Quantum yield enhancement of firefly bioluminescence with biomolecular condensates.

The enzymatic luminescence reactions of fireflies are accelerated in the presence of biomolecular condensates comprising a positively charged peptide and ATP. We revealed that this acceleration is caused by the enrichment of reaction elements, local pH changes, and promotion of inhibitory intermediate dissociation, improving the bioluminescence quantum yield by approximately 10%.

Spectrochemistry of Firefly Bioluminescence.

The chemical reactions underlying the emission of light in fireflies and other bioluminescent beetles are some of the most thoroughly studied processes by scientists worldwide. Despite these remarkable efforts, fierce academic arguments continue around even some of the most fundamental aspects of the reaction mechanism behind the beetle bioluminescence. In an attempt to reach a consensus, we made an exhaustive search of the available literature and compiled the key discoveries on the fluorescence and chemiluminescence spectrochemistry of the emitting molecule, the firefly oxyluciferin, and its chemical analogues reported over the past 50+ years. The factors that affect the light emission, including intermolecular interactions, solvent polarity, and electronic effects, were analyzed in the context of both the reaction mechanism and the different colors of light emitted by different luciferases. The collective data points toward a combined emission of multiple coexistent forms of oxyluciferin as the most probable explanation for the variation in color of the emitted light. We also highlight realistic research directions to eventually address some of the remaining questions related to firefly bioluminescence. It is our hope that this extensive compilation of data and detailed analysis will not only consolidate the existing body of knowledge on this important phenomenon but will also aid in reaching a wider consensus on some of the mechanistic details of firefly bioluminescence.

Intrinsic fluorescence from firefly oxyluciferin monoanions isolated in vacuo.

Fireflies, click beetles, and railroad worms glow in the dark. The color varies from green to red among the insects and is associated with an electronically excited oxyluciferin formed catalytically by the luciferase enzyme. The actual color tuning mechanism has been, and still is, up for much debate. One complication is that oxyluciferin can occur in different charge states and isomeric forms. We present here emission spectra of oxyluciferin monoanions in vacuo at both room temperature and at 100 K recorded with a newly developed and unique mass-spectroscopy setup specially designed for gas-phase ion fluorescence spectroscopy. Ions are limited to the phenolate-keto and phenolate-enol forms that account for natural bioluminescence. At 100 K, fluorescence band maxima are at 599 ± 2 nm and 563 ± 2 nm for the keto and enol forms, respectively, and at 300 K about 5 nm further to the red. The bare-ion spectra, free from solvent effects, serve as important references as they reveal whether a protein microenvironment redshifts or blueshifts the emission, and they serve as important benchmarks for nontrivial excited-state calculations.

Shining light on the electronic structure and relaxation dynamics of the isolated oxyluciferin anion.

Firefly bioluminescence is exploited widely in imaging in the biochemical and biomedical sciences; however, our fundamental understanding of the electronic structure and relaxation processes of the oxyluciferin that emits the light is still rudimentary. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate the electronic structure and relaxation of a series of model oxyluciferin anions. We find that changing the deprotonation site has a dramatic influence on the relaxation pathway following photoexcitation of higher lying electronically excited states. The keto form of the oxyluciferin anion is found to undergo internal conversion to the fluorescent S1 state, whereas we find evidence to suggest that the enol and enolate forms undergo internal conversion to a dipole bound state, possibly via the fluorescent S1 state. Partially resolved vibrational structure points towards the involvement of out-of-plane torsional motions in internal conversion to the dipole bound state, emphasising the combined electronic and structural role that the microenvironment plays in controlling the electronic relaxation pathway in the enzyme.

Elucidating the multi-configurational character of the firefly dioxetanone anion and its prototypes in the biradical region using full valence active spaces.

We analyzed the near-degenerate states of the firefly dioxetanone anion (FDO-) and its prototypes, especially in the biradical region, using multi-configurational approaches. The importance of utilizing full valence active spaces by means of density-matrix renormalization group self-consistent field (DMRG-SCF) calculations was described. Our results revealed that the neglect of some valence orbitals can affect the quantitative accuracy in later multi-reference calculations or the qualitative conclusion when optimizing conical intersections. Using all of the relevant valence orbitals of FDO-, we confirmed that there were two conical intersections, as reported in previous work, and that the intersecting states were changed when the active space was enlarged. Beyond these, we found that there were strong interactions between states in the biradical regions, in which the changes in entanglements can be used to visualize the interacting state evolution.

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis, Condensation and Chiral Inversion.

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.

Effect of Protein Conformation and AMP Protonation State on Fireflies' Bioluminescent Emission.

The emitted color in fireflies' bioluminescent systems depends on the beetle species the system is extracted from and on different external factors (pH, temperature…) among others. Controlling the energy of the emitted light (i.e., color) is of crucial interest for the use of such bioluminescent systems. For instance, in the biomedical field, red emitted light is desirable because of its larger tissue penetration and lower energies. In order to investigate the influence of the protein environment and the AMP protonation state on the emitted color, the emission spectra of the phenolate-keto and phenolate-enol oxyluciferin forms have been simulated by means of MD simulations and QM/MM calculations, considering: two different protein conformations (with an open or closed C-terminal domain with respect to the N-terminal) and two protonation states of AMP. The results show that the emission spectra when considering the protein characterized by a closed conformation are blue-shifted compared to the open conformation. Moreover, the complete deprotonation of AMP phosphate group (AMP2-) can also lead to a blue-shift of the emission spectra but only when considering the closed protein conformation (open form is not sensitive to changes of AMP protonation state). These findings can be reasoned by the different interactions (hydrogen-bonds) found between oxyluciferin and the surrounding (protein, AMP and water molecules). This study gets partial insight into the possible origin of the emitted color modulation by changes of the pH or luciferase conformations.

The role of solvation models on the computed absorption and emission spectra: the case of fireflies oxyluciferin.

Surrounding effects are crucial to successfully simulate the absorption and emission spectra of molecular systems. In this work we test different solvation models to compute transition energies and to simulate the spectra of oxyluciferin responsible for the light emission in fireflies and its derivatives. We demonstrate that, within the PCM model, the IBSF formalism is suitable for computing the transition energies of the oxyluciferin chemical forms characterized by a charge transfer character. On the other hand, the LR approach could be used for the chemical forms where an almost negligible charge transfer takes place. Moreover, we demonstrate that explicit solvation models, applied by QM/MM calculations, are needed to accurately reproduce the experimental shape of the spectra. Finally, the vibrationally resolved spectra using a solvation model (implicit or microsolvation) is computed. Some noticeable differences arise when considering the implicit solvation with respect to gas phase vibrational spectra, while small changes were found when explicit water molecules within a microsolvated model are considered.

Simulation and Analysis of the Spectroscopic Properties of Oxyluciferin and Its Analogues in Water.

Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions. In this work, we present the simulation of the absorption and emission spectra of the possible natural oxyluciferin forms in water and some of their analogues considering both the solvent/oxyluciferin interactions and the dynamical effects by using MD simulations and QM/MM methods. On the one hand, the absorption band shapes have been rationalized by analyzing the electronic nature of the transitions involved. On the other hand, the simulated and experimental emission spectra have been compared. In this case, an ultrafast excited state proton transfer (ESPT) occurs in oxyluciferin and its analogues, which impairs the detection of the emission from the protonated state by steady-state fluorescence spectroscopy. Transient absorption spectroscopy was used to evidence this ultrafast ESPT and rationalize the comparison between simulated and experimental steady-state emission spectra. Finally, this work shows the suitability of the studied oxyluciferin analogues to mimic the corresponding natural forms in water solution, as an elegant way to block the desired interexchange reactions allowing the study of each oxyluciferin form separately.

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging.

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC).

The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed 'bimolecular complementation' relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins. When these proteins interact, they effectively reconstitute the activity of the molecular reporter (typically leading to increased fluorescence or luminescence). This unit describes the selection and development of bimolecular luminescence complementation (BiLC) assays for reporting intracellular PPIs, and provides examples in which BiLC was used to identify small molecules that can modulate PPIs. © 2017 by John Wiley & Sons, Inc.

Bioluminescence emissions of female fireflies of the species Luciola praeusta.

Numerous investigations have been carried out on bioluminescence emissions from male fireflies. However, very few observations have been made on the emitted light from female specimens. Even in those, apart from observing responses from females to courtship flashes from conspecific males, detailed studies have not been performed. Here we present a first report on the light of female fireflies of the Indian species Luciola praeusta Kiesenwetter 1874 (Coleoptera:Lampyridae:Luciolinae). In the steady-state emission spectrum over the temperature range of 20-40°C, the peak wavelength is the same as, while the full width at half maximum is larger than, that of a male specimen of this species. Increase in temperature up to 45°C brings out a change in both the peak and FWHM values, shifting towards red. In the time-resolved measurement, duration of a flash, which is noticeably larger than that of a male, is found to decrease exponentially with temperature at 20-40°C. Further increase in the temperature produces a minimum flash duration at 41.5°C, and beyond this causes a considerable increase in duration for small increase in temperature. Additionally, lowering the temperature below 20°C makes a single flash appear as a combination of two or three flashes.

Identification and characterization of the Luc2-type luciferase in the Japanese firefly, Luciola parvula, involved in a dim luminescence in immobile stages.

Nocturnal Japanese fireflies, Luciola parvula, emit from their lanterns a yellow light, one of the most red-shifted colors found among fireflies. Previously, we isolated and characterized two different types of luciferase gene, Luc1 and Luc2, from the fireflies Luciola cruciata and Luciola lateralis; Luc1 is responsible for the green-yellow luminescence of larval and adult lanterns, whereas Luc2 is responsible for the dim greenish glow of eggs and pupal bodies. The biological role of firefly lanterns in adults is related to sexual communication, but why the eggs and pupae glow remains uncertain. In this study, we isolated the gene Luc2 from L. parvula, and compared its expression profiles and enzymatic characteristics with those of Luc1. A semi-quantitative reverse transcription polymerase chain reaction showed that Luc1 was predominantly expressed in larvae, prepupae, pupae and adults, whereas Luc2 was expressed in eggs, prepupae, pupae and adult females. Enzymatic analyses showed that the luminescent color of Luc1 matches the visual sensitivity of L. parvula eyes, whereas that of Luc2 is very different from it. These results suggest that the biological role of Luc2 expressed in immobile stages is not intraspecific communication.

Increase of segmental mobility through insertion of a flexible linker in split point of firefly luciferase.

The crystal structure of Photinus pyralis luciferase shows a unique molecular architecture consisting of a large N-terminal domain and a small C-terminal domain which is separated by a wide cleft. Protein engineering methods attempts to design the peptide linkers that make a connection between different protein domains or subunits to allow for separating domains and improve kinetics and structural features of proteins. In regard to this; introduction of a flexible linker at split point of luciferase which has a strong self-association activity, may leads to conformational change and improve general flexibility of protein. In this study, two flexible linkers in the split point of luciferase are introduced in order to test the effect of linker on flexibility of luciferase activity. Glycine-rich linkers are introduced into P. pyralis firefly luciferase to make two separate mutant enzymes. Enzymatic properties of mutant and native forms were measured using luminescence assay. Results show that lengthening of luciferase domains through insertion of a flexible linker did not affect bioluminescence emission spectra. Also adding linkers do not have remarkable effect on thermostability. The Km values of mutants were increased compared to native form, indicating lower affinity of mutants toward substrates.

In vivo bioluminescence emissions of the firefly Luciola praeusta at low temperatures.

Dependences of light emission from fireflies on external factors like temperature and magnetic field have been studied in recent times. Interesting conclusions have been drawn and hypotheses put forward in those studies. Here we report steady-state and time-resolved emissions of the Indian species of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae) at temperatures below 20°C. Intensity profiles of emission spectra remain the same as those recorded at normal or high temperatures. Two-flash combinations are frequently formed, giving the appearance of the resolution of a simple flash into two. Simple flashes also become abnormally broad with no uniformity in the increase of their durations. The flashes obtained from fireflies at low temperatures are compared and contrasted with the ones under a strong static magnetic field.

Mechanistic insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea firefly) lumophore.

Cypridina hilgendorfii (sea firefly) is a bioluminescent crustacean whose bioluminescence (BL) reaction is archetypal for a number of marine organisms, notably other bioluminescent crustaceans and coelenterates. Unraveling the mechanism of its BL is paramount for future applications of its strongly emissive lumophore. Cypridina produces light in a three-step reaction: First, the cypridinid luciferin is activated by an enzyme to produce a peroxide intermediate, cypridinid dioxetanone (CDO), which then decomposes to generate excited oxyluciferin (OxyCLnH*). Finally, OxyCLnH* deexcites to its ground state along with emission of bright blue light. Unfortunately, the detailed mechanism of the critical step, the thermolysis of CDO, remains unknown, and it is unclear whether the light emitter is generated from a neutral form (CDOH) or anionic form (CDO(-)) of the CDO precursor. In this work, we investigated the key step in the process by modeling the thermal decompositions of both CDOH and CDO(-). The calculated results indicate that the decomposition of CDO(-) occurs via the gradually reversible charge transfer (CT)-initiated luminescence (GRCTIL) mechanism, whereas CDOH decomposes through an entropic trapping mechanism without an obvious CT process. The thermolysis of CDO(-) is sensitive to solvent effects and is energetically favorable in polar environments compared with the thermolysis of CDOH. The thermolysis of CDO(-) produces the excited oxyluciferin anion (OxyCLn(-)*), which combines with a proton from the environment to form OxyCLnH*, the actual light emitter for the natural system.

The Theoretical Estimation of the Bioluminescent Efficiency of the Firefly via a Nonadiabatic Molecular Dynamics Simulation.

The firefly is famous for its high bioluminescent efficiency, which has attracted both scientific and public attention. The chemical origin of firefly bioluminescence is the thermolysis of the firefly dioxetanone anion (FDO(-)). Although considerable theoretical research has been conducted, and several mechanisms were proposed to elucidate the high efficiency of the chemi- and bioluminescence of FDO(-), there is a lack of direct experimental and theoretical evidence. For the first time, we performed a nonadiabatic molecular dynamics simulation on the chemiluminescent decomposition of FDO(-) under the framework of the trajectory surface hopping (TSH) method and theoretically estimated the chemiluminescent quantum yield. The TSH simulation reproduced the gradually reversible charge-transfer initiated luminescence mechanism proposed in our previous study. More importantly, the current study, for the first time, predicted the bioluminescence efficiency of the firefly from a theoretical viewpoint, and the theoretical prediction efficiency is in good agreement with experimental measurements.

Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

What Exactly Is the Light Emitter of a Firefly?

Firefly bioluminescence attracts people by its glaring beauty and fascinating applications, but what is the light emitter of a firefly? The answer to this question has been explored since before the 1960s. The unanimously accepted answer is that excited-state oxyluciferin is the light emitter. The complexity of this question arises from the existence of six chemical forms (keto, enol, keto-1, enol-1, enol-1', and enol-2) of oxyluciferin. After decades of experimental and theoretical efforts, a consistent conclusion was almost reached in 2011: excited-state keto-1 is the only light emitter in fireflies. However, the debate is raised again by the latest in vitro experimental results. This study will solve this contradiction via hybrid quantum mechanics and molecular mechanics (QM/MM) calculations combined with molecular dynamics (MD). The calculations were performed in the real protein for the six chemical forms of oxyluciferin and their corresponding analogues employed in the latest experiments. By considering the real environment, the pH value, and a possible equilibrium of the chemical forms of oxyluciferin in vivo, the calculated results indicate that the main emitter is still the excited-state keto-1 form.

Theoretical study of the nontraditional enol-based photoacidity of firefly oxyluciferin.

A theoretical analysis of the enol-based photoacidity of oxyluciferin in water is presented. The basis for this phenomenon is found to be the hydrogen-bonding network that involves the conjugated photobase of oxyluciferin. The hydrogen-bonding network involving the enolate thiazole moiety is stronger than that of the benzothiazole phenolate moiety. Therefore, enolate oxyluciferin should be stabilized versus the phenolate anion. This difference in strength is attributed to the fact that the thiazole moiety has more potential hydrogen-bond acceptors near the proton donor atom than the benzothiazole moiety. Moreover, the phenol-based excited-state proton transfer leads to a decrease in the hydrogen-bond acceptor potential of the thiazole atoms. The ground-state enol-based acidity of oxyluciferin is also studied. This phenomenon can be explained by stabilization of the enolate anion through strengthening of a bond between water and the nitrogen atom of the thiazole ring, in an enol-based proton-transfer-dependent way.