Firefly bioluminescence (BL) involves a series of chemical reactions that require several essential components: luciferin (L), luciferase, ATP, O2, and Mg2+. Luciferase consists of an N-terminal domain (N-domain) and a C-terminal domain (C-domain), which are linked by a flexible loop. Substrates bind mainly within the N-domain, suggesting that the N-domain alone should be capable of maintaining the BL. However, an experimental study indicated that when the C-domain of wild-type luciferase (WT) is removed, the truncated N-domain type luciferase (NT) retains only 0.03% of firefly BL activity compared with the WT system. This produces some fundamental questions: what is the role of the C-domain in firefly BL and why does the deletion of the C-domain cause such a drastic decrease in firefly BL activity? In this article, we address these questions in detail via multiscale theoretical calculations. Our results show that the C-domain plays a positive catalytic role by enhancing the affinity of substrates, promoting the formation of preorganized and compact conformations of critical intermediates, and sustaining the enzyme-generated internal electric field (IEF) favorable for the firefly BL reaction. In the absence of the C-domain, these BL-favorable factors are severely disrupted, leading to the suppression of the firefly BL efficiency. This study provides the first computational elucidation of the entire firefly BL process within a realistic protein environment and introduces the concept of IEF catalysis into the context of BL. Most importantly, we deeply identify and characterize the crucial role of the C-domain and some key residues (LYS439, LYS529, and ARG533) in firefly BL.

Abstract Development of precise, non-invasive, and dynamic methods for detecting amyloid-β (Aβ) aggregates is of vital importance for the early diagnosis and therapeutic evaluation of Alzheimer’s disease (AD). Recently, the Akalumine (AkaL)-based firefly bioluminescent probe has emerged as a promising tool for deep-tissue Aβ imaging. However, despite the successful experimental application of this probe, an understanding that links its microscopic mechanisms to the observed bioluminescence (BL) intensity, kinetics, and imaging performance is still lacking. In this work, a multiscale theoretical investigation combining classical molecular dynamics (cMD) simulations, absolute binding free energy (ABFE) calculations, quantum mechanics/molecular mechanics (QM/MM), and QM/MM metadynamics simulations was performed to comprehensively elucidate the mechanism of this bioluminescent system. Our cMD and ABFE results reveal that Aβ acts as a reservoir capable of storing AkaL via hydrophobic interactions, thereby dynamically modulating the concentration of free AkaL and the evolution of BL intensity. Changes in BL intensity can serve as an indicator of the concentration and distribution of Aβ. Furthermore, the complete BL process of AkaL catalyzed by firefly luciferase was systematically studied. The initial adenylation follows the SN2 mechanism. The water molecule was found to play a crucial catalytic role in the subsequent deprotonation by mediating proton transfer, significantly lowering the energy barrier. Then, oxygenation occurs via a triplet-to-singlet intersystem crossing (ISC) process to generate the dioxetanone intermediate. The decomposition of the dioxetanone intermediate follows the gradually reversible charge-transfer initiated luminescence (GRCTIL) mechanism that generates the light emitter Oxy-AkaL in the S1 state. The calculated emission wavelength is in reasonable agreement with the experimental BL wavelength. Overall, this work provides theoretical insights into the mechanism of firefly BL-based AD detection and offers guidance for the rational design of future bioluminescent probes for AD.

Theoretical simulations of vibrationally resolved electronic spectra not only allow direct comparison with experimental results but also enable simulations under conditions that are experimentally challenging, such as in the gas phase. For instance, firefly oxyluciferin (OL) exists in six neutral and ionic forms in equilibrium within a physiologically relevant pH range in aqueous solutions. Investigating the absorption and fluorescence spectra of OL helps in elucidating the mechanism of firefly bioluminescence and reveals its photophysical properties, thereby enabling control of the emission color. Recent experiments have investigated its absorption and fluorescence spectra in the gas phase, revealing that oxyluciferin anion (OL-) can exist in both phenolate-enol and phenolate-keto forms. In this study, we present a simulation of the vibrationally resolved absorption and fluorescence spectra of four OL- derivatives, namely, phenolate-enol, phenolate-keto, meo-OL- (enol-locked) and dm-OL- (keto-locked). Our spectra simulation employs several electronic structure methods, including time-dependent density functional theory (TDDFT), complete active space second-order perturbation theory (CASPT2), and density matrix renormalization group (DMRG) CASPT2. In addition, we studied the influence of quantum mechanics (QM) methods, active space size, the Herzberg-Teller effect, and the Duschinsky effect on the spectra simulation. The simulated spectra not only agree well with experimental results but also detail the experimental findings, revealing key vibronic transitions of OL- derivatives. The combination of CASPT2 and TDDFT calculated that the 0-0 transition energy can be directly used as a reference for the band origin when it is not reported experimentally. Our findings can also be extended to simulate the absorption and fluorescence spectra of other semirigid organic molecules.

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 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 electronic structure of oLu 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.

Bioluminescence (BL) is a unique chemiluminescent process in living organisms that has inspired extensive applications in bioimaging and biosensing due to its high sensitivity, non-invasiveness, and absence of background autofluorescence. Most luminescent organisms emit light through a set of luminescent systems. Some luminescent organisms possess an additional antenna protein that can receive energy from the former luminescent system. And the BL is emitted by the antenna protein. Inspired by these natural phenomena, bioluminescence resonance energy transfer (BRET) has been widely exploited to overcome the limitations of native BL, particularly the intensity and short-wavelength emission that restrict deep-tissue imaging. This review summarizes the fundamental mechanisms of ET in representative natural BL systems, combining insights from experimental and theoretical studies. We then conclude recent advances in engineering BRET systems by coupling luciferases with fluorescent proteins, organic dyes, and nanomaterials to achieve brighter and red-shifted emissions, extending into the near-infrared II (NIR-II) region. Finally, we discuss current challenges and propose future directions for developing next-generation BL probes with improved brightness, spectral tunability, and stability. We anticipate that these developments will provide powerful tools for real-time imaging of biological processes with high resolution and penetration depth.

Diplocardia longa (D. longa), a bioluminescent earthworm native to North America, emits blue-green light. Experimental studies conducted in the 1970s-1980s established that its bioluminescence (BL) involves reactions of H2O2, luciferin (L) and Cu+-dependent luciferase. However, research stalled after 1981, leaving some fundamental gaps: the chemical mechanism, key intermediates, product, light emitter, and role of Cu+ remain undefined. To address these uncertainties, this combined theoretical and experimental study systematically investigated the corresponding chemiluminescence (CL) of D. longa BL, since the crystal structure of luciferase has not yet been determined. Using density functional theory (DFT) and mixed reference spin flip (MRSF) DFT methods, we calculated the complete reaction pathway from reactants to light emission. Integrated with experimental evidence, we confirm the catalytic function of Cu+, identify the molecular structure of the peroxide intermediate and product, and assign the light emitter. This study provides critical insights into natural BL systems, particularly as D. longa luciferin is a shared substrate across 13 bioluminescent earthworm species spanning six genera.

Fluorescence molecular imaging aims to enhance clarity in the region of interest, particularly in the near-infrared IIb window (NIR-IIb, 1500–1700 nm). To achieve this, we developed a novel small-molecule dye, named DA-5, based on classic cyanine dyes (heptamethine or pentamethine is essential for wavelengths beyond 1000 nm). By reducing excessive polymethine to a single methine and disrupting symmetry to form an asymmetric donor-π-acceptor (D-π-A) architecture, we enhanced the donor’s electron-donating capability, yielding emission at 1088 nm. DA-5 exhibits superior properties, including excellent chemo- and photostability, resistance against solvatochromism-caused quenching, and antiaggregation in aqueous solution. With a large Stokes shift (241 nm) and high brightness (321 M–1 cm–1), DA-5 enables high-performance imaging of the lymphatic system, intestinal vessels, whole-body angiography, and cerebral and hindlimb microvasculature in NIR-IIb. This molecular design strategy offers a promising platform for advancing in vivo biophotonics.

The most well-known hydroxyl radical (•OH)-generating system is the classic iron-mediated Fenton reaction. Thiourea has been considered as an efficient •OH scavenger and is frequently used to study the role of •OH in various biochemical and medical research studies. Here we found that the highly reactive •OH can be produced from thiourea and H2O2 through a metal-independent pathway, as measured by electron spin resonance (ESR) secondary radical spin-trapping and fluorescent methods. The major reaction intermediates from thiourea/H2O2 were identified as formamidinesulfenic acid and formamidinesulfinic acid, with urea and sulfate as the major final products. Taken together, the underlying molecular mechanism for the unprecedented •OH production from thiourea/H2O2 was proposed: thiourea is initially attacked by H2O2 to produce the transient intermediates formamidinesulfenic acid and then formamidinesulfinic acid, which further react with H2O2 to produce their corresponding hydroperoxyl intermediates, which can decompose homolytically to generate •OH and the final products. Analogous •OH production and oxidative DNA damage were also observed with other thiourea derivatives and H2O2. This is the first report on metal-independent •OH production from the well-known •OH scavenging thioureas and H2O2, which may have important biochemical, environmental, and medical implications for future study of thiourea compounds.

Firefly bioluminescence (BL) imaging holds significant promise for non-invasive, real-time monitoring of biological processes. To improve its efficiency, considerable efforts have been made to achieve red-shifting of the emission wavelength. In this work, we systematically investigated the fluorescence properties of the light emitter oxyluciferin (oLu) dependent on the electrostatic effects using the TD DFT method. oLu is a typical donor-acceptor (D-A) charge transfer system, with the benzothiazole group acting as the electron donor and the thiazole group as the electron acceptor. Therefore, we focused on the significant impact of external charges on the fluorescence wavelength (7F), where the negative charge around the benzothiazole (donor) and/or the positive charge around the thiazole (acceptor) side promotes a red-shift in 7F. Basing on these insights, we theoretically explored modifications to the oLu structure and mutations in luciferase to enhance D and A properties of oLu for facilitating the 7F red-shift. Besides, we also inserted conjugation structures between D and A of oLu to format D-pi-A systems for further red-shifting the 7F. By integrating oLu modifications with luciferase mutations, we successfully designed a new nova-I351D using MD and QM/MM methods, a bioluminescent system that emits brightly in the near-infrared II (NIR-II) region. This system demonstrates significant potential for deep tissue imaging, providing a promising advancement in bioluminescence-based imaging technologies.

Energy transfer (ET) complex is not rare in bioluminescence. Usually, the ET occurs from the donor with higher emission energy to the acceptor with lower absorption energy. However, a blue-shifted ET is observed in the bioluminescence (BL) of Photobacterium phosphoreum (PP). The luminophore, 4a-hydroxy-5-hydro-flavin mononucleotide at the first singlet excited state ($S_1$-HFOH), in solitary PP luciferase (PPLuc) emits light at 495 nm. When a proportional concentration of lumazine protein (LumP) with a substrate of 6,7-dimethyl-8-ribityllumazine (DLZ) is introduced, the emission wavelength changes to 475 nm, accompanied by a 2.1-fold enhancement in intensity. The blueshift is only an observation, whose ET mechanism has not been uncovered over fifty years of research. In the present article, we evidenced that the ET process occurs via a Förster resonance energy transfer (FRET) mechanism by protein-protein docking and molecular dynamics (MD) simulations. Moreover, utilizing the combined quantum mechanics and molecular mechanics (QM/MM) method, we calculated the FRET rate and fluorescence quantum yield. The small Stokes shift of DLZ as well as the strong vibronic couplings of HFOH allow the blue-shifted FRET process. The calculated FRET rate is larger than the radiative and non-radiative decay ones of $S_1$-HFOH, and the fluorescence quantum yield of $S_1$-DLZ is higher than the one of $S_1$-HFOH, which clearly explains the experimentally observed enhancement of the emission intensity. Simultaneously, the blue-shifted FRET mechanism firstly interpreted that the wild-type PP emits 475 nm BL rather than 490 nm one as the other species of bioluminescent bacteria do. This first-time deep investigation establishes a theoretical research paradigm for the theoretical study of ET and holds significance in color regulation in the BL field.

Maintaining a stable physiological pH is essential for the normal functioning of both whole organisms and individual cells. Ratiometric chemiluminescence probes have been widely employed to monitor pH in cells and living organisms due to their high sensitivity, resistance to external interferences, and noninvasiveness. In this study, the working mechanism of a specific ratiometric chemiluminescent probe, Ratio-pHCL-1, is investigated using (time-dependent) density functional theory. The mechanism can be divided into three stages. At first, pH influences the protonation state of Ratio-pHCL-1 in physiological pH range of 6.8-8.4. Subsequently, Ratio-pHCL-1 decomposes to generate the light emitter in the first excited state (S1) via a gradually reversible charge-transfer initiated luminescence mechanism. Finally, at higher pH values, the intramolecular energy transfer (ET) occurs, resulting in a redshift of the emission wavelength. The redshift of the emission wavelength effectively enhances the luminescence intensity and improves the imaging ability. While at lower pH values, the ET process does not occur. This is the first systematic study on the working mechanism of ratiometric chemiluminescent probes at the molecular and electronic-state levels. The findings can also be extended to understand the mechanism of a class of ratiometric chemiluminescent probes.

Understanding the molecular mechanism of inhibitor binding to prostate-specific membrane antigen (PSMA) is of fundamental importance for designing targeted drugs for prostate cancer. Here we designed a series of PSMA-targeting inhibitors with distinct molecular structures, which were synthesized and characterized using both experimental and computational approaches. Microsecond molecular dynamics simulations revealed the structural and thermodynamic details of PSMA-inhibitor interactions. Our findings emphasize the pivotal role of the inhibitor's P1 region in modulating binding affinity and selectivity and shed light on the binding-induced conformational shifts of two key loops (the entrance lid and the interface loop). Binding energy calculations demonstrate the enthalpy-entropy balance in the thermodynamic driving force of different inhibitors. The binding of inhibitors in monomeric form is entropy-driven, in which the solvation entropy from the binding-induced water restraints plays a key role, while the binding of inhibitors in dimeric form is enthalpy-driven, due to the promiscuous PSMA-inhibitor interactions. These insights into the molecular driving force of protein-ligand binding offer valuable guidance for rational drug design.

The development of long-wavelength near-infrared II (NIR-II, 900-1700 nm) dyes is highly desirable but challenging. To achieve both red-shifted absorption/emission and superior in vivo imaging capabilities, a donor-acceptor-donor (D-A-D) xanthene core was strategically modified by extending π-conjugated double bonds and enhancing electron-donating properties. Two dyes named VIX-1250 and VIX-1450 were synthesized and exhibited notably red-shifted absorption/emission peaks at 942/1250 and 1098/1450 nm, respectively. Among them, VIX-1450 demonstrated superior chemo- and photostability even at such long wavelengths. Fluorescent angiography using VIX-1450 micelles enabled high-clarity blood vessel imaging with a remarkable signal-to-noise ratio (SNR), underscoring that the dye's large Stokes shift (352 nm), good brightness (13 M-1 cm-1), and long wavelength served as key factors for high-quality in vivo biosensing. Additionally, VIX-1450 combined with ICG for dual-color imaging achieved near-zero optical cross talk, enabling different organ labeling. This study provides a new direction for the design of long-wavelength organic dyes.

Green fluorescent protein (GFP) is famous for noninvasively observing the internal biological processes of cells and organisms, revolutionizing the field of cell biology. GFP was first discovered in jellyfish Aequorea victoria (AV). The GFP bioluminescence (BL) in AV can be divided into three stages: the first singlet excited state coelenteramide (S1-CTD) is formed in aequorin; GFP acquires energy from S1-CTD via an energy transfer (ET) process; and GFP emits green light. The first and final stages have been well studied, whereas the detailed mechanism of the second stage remains unclear, with only sporadic experimental evidence. The purpose of this study is to clarify how GFP acquires energy before emitting green light in AV. Through protein-protein docking, molecular dynamics simulations, and combined quantum mechanics and molecular mechanics calculations, we demonstrate that the ET process occurs via the Förster resonance energy transfer (FRET) mechanism. The calculated FRET rate is faster than the radiative and nonradiative decay ones of S1-CTD, which means the ET process can occur efficiently. Additionally, the calculated fluorescence quantum yield explains the experimentally observed BL enhancement after the ET. This is the first theoretical report on the ET mechanism in BL. This study not only clearly interprets how GFP acquires energy for emitting light but also helps to understand the ET mechanism in other bioluminescent systems and sheds new light on bioluminescence resonance energy transfer.

We extended the symmetric quasi-classical (SQC) method based on the Meyer-Miller mapping Hamiltonian (MM) to treat the nonadiabatic dynamics simulation including spin-orbit couplings (SOCs). We studied the photoinduced ultrafast excited state dynamic involving intersystem crossing (ISC) process of the ReBr(CO)3bpy molecule by performing the dynamics based on a preconstructed model Hamiltonian as well as with the on-the-fly ab initio calculations. For the model system, the dynamics results obtained with the SQC/MM method compare very well with those obtained with the ML-MCTDH method. The SQC/MM method also outperforms the widely used trajectory surface hopping (TSH) method for the system studied here. For the realistic system, we employed the quasi-diabatic propagation scheme to enable on-the-fly ab initio dynamics with the SQC/MM method. In this case, the dynamics results obtained with the SQC/MM method are very similar to those obtained with the TSH method, and both of them show great discrepancy with the model system results, which implies it is necessary to perform the full atom on-the-fly dynamics for some molecular systems. Due to the great performance of the SQC/MM method in this work, we strongly recommend using it in the study of ISC processes in the future, for both model and realistic systems. The implementation of the SQC/MM method in the on-the-fly dynamics including the SOCs also paves the way to employing the SQC/MM or other more advanced semiclassical dynamics methods based on the mapping Hamiltonian to study the ISC processes for complex realistic molecular systems.

In this study, we perform on-the-fly nonadiabatic molecular dynamics (NAMD) simulations for three molecular systems (ethylene, DMABN, and fulvene), which are suggested as molecular versions of the Tully models, with the trajectory surface hopping method based on the mixed-reference spin-flip time-dependent density functional theory (MRSF-TDDFT). We employ several density functionals (CAM-B3LYP, M06-2X, BH&HLYP, and DTCAM-VAEE) in the MRSF-TDDFT calculations and compare the results with those obtained with the SA-CASSCF and MS-CASPT2 methods. For the ethylene molecule, the dynamics results obtained with MRSF-TDDFT compare very well with those obtained with MS-CASPT2, and the results with different functionals are similar. For the DMABN and fulvene molecules, the dynamics results with different functionals show certain differences, while the DTCAM-VAEE functional performs best among all functionals compared to MS-CASPT2. Moreover, for all molecules, MRSF-TDDFT outperforms SA-CASSCF for all functionals used in this work. We further explain the discrepancies of the dynamics results with different electronic structure methods through reaction pathway analysis. Overall, we strongly recommend the use of MRSF-TDDFT, especially with the DTCAM-VAEE functionals, in the NAMD simulations for complex molecular systems in the future, considering its good balance between accuracy and computational cost.

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It is well-known that highly reactive hydroxyl radicals (HO • ) can be produced by the classic Fenton system and our recently discovered haloquinone/H 2 O 2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO • can be generated from H 2 O 2 and thiourea dioxide (TUO 2 ), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO • production. Taken together, we proposed that the molecular mechanism for HO • generation by TUO 2 /H 2 O 2 : TUO 2 tautomerizes from sulfinic acid into ketone isomer (TUO 2 -K) through proton transfer, then a nucleophilic addition of H 2 O 2 on the S atom of TUO 2 -K, forming a S-hydroperoxide intermediate TUO 2 -OOH, which dissociates homolytically to produce HO • . Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO • production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

In vivo bioimaging using shortwave infrared (SWIR) (1000-2000 nm) molecular dyes enables deeper penetration and higher contrast compared to visible and near-infrared-I (NIR-I, 700-900 nm) dyes. Developing new SWIR molecules is still quite challenging. This study developed SRHCYs, a panel of fluorescent dyes based on hemicyanine, with adjustable absorbance (830-1144 nm) and emission (886-1217 nm) wavelength. The photophysical attributes of these dyes are precisely tailored by strengthening the donor parts and extending polymethine chains. SRHCY-3, with its clickable azido group, was chosen for high-performance imaging of blood vessels in living mice, enabling the precise detection of brain and lung cancer. The combination of these probes achieved in vivo multicolor imaging with negligible optical crosstalk. This report presents a series of SWIR hemicyanine dyes with promising spectroscopic properties for high-contrast bioimaging and multiplexing detection.