Mechanically Assisted Bioluminescence with Natural Luciferase

Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion‐controlled in the viscous matrix. The results suggest that luciferases could be used as light‐emissive reporters of mechanoenzymatic reactions.     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Current Status of Research on Fungal Bioluminescence: Biochemistry and Prospects for Ecotoxicological Application

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H‐dependent reductase and a membrane‐bound luciferase in a two‐step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross‐reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.     

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3‐hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3‐hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.     

Toxicity Assessment of Cyanide and Tetramethylene Disulfotetramine （Tetramine） Using Luminescent Bacteria Vibrio-qinghaiensis and PbO= Electrochemical Sensor

The toxicities of cyanide and tetramethylene disulfotetramine （tetramine） were evaluated by two methods of luminescent bacteria and PbO2 electrochemical sensor. Vibrio-qinghaiensis, a kind of luminescent bacteria, can produce bioluminescence and the bioluminescence was decreased with the addition of toxicants. The toxicities of cyanide and tetrarnine were expressed as 10 min-EC50 value, which was the concentration of chemical that reduces the light output by 50% after contact for 10 min. Nano PbO2 modified electrode, a rapid toxicity determination method was also described in this work. By the PbO2 modified electrode, the current responses of Escherichia coli （E. coli） were changed with the addition of toxicants. The value of 10 min-EC50 was also provided with the PbO2 electrochemical sensor. Compared with the 10 min-EC50 and detection limits （38.38 and 0.60 μg/mL for cyanide, 0.24 and 0.02 μg/mL for tetramine） with luminescent bacteria, the PbO2 sensor provided a simple and convenient method with lower 10 min-EC50 and detection limits （26.37 and 0.52 μg/mL for cyanide, 0.21 and 0.01 μg/mL for tetramine） and fast response time.     

SOME SPECTRAL CHARACTERISTICS OF THE LIGHT EMITTING SYSTEM OF THE POLYNOID WORMS

Abstract— The bioluminescence emission spectrum of the light emitted from the elytra of certain poly-noid worms is similar to the fluorescence of the scales and to the fluorescence of flavins. In extracts the fluorescence is confined to the particulate fraction. Stimulation of bioluminescence from such particles was observed upon the addition of Fe²⁺ ions.     

Cobalt nanoparticles anchored to porous silicon as a novel modifier for the construction of enzyme‐free hydrogen peroxide screen‐printed sensor

In this work, a screen‐printed carbon electrode (SPCE) was modified with a cobalt/porous silicon (Co@PSi) nanocomposite powder to develop a nonenzymatic sensor for the detection of hydrogen peroxide. The Co@PSi nanocomposite was synthesized through the chemical reaction between silicon powder in a HF/HNO₃ solution and cobalt cations. In this process, cobalt nanoparticles were anchored on the porous silicon. The structure and morphology of the synthesized nanocomposite were investigated by X‐ray diffraction, Fourier transform infrared spectroscopy, X‐ray photoemission spectroscopy, energy dispersive X‐ray spectroscopy, and field‐emission scanning electron microscopy. The constructed nonenzymatic, screen‐printed sensors based on the Co@PSi nanocomposite showed perfect electrocatalytic oxidation response to hydrogen peroxide over the range 1–170 and 170–3,770 μmol/L with the limit of detection of 0.8 μmol/L. In addition, the Co@PSi‐SPCE sensor exhibited good selectivity for the determination of H₂O₂ in the presence of common interfering species including glucose, ascorbic acid, uric acid, dopamine, nitrate, and nitrite ions. The constructed electrochemical sensor was successfully used for the determination of H₂O₂ in real samples.     

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Unlike the enchanting yellow‐green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H‐bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.     

Synthesis and bioluminescence of electronically modified and rotationally restricted colour-shifting infraluciferin analogues

Synthetic nIR emitting luciferins can enable clearer bioluminescent imaging in blood and tissue. A limiting factor for all synthetic luciferins is their reduced light output with respect to D-luciferin. In this work we explore a design feature of whether rigidification of an exceptionally red synthetic luciferin, infraluciferin, can increase light output through a reduction in the degrees of freedom of the molecule. A rigid analogue pyridobenzimidazole infraluciferin was prepared and its bioluminescence properties compared with its non-rigid counterpart benzimidazole infraluciferin, luciferin, infraluciferin and benzimidazole luciferin. The results support the concept that synthetic rigidification of π-extended luciferins can increase bioluminescence activity while maintaining nIR bioluminescence.     

Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc‐1 (s ulfinate e ster luc iferin), a chemoselective probe exhibiting a ratiometric and turn‐on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the “caged” luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc‐1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS‐7 cells and human breast adenocarcinoma MCF‐7 cells.     

Computational Investigation of the Effect of pH on the Color of Firefly Bioluminescence by DFT

In spite of recent advances towards understanding the mechanism of firefly bioluminescence, there is no consensus about which oxyluciferin (OxyLH₂) species are the red and yellow‐green emitters. The crystal structure of Luciola cruciata luciferase (LcLuc) revealed different conformations for the various steps of the bioluminescence reaction, with different degrees of polarity and rigidity of the active‐site microenvironment. In this study, these different conformations of luciferase (Luc) are simulated and their effects on the different chemical equilibria of OxyLH₂ are investigated as a function of pH by means of density functional theory with the PBE0 functional. In particular, the thermodynamic properties and the absorption spectra of each species, as well as their relative stabilities in the ground and excited states, were computed in the different conformations of Luc. From the calculations it is possible to derive the acid dissociation and tautomeric constants, and the corresponding distribution diagrams. It is observed that the anionic keto form of OxyLH₂ is both the red and the yellow‐green emitter. Consequently, the effect of Luc conformations on the structural and electronic properties of the Keto‐(?1) form are studied. Finally, insights into the Luc‐catalyzed light‐emitting reaction are derived from the calculations. The multicolor bioluminescence can be explained by interactions of the emitter with active‐site molecules, the effects of which on light emission are modulated by the internal dielectric constant of the different conformations. These interactions can suffer also from rearrangement due to entry of external solvent and changes in the protonation state of some amino acid residues and adenosine monophosphate (AMP).     

Fragment molecular orbital investigation of the role of AMP protonation in firefly luciferase pH-sensitivity

Firefly bioluminescence displays a sensitivity to pH changes through an alteration of the energy of the emitted photon leading to yellow-green light above ～pH 6.5 and red light below this value. Calculations using the fragment molecular orbital method have been performed on the active site of the luciferase enzyme from the Japanese firefly Luciola cruciata in order to investigate both the importance of different protonation states and tautomeric forms of the lumophore, oxyluciferin, and the role played by protonation of the active site AMP molecule. The results suggest that whilst an equilibrium between several protonation/tautomeric states of oxyluciferin is possible, a single oxyluciferin species (the phenolate-keto form) may be mostly responsible for both emission colours, with changes in polarization by the active site caused by protonation of the AMP molecule playing an important role in mediating the pH-dependent shift.     

Exploring Bioluminescence Function of the Ca2+‐regulated Photoproteins with Site‐directed Mutagenesis

Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca²⁺‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.     

传感器用镓掺杂氧化锌纳米盘/纳米花状结构催化剂的制备及表面表征

采用简便的旋涂过程和一步水热法在压电基片上制备了Ga掺杂的ZnO纳米薄膜(GZO)。在水热处理过程中,通过添加不同的聚合物可形成纳米盘和纳米花状形貌的薄膜。采用场发射扫描电镜(Fe-SEM)、X射线衍射(XRD)和Raman光谱表征了样品的形貌、微结构和组成。 XRD和FE-SEM结果证明,在AlN/Si压电基片上形成的纳米盘、纳米棒和纳米花状GZO均为纤维锌矿相。采用浸渍法进一步在所制GZO样品上固定了绿色的荧光蛋白质(GFP)。运用原子力显微镜和荧光光谱分析了GFP与GZO表面结合的性质,考察了其用于传感器和生物成像技术的可行性。痕量GFP的固定使该材料产生荧光响应,表明其用于紫外光传感器时具有较好活性。     

Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect

Chimeric proteins were produced using the green light-emitting luciferase of Phrixothrix vivianii (PxGr: lambda max = 548 nm) and the red light-emitting luciferase of Phrixothrix hirtus (PxRe: lambda max = 623 nm). Constructs containing residues 1-344 of the red light-emitting luciferase with residues 345-545 of the green light emitting one emitted red light (PxReGr; lambda max = 613 nm), while the reverse emitted green light (PxGrRe; lambda max = 552 nm). From these results we conclude that the region 1-344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light-emitting luciferase (PxGr) resulted in a approximately 40 nm redshift on the BL spectrum (lambda max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from lambda max = 623 to lambda max = 600 nm) and in PxGr R215S (from lambda max = 585 to lambda max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.     

PHOTOINHIBITION OF STIMULABLE BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

Abstract— Different genera of bioluminescent photosynthetic dinoflagellates exhibit different mechanisms for the inhibition of stimulable bioluminescence during daylight. These are (a) reduction in bioluminescence capacity, (b) increased refractoriness to mechanical stimulation, and (c) inhibition of transmission of signals from mechanical receptor sites to bioluminescence emission sites. The increase in stimulable bioluminescence that in nature mirrors the decrease in sunlight intensity prior to sunset is dependent upon the logarithm of the ambient irradiation intensity. Photoinhibition of bioluminescence in all species examined except Gonyaulax polyedra is the result of absorption of light in the blue region of the spectrum.     

Perspectives on Bioluminescence Mechanisms

The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns–ps) fluorescence spectroscopy, NMR, X‐ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high‐energy species that undergoes decarboxylation to form directly the product in its S₁ state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ “antenna proteins,” lumazine protein and the green‐fluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca²⁺‐regulated photoproteins and the antenna protein interactions.     

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH₂ assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.     

Larval and adult emission spectra of bioluminescence in three European firefly species

We studied the spectral characteristics of the larvae of three sympatric Belgian species of fireflies, Lampyris noctiluca, Phosphaenus hemipterus and Lamprohiza splendidula. An in vivo spectral study was performed to compare bioluminescence spectra. The emission spectrum of a laboratory reared female L. noctiluca was recorded by a different, more exact method. The mean peak wavelength (lambdamax = 546 nm) and shapes of the unimodal emission spectra are visually similar for the larvae of all three species. The emission spectrum of the adult female L. noctiluca peaked in the same range as the larval bioluminescence between 546 and 551 nm. The bandwidth at half-maximum intensity was slightly greater for larval L. noctiluca (77 +/- 4 nm) compared with P. hemipterus (70 +/- 10 nm). The bandwidth of larval L. splendidula (77 +/- 8 nm) was not different compared with the other larvae, whereas the females' bandwidth was somewhat narrower (68 nm). The ecological significance of the color of bioluminescence and conservancy of green emission in larval fireflies and other luminescent beetle larvae is discussed.