistry and Photobiology , 62 (4), 662–673. https://doi.org/10.1111/j.1751-1097.1995.tb08714.x Viviani, V. R. (2002). The origin, diversity, and structure function relationships of insect luciferases. Cellular and Molecular Life Sciences , 59 (11), 1833–1850. https://doi.org/10.1007/s00018-002-8545-z McElroy, W. D., & Seliger, H. H. (1964). The colors of firefly bioluminescence: Enzyme configuration and species specificity. Proceedings of the National Academy of Sciences of the United States of America , 52 (1), 75–81. https://doi.org/10.1073/pnas.52.1.75 Viviani, V. R., & Bechara, E. J. H. (1995). Bioluminescence of Brazilian fireflies (Coleoptera: Lampyridae): Spectral distribution and pH effect on luciferase-elicited colors. Photochemistry and Photobiology , 62 (3), 490–495. https://doi.org/10.1111/j.1751-1097.1995.tb02373.x Sundlov, J. A., Fontaine, D. M., Southworth, T. L., & Gulick, A. M. (2012). Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism. Biochemistry , 51 (33), 6493–6495. https://doi.org/10.1021/bi300934s Nakatsu, T., Ichiyama, S., Hiratake, J., Mizutani, H., Chu, W. C., Itoh, N., et al. (2006). Structural basis for the spectral difference in luciferase bioluminescence. Nature , 440 (7082), 372–376. https://doi.org/10.1038/nature04542 Carrasco-Lopez, C., Lui, N. M., Schramm, S., & Viviani, V. R. (2021). The elusive relationship between structure and colour emission in beetle luciferases. Nature Reviews Chemistry , 5 (1), 4–20. https://doi.org/10.1038/s41570-020-00238-1 Ghose, A., Rebarz, M., Maltsev, O. V., Hintermann, L., Ruckebusch, C., Fron, E., et al. (2014). Emission properties of oxyluciferin and its derivatives in water: Revealing the nature of the emissive species in firefly bioluminescence. The Journal of Physical Chemistry B , 119 (6), 2638–2649. https://doi.org/10.1021/jp508905m White, E. H., Rapaport, E., Hopkins, T. A., & Seliger, H. H. (1969). Chemi- and bioluminescence of firefly luciferin. Journal of the American Chemical Society , 91 (8), 2178–2180. https://doi.org/10.1021/ja01036a093 White, E. H., Seliger, H. H., & Hopkins, T. A. (1971). The chemi- and bioluminescence of firefly luciferin: An efficient chemical production of electronically excited states. Bioorganic Chemistry , 1 (1–2), 92–122. https://doi.org/10.1016/0045-2068(71)90010-8 Wang, Y., Akiyama, H., Terakado, K., & Nakatsu, T. (2013). Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence. Scientific Reports , 3 (1), 2490. https://doi.org/10.1038/srep02490 Wang, Y., Hayamizu, Y., & Akiyama, H. (2014). Spectroscopic study of firefly oxyluciferin in an enzymatic environment on the basis of stability monitoring. The Journal of Physical Chemistry B , 118 (8), 2070–2076. https://doi.org/10.1021/jp411476p Mochizuki, T., Wang, Y., Hiyama, M., & Akiyama, H. (2014). Robust red-emission spectra and yields in firefly bioluminescence against temperature changes. Applied Physics Letters , 104 (18), 183701. https://doi.org/10.1063/1.4880578 Ugarova, N. N., & Brovko, L. Y. (2001). Relationship between the structure of the protein globule and bioluminescence spectra of firefly luciferase. Russian Chemical Bulletin International Edition , 50 (10), 1752–1761. https://doi.org/10.1023/A:1012726300408 Hirano, T., Nagai, H., Matsuhashi, T., & Maki, S. (2012). Spectroscopic studies of the color modulation mechanism of firefly (beetle) bioluminescence with amino-analogs of luciferin and oxyluciferin. Photochemical & Photobiological Sciences , 11 (8), 1281–1284. https://doi.org/10.1039/c2pp25106j Gandelman, O. A., Brovko, L. Y., Ugarova, N. N., & Klyachko, N. L. (1993). Oxyluciferin fluorescence is a model of native bioluminescence in the firefly luciferin-luciferase system. Journal of Photochemistry and Photobiology B: Biology , 19 (3), 187–191. https://doi.org/10.1016/1011-1344(93)80108-C Kim, H. W., & Rhee, Y. M. (2013). On the pH dependent behavior of the firefly bioluminescence: Protein dynamics and water content in the active pocket. The Journal of Physical Chemistry B , 117 (24), 7260–7269. https://doi.org/10.1021/jp4024553 Koksharov, M. I., & Ugarova, N. N. (2013). Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence. Photochemical & Photobiological Sciences , 12 (11), 2016–2027. https://doi.org/10.1039/c3pp50242b Zhou, J. G., Williams, Q. L., Walters, W., & Koua, K. (2015). How does the local electrostatic field influence emitted wavelengths and bioluminescent intensities of modified heteroaromatic luciferins? The Journal of Physical Chemistry B , 119 (33), 10399–10405. https://doi.org/10.1021/acs.jpcb.5b03827 Cheng, Y. Y., & Liu, Y. J. (2015). What exactly is the light emitter of a firefly? Journal of Chemical Theory and Computation , 11 (11), 5360–5370. https://doi.org/10.1021/acs.jctc.5b00659 Yue, L., Lan, Z., & Liu, Y. J. (2015). The theoretical estimation of the bioluminescent efficiency of the firefly via a nonadiabatic molecular dynamics simulation. The Journal of Physical Chemistry Letters , 6 (3), 540–548. https://doi.org/10.1021/jz502305g Takakura, H. (2021). Molecular design of D-luciferin-based bioluminescence and 1,2-dioxetane-based chemiluminescence substrates for altered output wavelength and detecting various molecules. Molecules , 26 (6). Article 1618. https://doi.org/10.3390/molecules26061618 Navizet, I., Roca-Sanjuán, D., Yue, L., Ferré, N., Liu, Y. J., & Lindh, R. (2012). Are the bio- and chemiluminescence states of the firefly oxyluciferin the same as the fluorescence state? Photochemistry and Photobiology , 89 (2), 319–325. https://doi.org/10.1111/php.12007 Gabriel, G. V. M., & Viviani, V. R. (2016). Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals. Analytical and Bioanalytical Chemistry , 408 (30), 8881–8893. https://doi.org/10.1007/s00216-016-0011-1 Viviani, V. R., Simões, A., Bevilaqua, V. R., Gabriel, G. V. M., Arnoldi, F. G., et al. (2016). Glu311 and Arg337 stabilize a closed active-site conformation and provide a critical catalytic base and countercation for green bioluminescence in beetle luciferases. Biochemistry , 55 (34), 4764–4776. https://doi.org/10.1021/acs.biochem.6b00260 Viviani, V. R., Gabriel, G. V. M., Bevilaqua, V. R., & Matsushita, A. J. (2018). The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases. Scientific Reports , 8 (1). Article 15456. https://doi.org/10.1038/s41598-018-33252-x Pelentir, G. F., Bevilaqua, V. R., & Viviani, V. R. (2020). A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays. Photochemical & Photobiological Sciences , 18 (9), 2061–2070. https://doi.org/10.1039/c9pp00174c Viviani, V. R., Amaral, D., Prado, R., & Arnoldi, F. G. (2011). A new blue-shifted luciferase from the Brazilian (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties. Photochemical & Photobiological Sciences , 10 (12), 1879–1886. https://doi.org/10.1039/c1pp05210a Carrasco-López, C., Ferreira, J. C., Lui, N. M., & Viviani, V. R. (2018). Beetle luciferases with naturally red- and blue-shifted emission. Life Science Alliance , 1 (5), e201800072. https://doi.org/10.26508/lsa.201800072 Tian, C., Kasavajhala, K., Belfon, K. A. A., & Simmerling, C. L. (2019). ff19SB: Amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. Journal of Chemical Theory and Computation , 16 (1), 528–552. https://doi.org/10.1021/acs.jctc.9b00591 Wang, J. M., Wolf, R. M., Caldwell, J. W., Kollman, P. A., & Case, D. A. (2004). Development and testing of a general Amber force field. Journal of Computational Chemistry , 25 (9), 1157–1174. https://doi.org/10.1002/jcc.20035 Li, P., & Merz, K. M. (2016). MCPB.py: A Python based metal center parameter builder. Journal of Chemical Information and Modeling , 56 (4), 599–604. https://doi.org/10.1021/acs.jcim.5b00674 Izaguirre, J. A., Catarello, D. P., Wozniak, J. M., & Skeel, R. D. (2001). Langevin stabilization of molecular dynamics. The Journal of Chemical Physics , 114 (5), 2090–2098. https://doi.org/10.1063/1.1332996 Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., & Haak, J. R. (1984). Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics , 81 (8), 3684–3690. https://doi.org/10.1063/1.448118 Darden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. The Journal of Chemical Physics , 98 (12), 10089–10092. https://doi.org/10.1063/1.464397 Case, D. A., Aktulga, H. M., Belfon, K., Ben-Shalom, I. Y., Brozell, S. R., Cerutti, D. S., et al. (2021). Amber 2021 . University of California. Yanai, T., Tew, D. P., & Handy, N. C. (2004). A new hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP). Chemical Physics Letters , 393 (1–3), 51–57. https://doi.org/10.1016/j.cplett.2004.06.011 Wang, J., Hu, D., & Liu, Y. J. (2025). Theoretical regulation of firefly bioluminescence spectra: Exploring new frontiers in NIR-II imaging. Journal of Photochemistry and Photobiology A: Chemistry , 466 , 116393. https://doi.org/10.1016/j.jphotochem.2025.116393 Sherwood, P., de Vries, A. H., Guest, M. F., Schreckenbach, G., Catlow, C. R. A., et al. (2003). QUASI: A general purpose implementation of the QM/MM approach and its application to problems in catalysis. Journal of Molecular Structure: THEOCHEM , 632 (1–3), 1–28. https://doi.org/10.1016/s0166-1280(03)00285-9 Metz, S., Kästner, J., Sokol, A. A., Keal, T. W., & Sherwood, P. (2014). ChemShell—a modular software package for QM/MM simulations. WIREs Computational Molecular Science , 4 (2), 101–110. https://doi.org/10.1002/wcms.1163 Neese, F. (2022). Software update: The ORCA program system—Version 5.0. WIREs Computational Molecular Science , 12 (5). https://doi.org/10.1002/wcms.1606 . Article e1606. Smith, W., & Forester, T. R. (1996). DL_POLY_2.0: A general-purpose parallel molecular dynamics simulation package. Journal of Molecular Graphics , 14 (3), 136–141. https://doi.org/10.1016/S0263-7855(96)00043-4 Runge, E., & Gross, E. K. U. (1984). Density-functional theory for time-dependent systems. Physical Review Letters , 52 (12), 997–1000. https://doi.org/10.1103/PhysRevLett.52.997 Bakowies, D., & Thiel, W. (1996). Hybrid models for combined quantum mechanical and molecular mechanical approaches. The Journal of Physical Chemistry , 100 (25), 10580–10594. https://doi.org/10.1021/jp9536514 Pelentir, G. F., Tomazini, A., Bevilaqua, V. R., & Viviani, V. R. (2024). Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties. Photochemical & Photobiological Sciences , 23 (6), 997–1010. https://doi.org/10.1007/s43630-024-00570-1 Shannon, R. D. (1976). Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallographica Section A , 32 (5), 751–767. https://doi.org/10.1107/S0567739476001551 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SI20260117A.pdf floatimage1.png Scheme 1 A brief illustration of firefly BL process Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviews received at journal 09 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 01 Feb, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 17 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8629136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584501509,"identity":"6a78495f-84f4-498c-a624-58e334e27f21","order_by":0,"name":"Jinyu Wang","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"Wang","suffix":""},{"id":584501514,"identity":"7fd98274-4f55-45c9-9a9e-c1504e268d49","order_by":1,"name":"Deping Hu","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Deping","middleName":"","lastName":"Hu","suffix":""},{"id":584501515,"identity":"0bd4845b-77da-4a31-bf95-2964cf6cf942","order_by":2,"name":"Yajun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYHCCBCC24GFg7wHzGBuI1CLBw8BzhngtICABRDlEajE4fuCZdAGDhIzBzbfHHvMw2MhuOMD87AFeLWcS0qRnAB1mcDsv3ZiHIc14wwE2cwO8Wg4AtfCAteSYARmHEzcc4GGTwKvl/AOolptnQFr+E6HlBsyWGzwgLQcIa5G88SDZmsdAgkfyTF6a5ByDZOOZh9nM8GrhO5+TeJunwsae7/jZYxJvKuxk+443P8OrReEATwLQeXB3AjEzPvVAIN/AfoCAklEwCkbBKBjxAADF1UBv37zUtQAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yajun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-18 04:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8629136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8629136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101848976,"identity":"2e0b426e-e4e0-4623-afbb-d4ffc143d136","added_by":"auto","created_at":"2026-02-04 09:43:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80236,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of the anionic keto form of oxyluciferin (\u003cstrong\u003eoLu\u003c/strong\u003e) with the defined coordinate system for applying an external uniform electric field. The positive direction along X axis is oriented from C2′ to N3, the positive direction along Z axis from C2′ to S1′, and the positive direction along Y axis is perpendicular to the XZ plane, pointing inward\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/729930b7e55e9f38f9ba6d8a.png"},{"id":101848971,"identity":"c9426e8a-5c30-44f0-89bc-d4f0102b5a7f","added_by":"auto","created_at":"2026-02-04 09:43:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379666,"visible":true,"origin":"","legend":"\u003cp\u003eInitial structural models of \u003cem\u003eA. vivianii \u003c/em\u003efirefly luciferase: (a) Metal ion-free structure (\u003cstrong\u003eSystem I\u003c/strong\u003e) and (b) metal ion-bound structures (\u003cstrong\u003eSystems II–V\u003c/strong\u003e), in which Ag\u003csup\u003e+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, or Hg\u003csup\u003e2+\u003c/sup\u003e is positioned between internal (orange) and external (blue) salt bridges\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/d76954aaa8f00dccd77d9a5e.png"},{"id":101848862,"identity":"30270fe3-ca13-4c74-8784-3d72bccd0000","added_by":"auto","created_at":"2026-02-04 09:42:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":216663,"visible":true,"origin":"","legend":"\u003cp\u003eFrontier molecular orbital analysis of \u003cstrong\u003eoLu\u003c/strong\u003e in \u003cstrong\u003eSystem I\u003c/strong\u003e and \u003cstrong\u003eII-V\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/e3bd40bc57a969f10a20d62d.png"},{"id":101848922,"identity":"e2802e02-ea4d-44e9-ac75-e97ad60a840e","added_by":"auto","created_at":"2026-02-04 09:43:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25415,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparison of S\u003csub\u003e1\u003c/sub\u003e-state geometries of \u003cstrong\u003eoLu\u003c/strong\u003e across five systems, with systems \u003cstrong\u003eI–V \u003c/strong\u003erepresented by black, red, blue, green, and purple, respectively\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/944ffeffc7a3e2003454a53a.png"},{"id":101848861,"identity":"095405de-c0c3-4ae1-ad3e-5c890e9fa55e","added_by":"auto","created_at":"2026-02-04 09:42:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":297351,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of salt bridge distances over time for the five systems. Panels a–f correspond to the distances between: (a) H310NE2–E354OE1, (b) H310NE2–E354OE2, (c) E311OE1–R337NH1, (d) E311OE1–R337NH2, (e) E311OE2–R337NH1, (f) E311OE2–R337NH2\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/4d3669398931b7547d4e0695.png"},{"id":101848926,"identity":"5638f2b7-3b74-4432-b78b-5efed6e0b99e","added_by":"auto","created_at":"2026-02-04 09:43:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":393828,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of metal ion coordination patterns from equilibrated MD trajectories for (a) \u003cstrong\u003eSystem II\u003c/strong\u003e with Ag⁺, (b) \u003cstrong\u003eSystem III\u003c/strong\u003e with Zn\u003csup\u003e2+\u003c/sup\u003e, (c) \u003cstrong\u003eSystem IV\u003c/strong\u003e with Cd\u003csup\u003e2+\u003c/sup\u003e, and (d) \u003cstrong\u003eSystem V\u003c/strong\u003e with Hg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/f9669947e62f259613fd2b50.png"},{"id":101848916,"identity":"8e98e27d-de24-4d4d-9e69-271e8108e7c8","added_by":"auto","created_at":"2026-02-04 09:43:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":366653,"visible":true,"origin":"","legend":"\u003cp\u003eThe key molecular interactions of \u003cstrong\u003eoLu\u003c/strong\u003e in \u003cstrong\u003eSystem I\u003c/strong\u003e. The dashed circle highlights the inner and outer salt-bridge regions, and the red arrow indicates the IEF applied by the enzyme to \u003cstrong\u003eoLu\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/d9f3ca4d0bac8e158490037d.png"},{"id":101848966,"identity":"73e3bb5b-ee03-497c-a3fa-e1024f00d367","added_by":"auto","created_at":"2026-02-04 09:43:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":956558,"visible":true,"origin":"","legend":"\u003cp\u003eCoordination conformations of Ag\u003csup\u003e+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, and Hg\u003csup\u003e2+\u003c/sup\u003e with residues in the salt-bridge region, and the key molecular interactions of \u003cstrong\u003eoLu\u003c/strong\u003e in (a) \u003cstrong\u003eSystem II (Ag\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e, (b) \u003cstrong\u003eSystem III (Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e, (c) \u003cstrong\u003eSystem IV (Cd\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e, and (d) \u003cstrong\u003eSystem V (Hg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e. The dashed circle highlights the salt-bridge region on the left side, and the red arrow indicates the IEF applied by the enzyme to \u003cstrong\u003eoLu\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/83f7473502abebf74f5362f7.png"},{"id":101848910,"identity":"31423a5d-5dd9-4e5c-b866-e78a336518fa","added_by":"auto","created_at":"2026-02-04 09:42:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":215808,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e of \u003cstrong\u003eoLu\u003c/strong\u003e and the strength of EEF applied along the (a) X and (b) Z directions with fitted quantitative correlation\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/a0392cba2485d41a49a6006e.png"},{"id":101848909,"identity":"c02b56a3-ff50-4cd1-bbb8-47044cc6fd9a","added_by":"auto","created_at":"2026-02-04 09:42:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":238069,"visible":true,"origin":"","legend":"\u003cp\u003eResidue-specific electric field contributions projected along the (a) X-axis and (b) Z-axis. The dashed line at 0 indicates no contribution. The arrows indicate the direction of field effects on the spectral shift based on the EEF calibration. Black arrows indicate that the largest electric field contribution along the given direction arises from the metal-free system, whereas colored arrows indicate that the largest electric field contribution originates from the metal-bound system corresponding to the arrow color. The red bold arrow highlights Lys529 as the primary contributing residue\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/b021ffaeb5a9b901440b99d3.png"},{"id":101849061,"identity":"8f1d8628-8b9c-4614-b00d-47796f5fe277","added_by":"auto","created_at":"2026-02-04 09:43:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4263150,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/1a4c494d-0464-4c29-add4-666ffed52077.pdf"},{"id":101848928,"identity":"d924afe5-c39f-447a-8bfc-95d9bae90a94","added_by":"auto","created_at":"2026-02-04 09:43:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1620821,"visible":true,"origin":"","legend":"","description":"","filename":"SI20260117A.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/bb6920a3354b8cd8e4ebc61a.pdf"},{"id":101848921,"identity":"7009ef77-6cb3-4fe3-bfd5-c9125dd057b2","added_by":"auto","created_at":"2026-02-04 09:43:05","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":126653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e A brief illustration of firefly BL process\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8629136/v1/21f80ad97b8ced7b18887c91.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Theoretical Study of Metal Ion–Induced Modulation of Firefly Bioluminescence Spectra","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFirefly bioluminescence (BL) is an enzyme-catalyzed light-emitting reaction that has been widely used for in vivo imaging [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and cellular monitoring [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] owing to its high quantum yield and signal-to-noise ratio [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, in this system, D-luciferin undergoes adenylation followed by an oxygen reaction catalyzed by luciferase, ultimately generating oxyluciferin in its first singlet excited (S₁) state, which emits visible light upon relaxation to the ground (S\u003csub\u003e0\u003c/sub\u003e) state [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Remarkably, despite sharing a common substrate and reaction pathway, firefly BL exhibits pronounced color variability, spanning from yellow-green to red, depending on the species as well as environmental factors such as pH, temperature, and the presence of heavy metal ions [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these factors, the mechanisms driving spectral tuning by species, pH, and temperature are relatively well-understood [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, the molecular mechanisms for heavy metal ion-induced spectral shifts remain elusive. To address this issue, it is essential to revisit the fundamental determinants governing firefly BL color.\u003c/p\u003e \u003cp\u003eThe red shift in firefly BL is generally attributed to two critical factors: the structural form of the light emitter oxyluciferin [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and the regulatory effects of the luciferase microenvironment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The former involves keto\u0026ndash;enol tautomerism and modifications of oxyluciferin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], such as the introduction of various substituents or other structural alterations, while the latter encompasses a range of enzyme-associated local environmental factors, such as pocket compactness, hydrogen-bonding networks, polarity of the active site, and the electrostatic effects [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Regarding the structural form of the light emitter, in the native firefly BL system employing the unmodified oxyluciferin, the anionic keto form (\u003cb\u003eoLu\u003c/b\u003e), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, has been confirmed to be the primary light emitter [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Based on this structure, numerous red-shifted analogs have been developed to improve in vivo imaging [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Accordingly, the observed firefly BL color in native firefly system is closely associated with fluorescence wavelength (\u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e) of \u003cb\u003eoLu\u003c/b\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. On the basis of these considerations, it is hypothesized that the heavy metal ion-induced red shift in native firefly BL primarily originates from the luciferase microenvironment. Given that the enzyme and \u003cb\u003eoLu\u003c/b\u003e are intrinsically coupled\u0026mdash;where microenvironmental changes inevitably force conformational adjustments in \u003cb\u003eoLu\u003c/b\u003e\u0026mdash;the red shift is attributed to the complex interplay between the remodeled microenvironment and the conformational modulation of \u003cb\u003eoLu\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eHowever, detecting the subtle conformational changes of \u003cb\u003eoLu\u003c/b\u003e is experimentally challenging; thus, previous research has primarily targeted the protein architecture [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Focusing specifically on the role of critical amino acid residues, Viviani et al. conducted systematic studies that provided crucial insights into the mechanisms underlying the heavy metal ion-induced red shift [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Their studies have shown that an internal salt bridge (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) between residues Glu311 (E311) and Arg337 (R337)\u0026mdash;located near the benzothiazole moiety of \u003cb\u003eoLu\u003c/b\u003e\u0026mdash;is crucial for maintaining green emission, as this closed conformation stabilizes a low-polarity active site [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additional residues, such as His310 (H310) and Glu354 (E354), which can form an external salt-bridge (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), have also been shown to influence emission color, with mutations leading to red-shifted spectra [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Mutational analyses further identified H310, E311, and E354 as potential metal-binding sites, leading to the proposal that metal ion binding disrupts key salt-bridge interactions and increases the polarity of the substrate-binding pocket, thereby favoring red-shifted emission [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nevertheless, a detailed theoretical understanding connecting these structural perturbations to the spectral red shift is still absent.\u003c/p\u003e \u003cp\u003eTo disentangle this complexity and identify the fundamental drivers of the red shift, we focused on the Brazilian species \u003cem\u003eAmydetes vivianii\u003c/em\u003e(\u003cem\u003eA. vivianii\u003c/em\u003e)as an ideal model system. Unlike many other luciferases, this enzyme exhibits a selective response to heavy metal ions (such as Cd\u003csup\u003e2+\u003c/sup\u003e and Hg\u003csup\u003e2+\u003c/sup\u003e) while being pH-insensitive, making it a promising candidate for detecting metal ions quantitatively in both cellular and environmental contexts [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This distinctive behavior allows us to isolate pure metal ion-induced structural perturbations from confounding protonation effects. In this study, we systematically investigate the fluorescence properties of \u003cb\u003eoLu\u003c/b\u003e in \u003cem\u003eA. vivianii\u003c/em\u003e luciferase in the presence of Ag\u003csup\u003e+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, and Hg\u003csup\u003e2+\u003c/sup\u003e using molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations (Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e). Given that the spectral shift arises from the coupled effects of the \u003cb\u003eoLu\u003c/b\u003e's conformation and the luciferase microenvironment, we adopted a decoupled analytical strategy to systematically identify their specific contributions: First, to isolate the contribution of \u003cb\u003eoLu\u003c/b\u003e\u0026rsquo;s geometry, we investigated its intrinsic structural distortion induced by metal ion binding and the steric constraints of the enzyme pocket (Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e). Second, to clarify the role of the enzymatic microenvironment in the observed red shift\u0026mdash;including both steric and electrostatic effects\u0026mdash;we systematically investigate microenvironmental changes induced by metal ions, encompassing salt-bridge rearrangements, metal-ion coordination modes, displacements of key active-site residues, and the internal electric fields (IEF) generated by the enzyme. (\u003cb\u003eSection 3.3\u003c/b\u003e). Finally, to explicitly quantify the direct electrostatic influence, we examine how IEF regulates spectra, drawing an analogy to the modulation induced by uniform external electric fields (EEF) (\u003cb\u003eSection 3.4\u003c/b\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis work provides the first theoretical clarification of the mechanisms governing metal ion-regulated BL, offering a structural rationale for metal-ion specificity and establishing a general framework for electrostatic engineering in enzyme color tuning, protein electrostatics, and the rational design of bioluminescent biosensors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Computational Methods","content":"\u003cp\u003e \u003cem\u003eSetup of Systems.\u003c/em\u003e Five systems were constructed based on a homology model of \u003cem\u003eA. vivianii\u003c/em\u003e firefly luciferase, as the available experimental structure lacks the substrate and adopts a non-luminescent open or semi-open conformation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The model was built using \u003cem\u003ePhotinus pyralis\u003c/em\u003e (\u003cem\u003eP. pyralis\u003c/em\u003e) luciferase (PDB ID: 4G36) as the template, which shares 79% sequence identity and preserves a similar salt-bridge architecture near the active site [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The validated model was used to construct a complete wild-type luciferase complex containing \u003cb\u003eoLu\u003c/b\u003e and AMP (\u003cb\u003eSystem I\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Using this structure as a reference, four additional systems were generated by introducing Ag⁺, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, or Hg\u003csup\u003e2+\u003c/sup\u003e ions between the internal (E311\u0026ndash;R337) and external (H310\u0026ndash;E354) salt bridges, yielding metal ion-bound complexes (\u003cb\u003eSystems II\u0026ndash;V\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). All systems were described using the Amber ff19SB force field for proteins and metal ions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], with the GAFF force field applied for \u003cb\u003eoLu\u003c/b\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For \u003cb\u003eSystems II\u0026ndash;V\u003c/b\u003e, the metal ion-binding clusters formed by the metal ions and coordinating residues were parameterized using the \u0026ldquo;MCPB.py\u0026rdquo; tool [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMD Simulation of Systems.\u003c/em\u003e After proper setups, each system was equilibrated in several steps: (1) energy minimization was carried out using a combination of steepest descent and conjugate-gradient methods; (2) the system was then gradually heated from 0 K to 300 K over a 400 ps under an NVT ensemble, with a weak constraint applied to the protein backbone atoms; (3) the density was equilibrated for 1 ns at 300 K and 1.0 atm using an isothermal-isobaric Langevin thermostat [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and Berendsen barostat [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] with a collision frequency of 2 ps\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a pressure relaxation time of 1 ps; (4) a further 3 ns equilibration was subsequently carried out in the NPT ensemble to ensure stable pressure and temperature; (5) a 100 ns MD simulation was conducted. Nonbonded interactions were treated with the Particle Mesh Ewald method with a cutoff of 12 \u0026Aring; [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm, enabling a 2-fs integration step. All simulations were performed with the AMBER21 software [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eQM/MM Calculations.\u003c/em\u003e The equilibrated geometry from the MD trajectory was used as the initial input structure for the QM/MM calculations. For five systems, \u003cb\u003eoLu\u003c/b\u003e was put into the QM region, while other atoms were put into the MM region. For the geometry optimizations in S\u003csub\u003e1\u003c/sub\u003e states of five systems, the long-range-corrected functional CAM-B3LYP [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] was employed for a better description of the charge-transfer transitions of \u003cb\u003eoLu\u003c/b\u003e. Consequently, the TD CAM-B3LYP/6\u0026ndash;31\u0026thinsp;+\u0026thinsp;G** method, which has been shown to perform well in predicting the fluorescent properties of \u003cb\u003eoLu\u003c/b\u003e, was employed for the QM region [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Atoms within 15 \u0026Aring; of \u003cb\u003eoLu\u003c/b\u003e in the MM region were allowed to relax during the optimization process. Fluorescence-related single-point calculations were performed at the TD CAM-B3LYP/ma-def2-TZVP level for the QM region. All QM/MM calculations were carried out using the ChemShell software [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], which integrates ORCA 5.0 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] for the QM region and DL_POLY [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] for the MM region. The TD-DFT method [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] was employed for the QM region of QM/MM calculations, while the Amber ff19SB force field was used to describe the MM protein environment. To include the polarization effect of the MM region on the QM region, the electronic embedding scheme was employed in the QM/MM calculations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eEEF and IEF Calculations.\u003c/em\u003e For both EEF and IEF, the molecular coordinate axes were defined relative to the \u003cb\u003eoLu\u003c/b\u003e scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) as follows: the positive X-axis along C2\u0026prime;\u0026rarr;N3, the positive Z-axis along C2\u0026prime;\u0026rarr;S1\u0026prime;, and the positive Y-axis along perpendicular to the XZ plane and pointing inward. For both EEF and IEF, the positive field direction was defined along the positive axes. Uniform EEFs ranging from \u0026minus;\u0026thinsp;0.08 to +\u0026thinsp;0.08 a.u. were applied independently along each axis to isolated \u003cb\u003eoLu\u003c/b\u003e (\u003cb\u003eSystems\u003c/b\u003e \u003cb\u003ei\u0026ndash;v\u003c/b\u003e), which were extracted from the QM region of the optimized S₁-state QM/MM structures (\u003cb\u003eSystems I\u0026ndash;V\u003c/b\u003e). EEF calculations were performed using Gaussian 09 at the TD CAM-B3LYP/ma-def2-TZVP level, while IEFs generated by the enzyme environment were computed with a homemade Python script.\u003c/p\u003e \u003cp\u003eFurther methodological details are provided in the Supporting Information (SI).\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Fluorescence Properties and Electronic Structure of oLu in Five Systems.\u003c/h2\u003e \u003cp\u003eThe fluorescence properties of \u003cb\u003eoLu\u003c/b\u003e in five systems were investigated at the level of TD-CAM-B3LYP/ma-def2-TZVP//MM, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Across all five systems, the oscillator strengths (\u003cem\u003ef\u003c/em\u003e) for the S₁ \u0026rarr; S\u003csub\u003e0\u003c/sub\u003e transition are approximately 0.6, suggesting that \u003cb\u003eoLu\u003c/b\u003e maintains a strong fluorescence emission in each system. In \u003cb\u003eSystem I\u003c/b\u003e, the metal ion-free system, the calculated \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e of \u003cb\u003eoLu\u003c/b\u003e is 523 nm, which shows reasonable agreement with the experimental value of 543\u0026thinsp;\u0026plusmn;\u0026thinsp;5 nm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], supporting the reliability of both the constructed model and the chosen computational methods. In \u003cb\u003eSystems II\u0026ndash;V\u003c/b\u003e, the binding of metal ions consistently induces a red shift in the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e of \u003cb\u003eoLu\u003c/b\u003e. The magnitude of this shift follows the order: Hg\u003csup\u003e2+\u003c/sup\u003e \u0026gt; Cd\u003csup\u003e2+\u003c/sup\u003e \u0026gt; Ag⁺ \u0026gt; Zn\u003csup\u003e2+\u003c/sup\u003e, with the largest shift occurring in \u003cb\u003eSystem V\u003c/b\u003e (Hg\u003csup\u003e2+\u003c/sup\u003e bound) and the smallest in \u003cb\u003eSystem III\u003c/b\u003e (Zn\u003csup\u003e2+\u003c/sup\u003e bound), which agrees qualitatively with the experimental observations [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Notably, this observed sequence deviates significantly from the trend predicted by classical electrostatics. Given that all four ions share a common \u003cem\u003ed\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e electronic configuration, their polarizing power is governed strictly by ionic potential (charge-to-radius ratio). Consequently, ions with higher ionic potential possess stronger polarizing power and are theoretically expected to induce larger spectral shifts via electrostatic polarization. Based on this, the theoretically expected order should be: Zn\u003csup\u003e2+\u003c/sup\u003e \u0026gt; Cd\u003csup\u003e2+\u003c/sup\u003e \u0026gt; Hg\u003csup\u003e2\u003c/sup\u003e⁺ \u0026gt; Ag\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This striking discrepancy establishes that the metal ion does not act merely as a point source of electrostatic polarization. Instead, its primary role is to act as a structural trigger to modulate the electronic structure, as will be revealed through frontier molecular orbital analysis below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalculated fluorescence wavelengths (\u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF,\u003c/sub\u003e nm) and oscillator strengths (\u003cem\u003ef\u003c/em\u003e) of \u003cb\u003eoLu\u003c/b\u003e in \u003cb\u003eSystem I\u003c/b\u003e and \u003cb\u003eII-V\u003c/b\u003e at TD CAM-B3LYP/ma-def2-TZVP//MM level, along with their corresponding experimental emission wavelength (\u003cem\u003eExp\u003c/em\u003e. nm).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetal ion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ef\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eExp\u003c/em\u003e.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e543\u0026thinsp;\u0026plusmn;\u0026thinsp;5 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAg\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e556\u0026thinsp;~\u0026thinsp;563 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCd\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e568\u0026thinsp;~\u0026thinsp;584 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e571\u0026thinsp;~\u0026thinsp;584 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFrontier molecular orbital analysis in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the fluorescence emission in \u003cb\u003eSystems I\u003c/b\u003e and \u003cb\u003eII-V\u003c/b\u003e arises from the de-excitation process, where the electron relaxes from the lowest unoccupied molecular orbital (LUMO) to the highest occupied molecular orbital (HOMO). The LUMO is delocalized over the entire molecule, whereas the HOMO is primarily localized on the benzothiazole moiety (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating a charge-transfer character, consistent with the charge analysis results (see \u003cb\u003eTable S2\u003c/b\u003e). Compared with \u003cb\u003eSystem I\u003c/b\u003e, the binding of metal ions in \u003cb\u003eSystems II\u0026ndash;V\u003c/b\u003e stabilizes both HOMO and LUMO energies, with a greater stabilization of the LUMO. This differential stabilization narrows the HOMO\u0026ndash;LUMO gap, which directly accounts for the observed red shift at the electronic-structure level. These changes in the electronic structure can be traced back to metal ion-induced alterations in the molecular geometry of \u003cb\u003eoLu\u003c/b\u003e and enzyme microenvironment of \u003cb\u003eoLu\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Geometric Distortion and Intrinsic Electronic Modulation of the Emitter oLu.\u003c/h2\u003e \u003cp\u003eThe molecular geometry of \u003cb\u003eoLu\u003c/b\u003e plays an important role in determining its \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e by modulating its electronic structure. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the comparison of S\u003csub\u003e1\u003c/sub\u003e-state geometries of \u003cb\u003eoLu\u003c/b\u003e across five systems. In \u003cb\u003eSystem I\u003c/b\u003e, \u003cb\u003eoLu\u003c/b\u003e adopts a mostly planar conformation. However, upon metal ions binding to the luciferase, \u003cb\u003eoLu\u003c/b\u003e adopts slightly bent conformations with the S1' and N3' atoms (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) as the central axis in \u003cb\u003eSystems II-V\u003c/b\u003e. Detailed geometric parameters are listed in \u003cb\u003eTable S3\u003c/b\u003e. An analysis of these parameters reveals a clear structural trend: while the benzothiazole moiety remains rigid across all systems, the thiazole ring exhibits a critical distortion characterized by both geometric bending and bond elongation. Specifically, the dihedral angle C7\u0026prime;\u0026ndash;S1\u0026prime;\u0026ndash;N3\u0026prime;\u0026ndash;S1 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) deviate significantly from the \u003cb\u003eSystem I\u003c/b\u003e (e.g., shifts from \u0026minus;\u0026thinsp;177\u0026deg; to ~\u0026thinsp;171\u0026deg;), confirming the planar-to-curved transition. Accompanying this bending, the carbonyl C\u0026thinsp;=\u0026thinsp;O bond (R7) consistently elongates in all metal ion-bound systems. Although the magnitude of this elongation is subtle (\u0026lt;\u0026thinsp;0.02 \u0026Aring;), the trend is uniform, indicating a weakening of the C\u0026thinsp;=\u0026thinsp;O double-bond character.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe observed geometric alterations in \u003cb\u003eoLu\u003c/b\u003e lead to corresponding changes in its intrinsic electronic structure. To isolate this effect, we analyzed the extracted \u003cb\u003eoLu\u003c/b\u003e molecules from the five systems in the gas phase (\u003cb\u003eSystems\u003c/b\u003e \u003cb\u003ei\u0026ndash;v\u003c/b\u003e). Frontier molecular orbital analysis (\u003cb\u003eFig S3\u003c/b\u003e) reveals that the HOMO-LUMO gap of \u003cb\u003eoLu\u003c/b\u003e in \u003cb\u003eSystems\u003c/b\u003e \u003cb\u003eii\u0026ndash;v\u003c/b\u003e is smaller than that in \u003cb\u003eSystem\u003c/b\u003e \u003cb\u003ei\u003c/b\u003e. Crucially, the orbital distribution explains the mechanistic link: since the HOMO is localized on the benzothiazole moiety, whereas the LUMO is delocalized over the entire molecule, extending significantly to the carbonyl group of the thiazole ring, the elongation of the C\u0026thinsp;=\u0026thinsp;O bond (R7) on thiazole ring directly stabilizes LUMO, lowering its energy level. Consequently, this geometric relaxation narrows the HOMO-LUMO gap, resulting in a systematic red shift of \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e, from 499 nm for \u003cb\u003eSystem\u003c/b\u003e \u003cb\u003ei\u003c/b\u003e to 504, 503, 507, and 508 nm for \u003cb\u003eSystems\u003c/b\u003e \u003cb\u003eii\u0026ndash;v\u003c/b\u003e, respectively.\u003c/p\u003e \u003cp\u003eRemarkably, the red-shift trend in these isolated systems (Zn\u003csup\u003e2+\u003c/sup\u003e \u0026lt; Ag\u003csup\u003e+\u003c/sup\u003e \u0026lt; Cd\u003csup\u003e2\u003c/sup\u003e⁺ \u0026lt; Hg\u003csup\u003e2+\u003c/sup\u003e) is qualitatively consistent with that observed in the full protein complex (\u003cb\u003eSystems I\u0026ndash;V\u003c/b\u003e). However, the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e in isolation (~\u0026thinsp;505 nm) is notably shorter than those in the protein (~\u0026thinsp;530 nm). This indicates that the geometric distortion contributes to the red shift, while the enzymatic microenvironment further amplifies the shift magnitude. This amplification effect is rooted in the specific structural remodeling of the microenvironment described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Remodeling of the Enzyme Microenvironment.\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Metal Ion Coordination and Salt-Bridge Network Disruption\u003c/h2\u003e \u003cp\u003eThe remodeling of the microenvironment initiates with the disruption of the native salt-bridge network, which is caused by the coordination of metal ions. Experimental studies have suggested that both internal and external salt bridges play a role in determining the BL color of firefly luciferase (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents the changes in salt bridge distances across the five systems over the 100 ns MD simulation (the root mean square deviation and radius of gyration analyses indicate that the protein backbone and \u003cb\u003eoLu\u003c/b\u003e remained stable, confirming structural reliability; see \u003cb\u003eFig S4\u0026ndash;S8\u003c/b\u003e), specific distances are shown in \u003cb\u003eFig S9\u003c/b\u003e. In \u003cb\u003eSystem I\u003c/b\u003e (metal ion-free), both the external and internal salt bridges are generally maintained throughout the simulation. The external salt bridge is formed between the NE2 atom of H310 and the OE1 or OE2 atom of E354, while the internal salt bridge involves the NH1 atom of R337 and the OE1 or OE2 atom of E311 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, in \u003cb\u003eSystem II\u003c/b\u003e (Ag\u003csup\u003e+\u003c/sup\u003e-bound), the external salt bridge is completely disrupted. In \u003cb\u003eSystems III\u0026ndash;V\u003c/b\u003e (bound to Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, and Hg\u003csup\u003e2+\u003c/sup\u003e, respectively), the internal salt bridge is largely dismantled, while the external one is weakened. These results suggest that upon binding to luciferase, the metal ions effectively take over the key residues (H310, E311, E354) through specific coordination with their functional atoms (H310NE2, E311OE1/OE2, E354OE1/OE2), thereby preventing them from forming their original salt bridges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCoordination analysis clarifies the specific atomistic contacts between each metal ion and the potential coordinating atoms (H310NE2, E311OE1/OE2, and E354OE1/OE2), as detailed in \u003cb\u003eFig S10\u003c/b\u003e and \u003cb\u003eS11\u003c/b\u003e. Based on these data, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e summarizes the coordination patterns, revealing distinct geometries for each metal ion: Ag\u003csup\u003e+\u003c/sup\u003e primarily interacts with E311OE1 and E354OE2, forming a bidentate complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Zn\u003csup\u003e2+\u003c/sup\u003e and Cd\u003csup\u003e2+\u003c/sup\u003e adopt a pentacoordinate geometry, involving all five potential coordinating atoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Hg\u003csup\u003e2+\u003c/sup\u003e predominantly adopts a tetracoordinate geometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Collectively, these specific coordination patterns provide the mechanistic explanation for the salt-bridge disruptions observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Importantly, the distinct geometries adopted by each metal ion highlight their ion-specific effects, which drive the rearrangement of key residues and the remodeling of the local molecular interaction network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Repositioning of Key Residues and Intermolecular Interactions.\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows that the key molecular interactions of \u003cb\u003eoLu\u003c/b\u003e in \u003cb\u003eSystem I\u003c/b\u003e. Aligning with the stable network described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e3.3.1\u003c/span\u003e, the inner and outer salt bridges, formed by R337\u0026ndash;E311 and H310\u0026ndash;E354, are located on the phenolate side of \u003cb\u003eoLu\u003c/b\u003e, creating a relatively closed environment for \u003cb\u003eoLu\u003c/b\u003e. The Ser314\u0026ndash;Glu315\u0026ndash;Gly316 peptide segment is positioned above \u003cb\u003eoLu\u003c/b\u003e. On the left side of \u003cb\u003eoLu\u003c/b\u003e, a water molecule bridges the O10' atom and the side chain of Ser314, while on the right side, another water molecule connects the O6 atom to the side chain of Glu316, establishing two separate hydrogen-bond networks. Additionally, the positively charged residues Lys529 and Arg218 are positioned near the right-upper and left-lower sides of \u003cb\u003eoLu\u003c/b\u003e, respectively, contributing electrostatic attraction to \u003cb\u003eoLu\u003c/b\u003e. Collectively, these salt bridges, hydrogen bonds, and electrostatic interactions create a well-defined local environment that maintains the stable conformation of \u003cb\u003eoLu\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe key molecular interactions of \u003cb\u003eoLu\u003c/b\u003e in metal ion-bound \u003cb\u003eSystems II\u0026ndash;V\u003c/b\u003e are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. As detailed in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e3.3.1\u003c/span\u003e, the metal ions bind to the luciferase through specific coordination modes. This binding acts as a structural force that pulls the coordinating residues closer to the ions, thereby remodeling the local microenvironment and inducing further adjustments in the overall protein conformation (see \u003cb\u003eFig S12\u003c/b\u003e). Despite these structural perturbations, certain interactions remain conserved: the Ser314\u0026ndash;Gly316 segment remains positioned above \u003cb\u003eoLu\u003c/b\u003e, with the Ser314\u0026ndash;R337 hydrogen bond preserved, and O10' of \u003cb\u003eoLu\u003c/b\u003e continues to form a hydrogen bond with a water molecule. However, the most critical change involves the positively charged residue Lys529. While Arg218 remains near the lower-left side, Lys529 is repositioned significantly closer to the active center, forming a new hydrogen bond with the O6 atom of \u003cb\u003eoLu\u003c/b\u003e. This closer approach exerts a significantly stronger electrostatic attraction on the carbonyl oxygen. Crucially, this specific interaction provides the physical basis for the polarization of the carbonyl oxygen and the concomitant elongation of the C\u0026thinsp;=\u0026thinsp;O bond (R7) identified in the geometric analysis (Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e). Concurrently, the electrostatic potential of the active-site pocket also changes (\u003cb\u003eFig S13\u003c/b\u003e), reflecting an adjustment in the electrostatic environment. To quantify the effect of the electrostatic environment change, we evaluated the total IEF exerted on \u003cb\u003eoLu\u003c/b\u003e by the enzyme. The calculated field strengths for the five systems are 19.519, 43.366, 42.665, 45.176, and 49.232 MV/cm, respectively. This substantial enhancement of the IEF is likely attributed to the closer proximity of the positively charged Lys529. Consequently, these results suggest that the electric field may directly contribute to the red shift of the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Regulation by IEF: Comparison with Uniform EEFs.\u003c/h2\u003e \u003cp\u003eTo further elucidate the regulatory mechanism of the enzyme-generated IEF on λ\u003csub\u003eF\u003c/sub\u003e, a comparative strategy was employed through the application of uniform EEFs along specific directions. By analyzing \u003cb\u003eoLu\u003c/b\u003e\u0026rsquo;s response to EEFs in different directions and comparing it with the effect of IEF, the contributions from different directional components of IEF can be assessed, providing a clearer understanding of how the electrostatic effects of enzyme microenvironment modulate its λ\u003csub\u003eF\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe effects of EEF on \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e of \u003cb\u003eSystems\u003c/b\u003e \u003cb\u003ei\u0026ndash;v\u003c/b\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cb\u003eFig S14\u003c/b\u003e. For all five systems, \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e is not sensitive to the electric field applied along the Y-axis (\u003cb\u003eFig S14\u003c/b\u003e), but it shows sensitivity to the electric fields applied along the X-axis and Z-axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e). When a positive uniform electric field is applied along the negative X-axis (-X) or the positive Z-axis (+\u0026thinsp;Z), a red shift in \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e is observed. Conversely, applying a negative electric field in these directions induces a blue shift, and the displacement increases with the field strength.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCrucially, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e, the response trends and magnitudes of the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e to the EEF are generally consistent across the five systems. Therefore, using \u003cb\u003eSystem\u003c/b\u003e \u003cb\u003ei\u003c/b\u003e as a reference, a quantitative relationship between the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e and the strength of EEF was fitted as follows:\u003c/p\u003e \u003cp\u003e \u003cem\u003ey\u003c/em\u003e = -249162.8 \u003cem\u003ex\u003c/em\u003e\u0026sup2; \u0026minus;\u0026thinsp;4608.46 \u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;499.07\u003c/p\u003e \u003cp\u003e \u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5504.29 \u003cem\u003ez\u003c/em\u003e\u0026thinsp;+\u0026thinsp;500.27\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ey\u003c/em\u003e represents the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e (in nm), and \u003cem\u003ex\u003c/em\u003e and \u003cem\u003ez\u003c/em\u003e denote the electric field strengths (in a.u.) applied along the X and Z axes, respectively.\u003c/p\u003e \u003cp\u003eThe directions of the IEF acting on \u003cb\u003eoLu\u003c/b\u003e in \u003cb\u003eSystems I-V\u003c/b\u003e are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Crucially, all systems were found to exhibit IEF components along the -X or +\u0026thinsp;Z directions\u0026mdash;directions previously shown by EEF studies in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e to effectively induce red shifts in emission. This consistency indicates that that the modulation of \u003cb\u003eoLu\u003c/b\u003e's \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e by the enzyme-generated IEF follows the same physical mechanism as that induced by EEF. On this basis, and given that the purpose of this analysis is to establish the physical relevance and order of magnitude of the electrostatic effect\u0026mdash;namely, whether the protein-generated electrostatic field can meaningfully influence \u003cb\u003eoLu\u003c/b\u003e through its components along the \u0026minus;\u0026thinsp;X and/or +\u0026thinsp;Z directions identified above\u0026mdash; rather than to achieve system-by-system quantitative prediction, \u003cb\u003eSystem I (\u003c/b\u003eand its corresponding isolated \u003cb\u003eSystem\u003c/b\u003e \u003cb\u003ei\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e was selected as a representative case for quantitative illustration. The IEF components along the X and Z axes are \u0026minus;\u0026thinsp;16.362 and 6.699 MV/cm, respectively (1 au\u0026thinsp;\u0026asymp;\u0026thinsp;5140 MV/cm). Based on the derived relationship between the \u003cem\u003eλ\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e and EEF, these components are predicted to contribute red shifts of approximately 12 and 8 nm. After accounting for this IEF, the predicted λ\u003csub\u003eF\u003c/sub\u003e for \u003cb\u003eSystem I\u003c/b\u003e is approximately 519 nm, showing close agreement with the directly computed QM/MM value of 523 nm. This indicates that the IEF inherent to the protein microenvironment, operating through its specific directional components, acts as a key physical factor for the spectral tuning.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e presents the residue-specific electric field contributions projected along the X (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea) and Z (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb) axes. As established by our EEF calibration, a negative field along the X-axis (-X) and a positive field along the Z-axis (+\u0026thinsp;Z) are the effective drivers for the red shift. The data reveals a striking contrast in residue behavior. For the majority of active-site residues, metal ion binding induces only minor or inconsistent fluctuations in their field contributions. However, residue Lys529 (K529) exhibits a dramatic and distinct deviation. Along the highly sensitive X-axis (see Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea), the contribution of K529 plunges from approximately \u0026minus;\u0026thinsp;16 MV/cm in the metal ion-free system to ~ -40 MV/cm in all metal ion-bound systems. This corresponds to a net field enhancement of ~\u0026thinsp;25 MV/cm specifically favoring the red shift. Although the Z-axis projection (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb) indicates that K529 makes a minor contribution opposing the red shift (negative values), it is completely overwhelmed by the dominant red-shifting effect along the X-axis. This quantitative surge in electric field strength directly corroborates our structural analysis in \u003cb\u003eSection 3.3.3\u003c/b\u003e. It confirms that the metal ion-induced structural reorganization\u0026mdash;specifically bringing K529 into close proximity with \u003cb\u003eoLu\u003c/b\u003e\u0026mdash;is the physical origin of the enhanced IEF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we performed a comprehensive theoretical investigation combining MD simulations, QM/MM calculations, and electric field analysis to decode the molecular mechanism underlying the heavy metal ion-induced red shift in \u003cem\u003eA. vivianii\u003c/em\u003e firefly BL. Our results demonstrate that the magnitude of the red shift does not simply correlate with the intrinsic physical properties (e.g., ionic radius or charge) of the metal ions but is governed by the specific remodeling of the enzyme microenvironment.\u003c/p\u003e \u003cp\u003eWe identified that metal ions act as allosteric triggers that disrupt the native salt-bridge network (H310, E311, and E354) through specific coordination geometries. This structural perturbation reshapes the active site, inducing coupled steric and electrostatic changes that collectively regulate \u003cb\u003eoLu\u003c/b\u003e. Structurally, the altered spatial constraints force the \u003cb\u003eoLu\u003c/b\u003e to undergo a planar-to-curved geometric transition, specifically elongating the carbonyl C\u0026thinsp;=\u0026thinsp;O bond. While this geometric distortion contributes to the red shift, our analysis shows it is insufficient to account for the full magnitude of the shift. Electrostatically, the environmental remodeling repositions residue Lys529 into close proximity with the emitter. This rearrangement acts as an electrostatic amplifier, boosting the IEF exerted on \u003cb\u003eoLu\u003c/b\u003e along the sensitive axis (from ~\u0026thinsp;16 to ~\u0026thinsp;40 MV/cm).\u003c/p\u003e \u003cp\u003eBy drawing an analogy to the modulation induced by uniform EEF, we confirm that this enhanced IEF regulates the \u003cb\u003eoLu\u003c/b\u003e's electronic structure via an electrochromic effect. Therefore, the observed red shift arises from the combined effects of the environment-enforced geometric distortion and the direct electrostatic modulation.\u003c/p\u003e \u003cp\u003eIn summary, the heavy metal ion-induced red shift arises from a precise \"\u003cb\u003eoLu\u003c/b\u003e-Luciferase\" reorganization, where the metal ion coordinates the positioning of key electrostatic residues. These findings clarify the structural origins of the observed metal-ion specificity and provide a theoretical blueprint for rational biosensor design. Future efforts should focus on \"electrostatic engineering,\" where binding pockets are designed so that the target analyte drives charged residues (like Lys529) toward the emitter, allowing for fine-tuned control over the bioluminescent color.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupporting Information\u003c/h2\u003e \u003cp\u003eThe Supporting Information contains computational details, additional tables and figures, molecular dynamics analyses, electronic structure results, and Cartesian coordinates supporting the conclusions of this work.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interests:\u003c/strong\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**Jinyu Wang** : Writing \u0026ndash; original draft, Visualization, Methodology, Formal analysis, Conceptualization, Data curation. **Deping Hu** : Writing \u0026ndash; review \u0026amp; editing, Methodology, Software **Ya-Jun Liu** : Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Formal analysis, Resources, Conceptualization.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 22373010 and 22403008), the Beijing Natural Science Foundation (Grant No. 2244074) and the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2025A1515012183). Computing resources were provided by the Interdisciplinary Intelligence Supercomputer Center of Beijing Normal University at Zhuhai.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao, J. Y., Lin, S. X., \u0026amp; Huang, Y. (2013). Mechanism-based design of a photoactivatable firefly luciferase. \u003cem\u003eJournal of the American Chemical Society\u003c/em\u003e, \u003cem\u003e135\u003c/em\u003e(19), 7410\u0026ndash;7413. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ja4013535\u003c/span\u003e\u003cspan address=\"10.1021/ja4013535\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng, P., Zhang, H., Deng, Q., \u0026amp; Liu, Y. (2016). Real-time bioluminescence imaging of nitroreductase in mouse model. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e, \u003cem\u003e88\u003c/em\u003e(11), 5610\u0026ndash;5614. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.6b01160\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.6b01160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeda, Y., Saitoh, T., Niwa, K., \u0026amp; Amano, R. (2018). An allylated firefly luciferin analogue with luciferase specific response in living cells. \u003cem\u003eChemical Communications\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e(14), 1774\u0026ndash;1777. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c7cc09720d\u003c/span\u003e\u003cspan address=\"10.1039/c7cc09720d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndo, Y., Niwa, K., Yamada, N., Enomoto, T., Irie, T., Kubota, H., et al. (2007). Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. \u003cem\u003eNature Photonics\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(1), 44\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nphoton.2007.251\u003c/span\u003e\u003cspan address=\"10.1038/nphoton.2007.251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. J. (2022). Understanding the complete bioluminescence cycle from a multiscale computational perspective: A review. \u003cem\u003eJournal of Photochemistry and Photobiology C: Photochemistry Reviews\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e, 100537. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jphotochemrev.2022.100537\u003c/span\u003e\u003cspan address=\"10.1016/j.jphotochemrev.2022.100537\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcElroy, W. D., Seliger, H. H., White, E. H., \u0026amp; Copley, M. (1969). Mechanism of bioluminescence, chemi-luminescence and enzyme function in the oxidation of firefly luciferin. \u003cem\u003ePhotochemistry and Photobiology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(3), 153\u0026ndash;170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1751-1097.1969.tb05676.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1751-1097.1969.tb05676.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWood, K. V. (1995). The chemical mechanism and evolutionary development of beetle bioluminescence. \u003cem\u003ePhotochemistry and Photobiology\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e(4), 662\u0026ndash;673. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1751-1097.1995.tb08714.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1751-1097.1995.tb08714.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani, V. R. (2002). The origin, diversity, and structure function relationships of insect luciferases. \u003cem\u003eCellular and Molecular Life Sciences\u003c/em\u003e, \u003cem\u003e59\u003c/em\u003e(11), 1833\u0026ndash;1850. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00018-002-8545-z\u003c/span\u003e\u003cspan address=\"10.1007/s00018-002-8545-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcElroy, W. D., \u0026amp; Seliger, H. H. (1964). The colors of firefly bioluminescence: Enzyme configuration and species specificity. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(1), 75\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.52.1.75\u003c/span\u003e\u003cspan address=\"10.1073/pnas.52.1.75\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani, V. R., \u0026amp; Bechara, E. J. H. (1995). Bioluminescence of Brazilian fireflies (Coleoptera: Lampyridae): Spectral distribution and pH effect on luciferase-elicited colors. \u003cem\u003ePhotochemistry and Photobiology\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e(3), 490\u0026ndash;495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1751-1097.1995.tb02373.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1751-1097.1995.tb02373.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSundlov, J. A., Fontaine, D. M., Southworth, T. L., \u0026amp; Gulick, A. M. (2012). Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism. \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cem\u003e51\u003c/em\u003e(33), 6493\u0026ndash;6495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bi300934s\u003c/span\u003e\u003cspan address=\"10.1021/bi300934s\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakatsu, T., Ichiyama, S., Hiratake, J., Mizutani, H., Chu, W. C., Itoh, N., et al. (2006). Structural basis for the spectral difference in luciferase bioluminescence. \u003cem\u003eNature\u003c/em\u003e, \u003cem\u003e440\u003c/em\u003e(7082), 372\u0026ndash;376. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature04542\u003c/span\u003e\u003cspan address=\"10.1038/nature04542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrasco-Lopez, C., Lui, N. M., Schramm, S., \u0026amp; Viviani, V. R. (2021). The elusive relationship between structure and colour emission in beetle luciferases. \u003cem\u003eNature Reviews Chemistry\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(1), 4\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41570-020-00238-1\u003c/span\u003e\u003cspan address=\"10.1038/s41570-020-00238-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhose, A., Rebarz, M., Maltsev, O. V., Hintermann, L., Ruckebusch, C., Fron, E., et al. (2014). Emission properties of oxyluciferin and its derivatives in water: Revealing the nature of the emissive species in firefly bioluminescence. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e, \u003cem\u003e119\u003c/em\u003e(6), 2638\u0026ndash;2649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jp508905m\u003c/span\u003e\u003cspan address=\"10.1021/jp508905m\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite, E. H., Rapaport, E., Hopkins, T. A., \u0026amp; Seliger, H. H. (1969). Chemi- and bioluminescence of firefly luciferin. \u003cem\u003eJournal of the American Chemical Society\u003c/em\u003e, \u003cem\u003e91\u003c/em\u003e(8), 2178\u0026ndash;2180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ja01036a093\u003c/span\u003e\u003cspan address=\"10.1021/ja01036a093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite, E. H., Seliger, H. H., \u0026amp; Hopkins, T. A. (1971). The chemi- and bioluminescence of firefly luciferin: An efficient chemical production of electronically excited states. \u003cem\u003eBioorganic Chemistry\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(1\u0026ndash;2), 92\u0026ndash;122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0045-2068(71)90010-8\u003c/span\u003e\u003cspan address=\"10.1016/0045-2068(71)90010-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., Akiyama, H., Terakado, K., \u0026amp; Nakatsu, T. (2013). Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), 2490. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep02490\u003c/span\u003e\u003cspan address=\"10.1038/srep02490\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., Hayamizu, Y., \u0026amp; Akiyama, H. (2014). Spectroscopic study of firefly oxyluciferin in an enzymatic environment on the basis of stability monitoring. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e(8), 2070\u0026ndash;2076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jp411476p\u003c/span\u003e\u003cspan address=\"10.1021/jp411476p\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMochizuki, T., Wang, Y., Hiyama, M., \u0026amp; Akiyama, H. (2014). Robust red-emission spectra and yields in firefly bioluminescence against temperature changes. \u003cem\u003eApplied Physics Letters\u003c/em\u003e, \u003cem\u003e104\u003c/em\u003e(18), 183701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.4880578\u003c/span\u003e\u003cspan address=\"10.1063/1.4880578\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUgarova, N. N., \u0026amp; Brovko, L. Y. (2001). Relationship between the structure of the protein globule and bioluminescence spectra of firefly luciferase. \u003cem\u003eRussian Chemical Bulletin International Edition\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(10), 1752\u0026ndash;1761. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1012726300408\u003c/span\u003e\u003cspan address=\"10.1023/A:1012726300408\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirano, T., Nagai, H., Matsuhashi, T., \u0026amp; Maki, S. (2012). Spectroscopic studies of the color modulation mechanism of firefly (beetle) bioluminescence with amino-analogs of luciferin and oxyluciferin. \u003cem\u003ePhotochemical \u0026amp; Photobiological Sciences\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(8), 1281\u0026ndash;1284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c2pp25106j\u003c/span\u003e\u003cspan address=\"10.1039/c2pp25106j\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGandelman, O. A., Brovko, L. Y., Ugarova, N. N., \u0026amp; Klyachko, N. L. (1993). Oxyluciferin fluorescence is a model of native bioluminescence in the firefly luciferin-luciferase system. \u003cem\u003eJournal of Photochemistry and Photobiology B: Biology\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(3), 187\u0026ndash;191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/1011-1344(93)80108-C\u003c/span\u003e\u003cspan address=\"10.1016/1011-1344(93)80108-C\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, H. W., \u0026amp; Rhee, Y. M. (2013). On the pH dependent behavior of the firefly bioluminescence: Protein dynamics and water content in the active pocket. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e, \u003cem\u003e117\u003c/em\u003e(24), 7260\u0026ndash;7269. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jp4024553\u003c/span\u003e\u003cspan address=\"10.1021/jp4024553\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoksharov, M. I., \u0026amp; Ugarova, N. N. (2013). Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence. \u003cem\u003ePhotochemical \u0026amp; Photobiological Sciences\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(11), 2016\u0026ndash;2027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c3pp50242b\u003c/span\u003e\u003cspan address=\"10.1039/c3pp50242b\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, J. G., Williams, Q. L., Walters, W., \u0026amp; Koua, K. (2015). How does the local electrostatic field influence emitted wavelengths and bioluminescent intensities of modified heteroaromatic luciferins? \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e, \u003cem\u003e119\u003c/em\u003e(33), 10399\u0026ndash;10405. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jpcb.5b03827\u003c/span\u003e\u003cspan address=\"10.1021/acs.jpcb.5b03827\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, Y. Y., \u0026amp; Liu, Y. J. (2015). What exactly is the light emitter of a firefly? \u003cem\u003eJournal of Chemical Theory and Computation\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(11), 5360\u0026ndash;5370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jctc.5b00659\u003c/span\u003e\u003cspan address=\"10.1021/acs.jctc.5b00659\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYue, L., Lan, Z., \u0026amp; Liu, Y. J. (2015). The theoretical estimation of the bioluminescent efficiency of the firefly via a nonadiabatic molecular dynamics simulation. \u003cem\u003eThe Journal of Physical Chemistry Letters\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(3), 540\u0026ndash;548. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jz502305g\u003c/span\u003e\u003cspan address=\"10.1021/jz502305g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakakura, H. (2021). Molecular design of D-luciferin-based bioluminescence and 1,2-dioxetane-based chemiluminescence substrates for altered output wavelength and detecting various molecules. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eArticle 1618. https://doi.org/10.3390/molecules26061618\u003c/span\u003e\u003cspan address=\"Article 1618. 10.3390/molecules26061618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavizet, I., Roca-Sanju\u0026aacute;n, D., Yue, L., Ferr\u0026eacute;, N., Liu, Y. J., \u0026amp; Lindh, R. (2012). Are the bio- and chemiluminescence states of the firefly oxyluciferin the same as the fluorescence state? \u003cem\u003ePhotochemistry and Photobiology\u003c/em\u003e, \u003cem\u003e89\u003c/em\u003e(2), 319\u0026ndash;325. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/php.12007\u003c/span\u003e\u003cspan address=\"10.1111/php.12007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabriel, G. V. M., \u0026amp; Viviani, V. R. (2016). Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals. \u003cem\u003eAnalytical and Bioanalytical Chemistry\u003c/em\u003e, \u003cem\u003e408\u003c/em\u003e(30), 8881\u0026ndash;8893. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-016-0011-1\u003c/span\u003e\u003cspan address=\"10.1007/s00216-016-0011-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani, V. R., Sim\u0026otilde;es, A., Bevilaqua, V. R., Gabriel, G. V. M., Arnoldi, F. G., et al. (2016). Glu311 and Arg337 stabilize a closed active-site conformation and provide a critical catalytic base and countercation for green bioluminescence in beetle luciferases. \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cem\u003e55\u003c/em\u003e(34), 4764\u0026ndash;4776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.biochem.6b00260\u003c/span\u003e\u003cspan address=\"10.1021/acs.biochem.6b00260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani, V. R., Gabriel, G. V. M., Bevilaqua, V. R., \u0026amp; Matsushita, A. J. (2018). The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eArticle 15456. https://doi.org/10.1038/s41598-018-33252-x\u003c/span\u003e\u003cspan address=\"Article 15456. 10.1038/s41598-018-33252-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelentir, G. F., Bevilaqua, V. R., \u0026amp; Viviani, V. R. (2020). A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays. \u003cem\u003ePhotochemical \u0026amp; Photobiological Sciences\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(9), 2061\u0026ndash;2070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c9pp00174c\u003c/span\u003e\u003cspan address=\"10.1039/c9pp00174c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani, V. R., Amaral, D., Prado, R., \u0026amp; Arnoldi, F. G. (2011). A new blue-shifted luciferase from the Brazilian (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties. \u003cem\u003ePhotochemical \u0026amp; Photobiological Sciences\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(12), 1879\u0026ndash;1886. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c1pp05210a\u003c/span\u003e\u003cspan address=\"10.1039/c1pp05210a\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrasco-L\u0026oacute;pez, C., Ferreira, J. C., Lui, N. M., \u0026amp; Viviani, V. R. (2018). Beetle luciferases with naturally red- and blue-shifted emission. \u003cem\u003eLife Science Alliance\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(5), e201800072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.26508/lsa.201800072\u003c/span\u003e\u003cspan address=\"10.26508/lsa.201800072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, C., Kasavajhala, K., Belfon, K. A. A., \u0026amp; Simmerling, C. L. (2019). ff19SB: Amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. \u003cem\u003eJournal of Chemical Theory and Computation\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(1), 528\u0026ndash;552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jctc.9b00591\u003c/span\u003e\u003cspan address=\"10.1021/acs.jctc.9b00591\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, J. M., Wolf, R. M., Caldwell, J. W., Kollman, P. A., \u0026amp; Case, D. A. (2004). Development and testing of a general Amber force field. \u003cem\u003eJournal of Computational Chemistry\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(9), 1157\u0026ndash;1174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcc.20035\u003c/span\u003e\u003cspan address=\"10.1002/jcc.20035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, P., \u0026amp; Merz, K. M. (2016). MCPB.py: A Python based metal center parameter builder. \u003cem\u003eJournal of Chemical Information and Modeling\u003c/em\u003e, \u003cem\u003e56\u003c/em\u003e(4), 599\u0026ndash;604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jcim.5b00674\u003c/span\u003e\u003cspan address=\"10.1021/acs.jcim.5b00674\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzaguirre, J. A., Catarello, D. P., Wozniak, J. M., \u0026amp; Skeel, R. D. (2001). Langevin stabilization of molecular dynamics. \u003cem\u003eThe Journal of Chemical Physics\u003c/em\u003e, \u003cem\u003e114\u003c/em\u003e(5), 2090\u0026ndash;2098. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.1332996\u003c/span\u003e\u003cspan address=\"10.1063/1.1332996\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., \u0026amp; Haak, J. R. (1984). Molecular dynamics with coupling to an external bath. \u003cem\u003eThe Journal of Chemical Physics\u003c/em\u003e, \u003cem\u003e81\u003c/em\u003e(8), 3684\u0026ndash;3690. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.448118\u003c/span\u003e\u003cspan address=\"10.1063/1.448118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarden, T., York, D., \u0026amp; Pedersen, L. (1993). Particle mesh Ewald: An N\u0026middot;log(N) method for Ewald sums in large systems. \u003cem\u003eThe Journal of Chemical Physics\u003c/em\u003e, \u003cem\u003e98\u003c/em\u003e(12), 10089\u0026ndash;10092. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.464397\u003c/span\u003e\u003cspan address=\"10.1063/1.464397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCase, D. A., Aktulga, H. M., Belfon, K., Ben-Shalom, I. Y., Brozell, S. R., Cerutti, D. S., et al. (2021). \u003cem\u003eAmber 2021\u003c/em\u003e. University of California.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYanai, T., Tew, D. P., \u0026amp; Handy, N. C. (2004). A new hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP). \u003cem\u003eChemical Physics Letters\u003c/em\u003e, \u003cem\u003e393\u003c/em\u003e(1\u0026ndash;3), 51\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cplett.2004.06.011\u003c/span\u003e\u003cspan address=\"10.1016/j.cplett.2004.06.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, J., Hu, D., \u0026amp; Liu, Y. J. (2025). Theoretical regulation of firefly bioluminescence spectra: Exploring new frontiers in NIR-II imaging. \u003cem\u003eJournal of Photochemistry and Photobiology A: Chemistry\u003c/em\u003e, \u003cem\u003e466\u003c/em\u003e, 116393. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jphotochem.2025.116393\u003c/span\u003e\u003cspan address=\"10.1016/j.jphotochem.2025.116393\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSherwood, P., de Vries, A. H., Guest, M. F., Schreckenbach, G., Catlow, C. R. A., et al. (2003). QUASI: A general purpose implementation of the QM/MM approach and its application to problems in catalysis. \u003cem\u003eJournal of Molecular Structure: THEOCHEM\u003c/em\u003e, \u003cem\u003e632\u003c/em\u003e(1\u0026ndash;3), 1\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0166-1280(03)00285-9\u003c/span\u003e\u003cspan address=\"10.1016/s0166-1280(03)00285-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetz, S., K\u0026auml;stner, J., Sokol, A. A., Keal, T. W., \u0026amp; Sherwood, P. (2014). ChemShell\u0026mdash;a modular software package for QM/MM simulations. \u003cem\u003eWIREs Computational Molecular Science\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(2), 101\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/wcms.1163\u003c/span\u003e\u003cspan address=\"10.1002/wcms.1163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeese, F. (2022). Software update: The ORCA program system\u0026mdash;Version 5.0. \u003cem\u003eWIREs Computational Molecular Science\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/wcms.1606\u003c/span\u003e\u003cspan address=\"10.1002/wcms.1606\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Article e1606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, W., \u0026amp; Forester, T. R. (1996). DL_POLY_2.0: A general-purpose parallel molecular dynamics simulation package. \u003cem\u003eJournal of Molecular Graphics\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(3), 136\u0026ndash;141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0263-7855(96)00043-4\u003c/span\u003e\u003cspan address=\"10.1016/S0263-7855(96)00043-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRunge, E., \u0026amp; Gross, E. K. U. (1984). Density-functional theory for time-dependent systems. \u003cem\u003ePhysical Review Letters\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(12), 997\u0026ndash;1000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1103/PhysRevLett.52.997\u003c/span\u003e\u003cspan address=\"10.1103/PhysRevLett.52.997\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBakowies, D., \u0026amp; Thiel, W. (1996). Hybrid models for combined quantum mechanical and molecular mechanical approaches. \u003cem\u003eThe Journal of Physical Chemistry\u003c/em\u003e, \u003cem\u003e100\u003c/em\u003e(25), 10580\u0026ndash;10594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jp9536514\u003c/span\u003e\u003cspan address=\"10.1021/jp9536514\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelentir, G. F., Tomazini, A., Bevilaqua, V. R., \u0026amp; Viviani, V. R. (2024). Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties. \u003cem\u003ePhotochemical \u0026amp; Photobiological Sciences\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(6), 997\u0026ndash;1010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s43630-024-00570-1\u003c/span\u003e\u003cspan address=\"10.1007/s43630-024-00570-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShannon, R. D. (1976). Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. \u003cem\u003eActa Crystallographica Section A\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(5), 751\u0026ndash;767. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1107/S0567739476001551\u003c/span\u003e\u003cspan address=\"10.1107/S0567739476001551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"firefly bioluminescence, heavy metal ions, red-shift mechanism, electric field effect, QM/MM","lastPublishedDoi":"10.21203/rs.3.rs-8629136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8629136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFirefly 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\u003csup\u003e+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, and Hg\u003csup\u003e2+\u003c/sup\u003e 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 (\u003cb\u003eoLu\u003c/b\u003e) 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 \u003cb\u003eoLu\u003c/b\u003e, 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 \u003cb\u003eoLu\u003c/b\u003e'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.\u003c/p\u003e","manuscriptTitle":"Theoretical Study of Metal Ion–Induced Modulation of Firefly Bioluminescence Spectra","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 09:40:55","doi":"10.21203/rs.3.rs-8629136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-18T16:43:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T12:00:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1603182742650265676190394096884365906","date":"2026-03-10T08:56:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T17:55:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303131969829137290878384661189082294517","date":"2026-02-02T16:31:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T08:54:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-01T06:11:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T16:11:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photochemical \u0026 Photobiological Sciences","date":"2026-01-18T04:12:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"afb35edf-15a6-4aa7-b1fb-b2bb78f28854","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T16:54:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 09:40:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8629136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8629136","identity":"rs-8629136","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}