Optical steady-state and time-resolved spectroscopic methods were used to study the photoprotolytic reaction of oxyluciferin, the active bioluminescence chromophore of the firefly's luciferase-catalyzed reaction. We found that like D-luciferin, the substrate of the firefly bioluminescence reaction, oxyluciferin is a photoacid with pK(a)* value of ∼0.5, whereas the excited-state proton transfer (ESPT) rate coefficient is 2.2 × 10(10) s(-1), which is somewhat slower than that of D-luciferin. The kinetic isotope effect (KIE) on the fluorescence decay of oxyluciferin is 2.5 ± 0.1, the same value as that of D-luciferin. Both chromophores undergo fluorescence quenching in solutions with a pH value below 3.
The bioluminescent reaction of the "sea firefly" Cypridina hilgendorfii is a prototypical system for marine bioluminescence, as its substrate possesses an imidazopyrazinone core that is a common link among organisms of eight phyla. The elucidation of the mechanism behind Cypridina bioluminescence is essential for future applications in bioimaging, biomedicine, and bioanalysis. In this study we have investigated the key step of chemiexcitation with a combined experimental and theoretical approach. The obtained results indicate that neutral dioxetanone is responsible for efficient chemiexcitation, as the thermolysis of this species gives access to a long region of the potential energy surface (PES), where the ground and excited singlet states are degenerated. Contrary to expected, neither chemically induced electron-exchange luminescence (CIEEL) nor charge transfer-initiated luminescence (CTIL) can be used to explain imidazopyrazinone-based bioluminescence, as there is no clear relationship between electron (ET)/charge (CT) transfer (occurring between the electron-rich moiety and dioxetanone) and chemiexcitation. Attractive electrostatic interactions between the CO and oxyluciferin moieties allow neutral dioxetanone to spend time in the PES region of degeneracy, while repulsive interactions for anionic dioxetanone lead to a quicker CO detachment.
In spite of recent advances towards understanding the mechanism of firefly bioluminescence, there is no consensus about which oxyluciferin (OxyLH(2)) 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 OxyLH(2) 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 OxyLH(2) 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).
Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.
Coelenterazine, a member of the imidazopyrazinone class of chemiluminescent substrates, presents significant potential as a dynamic probe of reactive oxygen species in a biological environment, such as a superoxide anion, in which these species are important in cellular biology and pathology. The objective of the current study was to understand in what way the efficiency of singlet and triplet chemiexcitation could be modulated, towards a more efficient use of imidazopyrazinone-based compounds as dynamic chemiluminescent probes. To this end the thermolysis of imidazopyrazinone dioxetanone, substituted at the C-position with electron-donating or electron-withdrawing groups, was characterized with a theoretical approach based on density functional theory. Substituents with different electron-donating/withdrawing characters have only a limited effect on the singlet chemiexcitation of anionic dioxetanone. For neutral dioxetanone, both electron-withdrawing and weak electron-donating substituents increase singlet chemiexcitation, to the contrary of strong electron-donating groups. During their thermolysis reaction, all molecules presented regions of degeneracy with triplet states, thereby indicating the possibility of triplet chemiexcitation.
Firefly luciferase is the most studied bioluminescence system, and its catalyzed reactions have been relatively well characterized. However, the color tuning mechanism that leads to firefly multicolor bioluminescence is still unknown, nor is consensual which is the yellow-green and red emitters. Computational studies have been essential in the study of oxyluciferin (OxyLH2) chemi- and bioluminescence and are responsible for most of our knowledge of this natural phenomenon. The objective of this manuscript is the analysis of the benefits and the conclusions derived from the theoretical studies of the light emitter, OxyLH2, and its applications on bioluminescence research.
Firefly luciferase catalyzes a light-emitting reaction in which an excited-state product is formed. Both experimental and theoretical methodologies are used to study this system, and the reactions catalyzed by luciferase are relatively well characterized. However, the mechanism by which an excited-state product is formed is still unknown. This Minireview deals with the current understanding of firefly bioluminescence and chemiluminescence. Thermal decomposition of simple 1,2-dioxetanes is also discussed, due to their role in formation of the excited-state bioluminophore.
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