Bioluminescence Process Research Articles

Exploring the Light-Emitting Agents in Renilla Luciferases by an Effective QM/MM Approach.

Bioluminescence is a fascinating natural phenomenon, wherein organisms produce light through specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc) derived from the sea pansy Renilla reniformis is notable for its blue light emission and has potential applications in bioluminescent tagging. Our study focuses on RLuc8, a variant of RLuc with eight amino acid substitutions. Recent studies have shown that the luminescent emitter coelenteramide can adopt multiple protonation states, which may be influenced by nearby residues at the enzyme's active site, demonstrating a complex interplay between protein structure and bioluminescence. Herein, using the quantum mechanical consistent force field method and the semimacroscopic protein dipole-Langevin dipole method with linear response approximation, we show that the phenolate state of coelenteramide in RLuc8 is the primary light-emitting species in agreement with experimental results. Our calculations also suggest that the proton transfer (PT) from neutral coelenteramide to Asp162 plays a crucial role in the bioluminescence process. Additionally, we reproduced the observed emission maximum for the amide anion in RLuc8-D120A and the pyrazine anion in the presence of a Na+ counterion in RLuc8-D162A, suggesting that these are the primary emitters. Furthermore, our calculations on the neutral emitter in the engineered AncFT-D160A enzyme, structurally akin to RLuc8-D162A but with a considerably blue-shifted emission peak, aligned with the observed data, possibly explaining the variance in emission peaks. Overall, this study demonstrates an effective approach to investigate chromophores' bimolecular states while incorporating the PT process in emission spectra calculations, contributing valuable insights for future studies of PT in photoproteins.

Behind the glow: unveiling the nature of NanoLuc reactants and products.

Due to the largely recognized utility of bioluminescence in many fields, a wide variety of luciferase-luciferin systems have been investigated in order to find the best-suited for a number of different applications. The collected knowledge has allowed the identification of a few necessary, or at least desirable, properties, such as bright luminescence, low background signal and small dimension of the enzyme that must exhibit structural stability at operating conditions. The NanoLuc-furimazine pair seems to meet all these requirements, but the mechanism of the reaction and the characteristics of the species responsible for the emission remain unknown. The aim of this study is to identify the luminescent product among the possible forms of oxidized furimazine and to understand how the chemical form and structure of the system, before and after the oxidation, are involved into the reaction mechanism and determine emission. To do this, we consider two possible forms of furimazine, the keto and the enol one, and test which of them is the most plausible candidate in the bioluminescence process on the basis of enzyme-substrate interactions from docking calculations. A similar procedure is repeated for three possible forms of the furimamide luminescent product, and their properties in the protein environment are then evaluated via QM/MM calculations. In contrast with previous indications, our simulations well support the involvement of the enol form of furimazine as reagent and point to the zwitterionic forms of furimamide as emissive species.

Bioluminescence in cephalopods: biodiversity, biogeography and research trends

Numerous terrestrial and marine organisms, including cephalopods, are capable of light emission. In addition to communication, bioluminescence is used for attraction and defense mechanisms. The present review aims to: (i) present updated information on the taxonomic diversity of luminous cephalopods and morphological features, (ii) describe large-scale biogeographic patterns, and (iii) show the research trends over the last 50 years on cephalopod bioluminescence. According to our database (834 species), 32% of all known cephalopod species can emit light, including oegopsid and myopsid squids, sepiolids, octopuses, and representatives of several other smaller orders (bathyteuthids, and the monotypic vampire “squid”, Vampyroteuthis infernalis and ram’s horn “squid”, Spirula spirula). Most species have a combination of photophores present in different locations, of which light organs on the head region are dominant, followed by photophores associated with the arms and tentacles and internal photophores. Regarding the biogeographic patterns of cephalopod species with light organs, the most diverse ocean is the Pacific Ocean, followed by the Atlantic and Indian Oceans. The least diverse are the Southern and the Arctic Oceans. Regarding publication trends, our systematic review revealed that, between 1971 and 2020, 277 peer-reviewed studies were published on bioluminescent cephalopods. Most research has been done on a single species, the Hawaiian bobtail squid Euprymna scolopes. The interest in this species is mostly due to its species-specific symbiotic relationship with the bacterium Vibrio fischeri, which is used as a model for the study of Eukaryote–Prokaryote symbiosis. Because there are many knowledge gaps about the biology and biogeography of light-producing cephalopods, new state-of-the-art techniques (e.g., eDNA for diversity research and monitoring) can help achieve a finer resolution on species’ distributions. Moreover, knowledge on the effects of climate change stressors on the bioluminescent processes is nonexistent. Future studies are needed to assess such impacts at different levels of biological organization, to describe the potential broad-scale biogeographic changes, and understand the implications for food web dynamics.

Morphological changes in the tracheal system associated with light organs of the firefly Photinus pyralis (Coleoptera: Lampyridae) across life stages.

Oxygen is an important and often limiting reagent of a firefly's bioluminescent chemical reaction. Therefore, the development of the tracheal system and its subsequent modification to support the function of firefly light organs are key to understanding this process. We employ micro-CT scanning, 3D rendering, and confocal microscopy to assess the abdominal tracheal system in Photinus pyralis from the external spiracles to the light organ's internal tracheal brush, a feature named here for the first time. The abdominal spiracles in firefly larvae and pupae are of the biforous type, with a filter apparatus and appear to have an occlusor muscle to restrict airflow. The first abdominal spiracle in the adult firefly is enlarged and bears an occlusor muscle, and abdominal spiracles two through eight are small, with a small atrium and bilobed closing apparatus. Internal tracheal system features, including various branches, trunks, and viscerals, were homologized across life stages. In adults, the sexually dimorphic elaboration and increase in volume associated with tracheal features of luminous segments emphasizes the importance of gas exchange during the bioluminescent process.

Similarities and Differences in Quorum Sensing-Controlled Bioluminescence between Photobacterium phosphoreum T3 and Vibrio qinghaiensis sp.-Q67

Quorum sensing is a density-dependent mechanism using chemical signal molecules termed autoinducers to regulate diverse biological processes in bacteria, including bioluminescence. However, the correlation between growth and light emission of two typical luminescent bacteria, Photobacterium phosphoreum T3 and Vibrio qinghaiensis sp.-Q67, is still unclear. This study investigates the variations of bioluminescence and the light-emission-involved gene expression of the above two strains, respectively, showing that bioluminescence is population density-dependent. Furthermore, the effect of crude extracts (175, 350, 700 and 1750 mg/L) from the bacterial culture that contains the potential autoinducers on the bioluminescence is explored. At the exponential and the early stationary growth phase, T3 did not exhibit an obvious light intensity and cell density change after adding crude extracts at 175 and 350 mg/L, while the light intensity decreased at 700 and 1750 mg/L, showing a luminescence inhibition. For Q67, the light intensity increased dramatically with crude extract concentration. These results suggest that the bioluminescence process of both T3 and Q67 is controlled by quorum sensing. Furthermore, the different response modes of these two strains to autoinducers imply that the two strains could be applied to different compounds for toxicity assesses.

Bacterial bioluminescence assay for bioanalysis and bioimaging.

Bioluminescence occurs through a chemical reaction in organisms that spontaneously produce light. Luminescent bacteria are unique among bioluminescent organisms. Their bioluminescence intensity is an indicator of their metabolic activity, which can directly reflect the influence of environmental factors on cell viability. Moreover, the whole bioluminescence process is totally gene encoded without the addition of extra substrates. As a result, bacterial bioluminescence has been a powerful tool for whole-cell biosensors and bio-reporters in bioanalysis and bioimaging. This review aims to cover the applications of wild-type and recombinant luminescent bacteria to detect the toxicity of environmental pollutants and biological molecules. The bacterial bioluminescence analytical assay has characteristics such as high sensitivity, short-term detection, and easy operation. Meanwhile, due to the development of gene engineering and optical technology, bacterial luciferase as a reporter protein has been successfully expressed in prokaryotic and eukaryotic cells, tissues, and organs of animals. The major applications for bacterial luciferase-based bioluminescence imaging, such as infectious diseases, cancer therapy, and stem cell tracing, are discussed in this review.

Exposure of Metal Oxide Nanoparticles on the Bioluminescence Process of Pu- and Pm-lux Recombinant P. putida mt-2 Strains.

Comparison of the effects of metal oxide nanoparticles (NPs; CuO, NiO, ZnO, TiO2, and Al2O3) on different bioluminescence processes was evaluated using two recombinant (Pm-lux and Pu-lux) strains of Pseudomonas putida mt-2 with same inducer exposure. Different sensitivities and responses were observed according to the type of NPs and recombinant strains. EC50 values were determined. The negative effects on the bioluminescence activity of the Pm-lux strain was greater than for the Pu-lux strains for all NPs tested. EC50 values for the Pm-lux strain were 1.7- to 6.2-fold lower (corresponding to high inhibition) than for Pu-lux. ZnO NP caused the greatest inhibition among the tested NPs in both strains, showing approximately 11 times less EC50s of CuO, which appeared as the least inhibited. Although NPs showed different sensitivities depending on the bioluminescence process, similar orders of EC50s for both strains were observed as follows: ZnO > NiO, Al2O3 > TiO2 > CuO. More detailed in-depth systematic approaches, including in the field of molecular mechanisms, is needed to evaluate the accurate effect mechanisms involved in both bioluminescence metabolic processes.

Optimized methodology for obtention of high-yield and -quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna-like vegetation) in Northeast Brazil, especially in Piauí State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and -quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy® kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of ~130% in comparison to the RNeasy® method and of ~40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy® method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and -yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes.

A QM/MM Study on the Initiation Reaction of Firefly Bioluminescence-Enzymatic Oxidation of Luciferin.

Among all bioluminescent organisms, the firefly is the most famous, with a high luminescent efficiency of 41%, which is widely used in the fields of biotechnology, biomedicine and so on. The entire bioluminescence (BL) process involves a series of complicated in-vivo chemical reactions. The BL is initiated by the enzymatic oxidation of luciferin (LH2). However, the mechanism of the efficient spin-forbidden oxygenation is far from being totally understood. Via MD simulation and QM/MM calculations, this article describes the complete process of oxygenation in real protein. The oxygenation of luciferin is initiated by a single electron transfer from the trivalent anionic LH2 (L3−) to O2 to form 1[L•2−…O2•−]; the entire reaction is carried out along the ground-state potential energy surface to produce the dioxetanone (FDO−) via three transition states and two intermediates. The low energy barriers of the oxygenation reaction and biradical annihilation involved in the reaction explain this spin-forbidden reaction with high efficiency. This study is helpful for understanding the BL initiation of fireflies and the other oxygen-dependent bioluminescent organisms.

Bioluminescence of the Largest Luminous Vertebrate, the Kitefin Shark, Dalatias licha: First Insights and Comparative Aspects

Bioluminescence has often been seen as a spectacular yet uncommon event at sea but considering the vastness of the deep sea and the occurrence of luminous organisms in this zone, it is now more and more obvious that producing light at depth must play an important role structuring the biggest ecosystem on our planet. Three species of deepwater sharks (Dalatias licha,Etmopterus lucifer, andEtmopterus granulosus) were collected from the Chatham Rise, off New Zealand, and for the first time, we documented their luminescence. Comparison of glowing shark pictures, combined with histological description of light organs and hormonal control analysis, highlight the evolutive conservation of the bioluminescence process within Dalatiidae and Etmopteridae. A special emphasis is placed on the luminescence ofD. licha, the largest known luminous vertebrate. This first experimental study of three luminous shark species from New Zealand provides an insight into the diversity of shark bioluminescence and highlights the need for more research to help understand these unusual deep-sea inhabitants: the glowing sharks.

Chemistry in Fungal Bioluminescence: A Theoretical Study from Luciferin to Light Emission.

As the only genetically encodable bioluminescent system among eukaryotes to date, bioluminescent fungi can unceasingly emit green light for days. Cross-reactions among four lineages of luminescent fungi suggest that all of them share a common bioluminescent mechanism. A series of excellent experiments by Yampolsky's group have revealed the key components in fungal bioluminescence (BL) from luciferin to light emission. However, the detailed underlying mechanism and processes remain unknown. By performing multireference and (time dependent) density functional theory calculations, we clearly described the bioluminescent process at the molecular and electronic state level. The fungal BL is initiated by the cycloaddition of O2 to luciferin to form an α-pyrone endoperoxide high-energy intermediate (II). This oxygenation is not initiated by a single-electron transfer as it is in firefly BL, but it is explained by a charge transfer followed by a spin inversion mechanism. The thermolysis of II generates oxyluciferin at the first singlet excited state (S1) through a zwitterion intermediate (III). A chemical form of the S1-state oxyluciferin, E-V(3)*, has the potential to be a light emitter. The current theoretical calculation provides great detail for deeply understanding the chemical processes in fungal BL and in chemiluminescence involving α-pyrone endoperoxide.

Cyber Firefly Algorithm Based on Adaptive Memory Programming for Global Optimization

Recently, two evolutionary algorithms (EAs), the glowworm swarm optimization (GSO) and the firefly algorithm (FA), have been proposed. The two algorithms were inspired by the bioluminescence process that enables the light-mediated swarming behavior for mating or foraging. From our literature survey, we are convinced with much evidence that the EAs can be more effective if appropriate responsive strategies contained in the adaptive memory programming (AMP) domain are considered in the execution. This paper contemplates this line and proposes the Cyber Firefly Algorithm (CFA), which integrates key elements of the GSO and the FA and further proliferates the advantages by featuring the AMP-responsive strategies including multiple guiding solutions, pattern search, multi-start search, swarm rebuilding, and the objective landscape analysis. The robustness of the CFA has been compared against the GSO, FA, and several state-of-the-art metaheuristic methods. The experimental result based on intensive statistical analyses showed that the CFA performs better than the other algorithms for global optimization of benchmark functions.

Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase.

Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.

Why does the bioluminescent fungus Armillaria mellea have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin-the key component of fungal bioluminescent system-was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.

Bioluminescence of Firefly Squid via Mechanism of Single Electron-Transfer Oxygenation and Charge-Transfer-Induced Luminescence

Watasenia scintillans (W.scintillans) is a deep-sea luminescent squid with a popular name of firefly squid. It produces flashes of blue light via a series of complicated luciferin-luciferase reactions involving ATP, Mg2+, and molecular oxygen. Tsuji has proposed a hypothetical scheme for this mysterious bioluminescence (BL) process, but the proposal is short of strong evidence experimentally or theoretically, especially for two key steps. They are the addition of molecular oxygen to luciferin and the formation of light emitter. For the first time, the present study investigates the two steps by reliable density functional theory (DFT) and time-dependent DFT. The results of calculated energetics, charge transfer process, electronic structures, and molecular dynamics give convincing support for Tsuji's proposal. The oxygenation reaction occurs with a single electron-transfer (SET) mechanism, and the light emitter is produced via the mechanism of gradually reversible charge-transfer-induced luminescence (GRCTIL). The simulation of nonadiabatic molecular dynamics further confirms the GRCTIL mechanisms and evaluates the quantum yield of the light emitter to be 43%. The knowledge obtained in the current study will help to understand a large amount of BL systems in nature, since the core structure of W.scintillans luciferin, imidazopyrazinone, is common in the luciferins of about eight phyla of luminescent organisms.

A combined theoretical and experimental study on the mechanism of spiro-adamantyl-1,2-dioxetanone decomposition

Our study on the unimolecular decomposition of a relatively stable 1,2-dioxetanone derivative, model compound for bioluminescence processes, indicates the existence of different reaction pathways for ground and excited state formation.

Fast and high resolution single-cell BRET imaging.

Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways.

On the mechanism of biological activation by tritium

The mechanism of biological activation by beta-emitting radionuclide tritium was studied. Luminous marine bacteria were used as a bioassay to monitor the biological effect of tritium with luminescence intensity as the physiological parameter tested. Two different types of tritium sources were used: HTO molecules distributed regularly in the surrounding aqueous medium, and a solid source with tritium atoms fixed on its surface (tritium-labeled films, 0.11, 0.28, 0.91, and 2.36 MBq/cm2). When using the tritium-labeled films, tritium penetration into the cells was prevented. The both types of tritium sources revealed similar changes in the bacterial luminescence kinetics: a delay period followed by bioluminescence activation. No monotonic dependences of bioluminescence activation efficiency on specific radioactivities of the films were found. A 15-day exposure to tritiated water (100 MBq/L) did not reveal mutations in bacterial DNA. The results obtained give preference to a “non-genomic” mechanism of bioluminescence activation by tritium. An activation of the intracellular bioluminescence process develops without penetration of tritium atoms into the cells and can be caused by intensification of trans-membrane cellular processes stimulated by ionization and radiolysis of aqueous media.

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.

Chemiluminescence efficiency of catalyzed 1,2-dioxetanone decomposition determined by steric effects.

The chemiluminescent decomposition of 1,2-dioxetanones (α-peroxylactones), catalyzed by an appropriate fluorescent activator, is an important simple model for efficient bioluminescent transformations. In this work, we report experimental data on the catalyzed decomposition of two spiro-substituted 1,2-dioxetanone derivatives, which support the occurrence of an intermolecular electron transfer from the activator to the peroxide. The low efficiency of the studied systems is associated with steric hindrance during the chemiexcitation sequence, rationalized using the concept of supermolecule formation between the peroxide and the catalyst. This approach explains the difference in the chemiexcitation efficiencies in the decomposition of four-membered cyclic peroxide derivatives: 1,2-dioxetanes, 1,2-dioxetanones, and 1,2-dioxetanedione (the intermediate in the peroxyoxalate reaction), which are the most important model compounds for excited-state formation in chemiluminescence and bioluminescence processes.