MECHANISM OF BIOLUMINESCENCE, CHEMI-LUMINESCENCE AND ENZYME FUNCTION IN THE OXIDATION OF FIREFLY LUCIFERIN*,†

Abstract— The chemical steps and the products of the bioluminescent and chemiluminescent oxidations of firefly luciferin are elucidated. The colors of firefly bioluminescence can be explained in terms of different ionic excited states and spectral shifts due to changes in molecular environment. Firefly luciferase undergoes conformational changes during catalysis. There are two sites for light production per 100,000 mW. A regulatory mechanism involving dehydro-luciferin is proposed for control of firefly flashing.     

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.     

Luciferins, bioluminescent substances

The bioluminescence of the American firefly Photinus is due to the reaction of 2-(6-hydroxybenzothiazol-2-yl)-Δ²-1,3-thiazoline-4-carboxylic acid (“firefly luciferin”) with the enzyme luciferase in the presence of ATP and magnesium ion. In the crustacean Cypridina, on the other hand, the bioluminescence is due to the reaction of a luciferase with 8-(3-guanidinopropyl)-6-indol-3-yl-2-(1-methylpropyl)-3,7-dihydroimidazo[1,2-a]-pyrazin-3-one (“Cypridina luciferin”). The luciferin in Latia is 1,3,3-trimethyl-2-(4-formyloxy-3-methyl-3-butenyl)-1-cyclohexene and that in Renilla is a tryptamine derivative that has not yet been accurately identified; the luciferins of other luminescent organisms are not yet known. A review is given of the investigations which have been carried out on the above luciferins and the course of the luciferin-luciferase reaction is examined. Numerous spectral data obtained during the examination of these compounds are included in the text.     

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.     

Synthesis and evaluation of new caged compound with thiochromone derivative

A new caged firefly luciferin (luciferin) with a thiochromone S,S-dioxide (TSSDO) as a photolabile protecting group was synthesized. Photodeprotection of the caged compound proceeded smoothly under photoirradiation at 365 nm in aqueous solution. The bioluminescence of the regenerated luciferin after uncaging was detected using a typical luciferin–luciferase reaction. These results indicated that TSSDO could be an attractive chemical tool for regulating biological phenomena.     

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

ANALOGS AND DERIVATIVES OF FIREFLY OXYLUCIFERIN, THE LIGHT EMITTER IN FIREFLY BIOLUMINESCENCE

Abstract— The 5-methyl analog of firefly oxyluciferin, two isomeric O-methyl ether derivatives of it and an O, O Ó-dimethyl ether derivative were synthesized and their UV absorption and fluorescence emission spectra were determined. Comparisons of the emission data with the emission wavelength in bioluminescence indicate that the mono-anions of firefly oxyluciferin are candidates for the light-emitters in bioluminescence. Further, we have found that the chemiluminescence of active esters of firefly luciferin produces (from the keto form of oxyluciferin) only red light emission under a variety of conditions; a yellow-green light emission (from the enolic forms of the oxyluciferin product) could not be elicited.     

Toward bioluminescence in the near-infrared region: Tuning the emission wavelength of firefly luciferin analogues by allyl substitution

The synthesis and bioluminescence of allyl-substituted luciferin derivatives as substrates for firefly luciferase are reported. The allylation of luciferins induced bathochromic shift (15–40?nm) of the bioluminescence emission. Upon combination with other chemical modifications for bioluminescence wavelength tuning, novel red emitting luciferin analogues were obtained with emission maxima at 685 and 690?nm.     

NAPHTHYL- AND QUINOLYLLUCIFERIN: GREEN AND RED LIGHT EMITTING FIREFLY LUCIFERIN ANALOGUES

In the course of investigations on the possible involvement of the CIEEL (chemically initiated electron-exchange luminescence) mechanism in firefly bioluminescence, we have synthesized two novel firefly luciferin substrate analogues. D-Naphthylluciferin and D-quinolylluciferin were prepared by condensing D-cysteine with 2-cyano-6-hydroxynaphthalene and 2-cyano-6-hydroxyquinoline, respectively. These analogues are the first examples of bioluminescent substrates for firefly luciferase that do not contain a benzothiazole moiety. Firefly luciferase-catalyzed bioluminescence emission spectra revealed that compared to the normal yellow-green light of luciferin (lambda max = 559 nm), the emission from naphthylluciferin is significantly blue-shifted (lambda max = 524 nm); whereas quinolylluciferin emits orange-red light (lambda max = 608 nm). The fluorescence emission spectra, reaction pH optima, relative light yields, light emission kinetics and KM values of the analogues also were measured and compared to those of luciferin. Neither of the analogues produced the characteristic flash kinetics observed for the natural substrate. Instead, slower rise times to peak emission intensity were recorded. It appears that the formation of an intermediate from the analogue adenylates prior to the addition of oxygen is responsible for the slow rise times. The synthetic substrate analogues described here should be useful for future mechanistic studies.     

Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg²⁺. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family ( Photinus pyralis and Lampyris turkestanicus ) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.     

Understanding the complete bioluminescence cycle from a multiscale computational perspective: A review

Bioluminescence (BL) is an amazing natural phenomenon whose visible light is produced by living organisms. BL phenomenon is quite pervasive and has been observed in 17 phyla of 4 kingdoms. This fascinating natural phenomenon has unceasingly attracted people’s curiosity from ancient era to today. For a very long time, we can only receive some sporadic and static information from experimental observations, the mechanism of most BL remains is unclear. How the chemical reaction of BL process is initiated? Where the energy for light emission comes from? How does the light emitter produce? What is the light emitter for a wild bioluminescent organism? How to regain luciferin for next bioluminescence when it is used up? The luciferin is utilized forthwith or stored and release for subsequent light emission? What factors affect the color and strength of a bioluminescence? How to artificially tune the bioluminescence for special application? Computational BL plays unreplaceable role in answering these mechanistic questions. In contrast with experimental BL, computational BL came very late. In the past two decades, computational BL has touched nearly all the bioluminescent systems with chemical bases via the method of multiscale simulation. In this review, the author firstly introduced the history, types and general chemical process of BL. Then, the computational scheme on BL was briefly epitomized. Using firefly BL as a paradigmatic case, the author summarized theoretical investigation on the six stages of general chemical process in a BL cycle: luciferin oxidation, peroxide thermolysis, light emission, luciferin regeneration, luciferin storage and luciferin release. At each stage, the available theoretical studies of other bioluminescent organisms are briefly introduced and compared with the firefly system. Basing on the mechanistic understanding, the author reviewed the up-to-date theoretical design on bioluminescent systems. Again, the firefly was mainly focused on, and the other possible systems were just briefly introduced. This review summarized the theoretical studies to date on BL and addressed the status, critical challenges and future prospects of computational BL.     

Theoretical Study of Firefly Luciferin pKa Values—Relative Absorption Intensity in Aqueous Solutions

Ground‐state vibrational analyses of firefly luciferin and its conjugate acids and bases are performed. The Gibbs free energies obtained from these analyses are used to estimate pK_a values for phenolic hydroxy and carboxy groups and the N–H⁺ bond in the N‐protonated thiazoline or benzothiazole ring of firefly luciferin. The theoretical pK_a values are corrected using the experimental values. The concentrations of these chemical species in solutions with different pH values are estimated from their corrected pK_a values, and the pH dependence of their relative absorption intensities is elucidated. With the results obtained we assign the experimental spectra unequivocally. Especially, the small peak near 400 nm at pH 1–2 in experimental absorption spectra is clarified to be due to the excitation of carboxylate anion with N‐protonated thiazoline ring of firefly luciferin. Our results show that the pK_a values of chemical species, which are contained in the aqueous solutions, are effective to assign experimental absorption spectra.     

Quenching of tryptophan fluorescence of firefly luciferase by substrates

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient quenching of tryptophan fluorescence of the active enzyme takes place upon its binding with substrates. In the presence of ATP the quenching is of dynamic type and is caused by structural changes in the protein molecule upon ATP binding. A model is proposed in which the complex has smaller fluorescence quantum yield than the free enzyme because of partial quenching of tryptophan fluorescence by the new microenvironment. Quenching of tryptophan fluorescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luciferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 A.     

Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

Spectroscopic Properties of Amine‐substituted Analogues of Firefly Luciferin and Oxyluciferin

Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an amine substituent (NH₂, NHMe and NMe₂) at the C6' position were studied based on absorption and fluorescence measurements. Their π‐electronic properties were investigated by DFT and TD‐DFT calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An increase in the electron‐donating strength of the substituent led to the bathochromic shift of the fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding oxyluciferin analogues in a solvent were well correlated with each other. Based on the obtained data, the polarity of a luciferase active site was explained. As a result, the maximum wavelength of bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence of the luciferin analogue in place of that of the corresponding oxyluciferin analogue.     

Analysis of Photoexcitation Energy Dependence in the Photoluminescence of Firefly Luciferin

The whole pathways for photoluminescence, which include absorption, relaxation and emission, of firefly luciferin in aqueous solutions of different pH values with different photoexcitation energies were theoretically investigated by considering protonation/deprotonation. It is experimentally known that the color of fluorescence changes from green to red with a decrease in the photoexcitation energy. We confirmed with the theoretical analysis that the peak energy shift in the fluorescence spectra with varying photoenergies is due to a change in photoluminescence pathway. When the photoexcitation energy is decreased, the red emission from a monoanion form of firefly luciferin with carboxylate and phenolate groups and N‐protonated thiazoline ring occurs irrespective of the pH values. However, because the species abundant in the solution and those excited by the photon depend on the solution pH, the pathway leading to the monoanion form changes with the solution pH.     

TDDFT Study of the Electronic Structure,Absorption and Emission Spectra of the Light Emitters of the Amazing Firefly Bioluminescence and Solvation Effects on the Spectra

The ground and excited state properties of luciferin (LH₂) and oxyluciferin (OxyLH₂), the bioluminescent chemicals in the firefly, have been characterized using density functional theory (DFT) and time dependent DFT (TDDFT) methods. The effects of solvation on the electronic absorption and emission spectra of luciferin and oxyluciferin were predicted with a self‐consistent isodensity polarized continuum model of the solvent using TDDFT. The S₀→S₁ vertical excitation energies in the gas phase and in water were obtained. Optimizations of the excited state geometries permitted the first predictions of the fluorescence spectra for these biologically important molecules. Shifts in both of the absorption and emission spectra on proceeding from the gas phase to aqueous solution were also predicted.     

Vibrationally Resolved Absorption and Fluorescence Spectra of Firefly Luciferin: A Theoretical Simulation in the Gas Phase and in Solution

Firefly bioluminescence has been applied in several fields. However, the absorption and fluorescence spectra of the substrate, luciferin, have not been observed at the vibrational level. In this study, the vibrationally resolved absorption and fluorescence spectra of firefly luciferin (neutral form LH₂, phenolate ion form LH^? and dianion form L^2?) are simulated using the density functional method and convoluted by a Gaussian function, with displacement, distortion and Duschinsky effects in the framework of the Franck–Condon approximation. Both neutral and anionic forms of the luciferin are considered in the gas phase and in solution. The simulated spectra have desired band maxima with the experimental ones. The vibronic structure analysis reveals that the features of the most contributive vibrational modes coincide with the key geometry‐changing region during transition between the ground state and the first singlet excited state.     

Synthesis and luminescence properties of biphenyl-type firefly luciferin analogs with a new,near-infrared light-emitting bioluminophore

New firefly luciferin analogs of the 4,4′-substituted biphenyl-type were synthesized. One analog with a 4′-dimethylamino group possessed bioluminescence activity, emitting near-infrared biological window light at 675 nm suitable for deep-site bioimaging of living animals. The chemiluminescence light-emission maximum of the corresponding methyl ester of the bioluminescence active analog was 500 nm, implying that biphenyl and thiazolinone rings in the light emitter might be placed in a coplanar conformation at the polar luciferase active site.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.