Joaquim C. G. Esteves da Silva

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two‐step process: the first corresponds to the reaction of luciferases substrate, luciferin (LH2), with ATP‐Mg2+ generating inorganic pyrophosphate and an intermediate luciferyl‐adenylate (LH2‐AMP); the second is the oxidation and decarboxylation of LH2‐AMP to oxyluciferin, the light emitter, producing CO2, AMP, and photons of yellow‐green light (550– 570 nm). In a dark reaction LH2‐AMP is oxidized to dehydroluciferyl‐adenylate (L‐AMP). Luciferase also shows acyl‐coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl‐coenzyme A (L‐CoA), luciferyl‐coenzyme A (LH2‐CoA), and fatty acyl‐CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3′‐phosphate chain plus an intact terminal pyrophosphate moiety. The LH2 stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D‐LH2 or L‐LH2, has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

Optical fiber sensor for Hg(II) based on carbon dots

An optical fiber sensor for Hg(II) in aqueous solution based on sol-gel immobilized carbon dots nanoparticles functionalized with PEG(200) and N-acetyl-L-cysteine is described. This sol-gel method generated a thin (about 750 nm), homogenous and smooth (roughness of 2.7±0.7 Å) film that immobilizes the carbon dots and allows reversible sensing of Hg(II) in aqueous solution. A fast (less than 10 s), reversible and stable (the fluorescence intensity measurements oscillate less than 1% after several calibration cycles) sensor system was obtained. The sensor allow the detection of submicron molar concentrations of Hg(II) in aqueous solution. The fluorescence intensity of the immobilized carbon dots is quenched by the presence of Hg(II) with a Stern-Volmer constant (pH=6.8) of 5.3×10(5) M(-1).

Fluorescence quenching of anthropogenic fulvic acids by Cu(II), Fe(III) and UO22+

The quenching of the fluorescence of three anthropogenic fulvic acids (FA) provoked by Cu(II) (pH 6.0), Fe(III) (pH 4.0) and UO(2)(2+) (pH 3.5), was analyzed by a non-linear method and by Stern-Volmer plots. The FA samples were extracted from composted sewage sludges (csFA), composted municipal wastes (mwFA) and composted livestock wastes (lsFA). Synchronous-scan fluorescence (SyF) spectra were collected as a function of metal ion concentration. Spectral data were treated by a self-modeling mixture analysis method (SIMPLISMA) to detect the SyF spectral band with the strongest quenching and to calculate the corresponding quenching profile. The analysis of these profiles by a non-linear method allowed the estimation of conditional stability constants (K) and of the percentage of non-complexing fluorophores. The same quantitative information was obtained by the modified Stern-Volmer equation taking into account the existence of fluorophores that do not participate in the complexation. Good agreement was found between the results of the two procedures. The logK calculated by the non-linear method were (standard deviation in parenthesis): csFA, Cu(II), 4.22 (5); Fe(III), 5.0 (1); UO(2)(2+), 5.2 (2); mwFA, Cu(II), 4.21 (3); Fe(III), 5.6 (2); UO(2)(2+), 4.7 (3); lsFA, Cu(II), 4.51 (8); Fe(III), 5.5 (2); UO(2)(2+), 3.6 (2).

The degradation products of UV filters in aqueous and chlorinated aqueous solutions

Ultraviolet (UV) filters are vital constituents of sunscreens and other personal care products since they absorb, reflect and/or scatter UV radiation, therefore protecting us from the suns deleterious UV radiation and its effects. However, they suffer degradation, mainly through exposure towards sunlight and from reactions with disinfectant products such as chlorine. On the basis of their increasing production and use, UV filters and their degradation products have already been detected in the aquatic environment, especially in bathing waters. This paper presents a comprehensive review on the work done so far as to identify and determine the by-products of UV filter photodegradation in aqueous solutions and those subsequent to disinfection-induced degradation in chlorinated aqueous solutions, namely swimming pools.

Seasonal variations of heavy metals in sediments and aquatic mosses from the Cávado river basin (Portugal)

Abstract Concentrations of Cd, Cr, Cu, Ni, Pb and Zn in surface sediments and aquatic mosses from the Cavado river basin were determined in order to evaluate the overall metal contamination and trace the main pollution sources. The natural background levels were calculated for both plants and sediments (fraction μ m) collected at uncontaminated sites and concentrations were normalized to the natural levels. The maximum resulting contamination factors in the aquatic mosses ranged from 6 (Zn) to 101 (Cr). In the sediments the accumulation rates are lower, between 3 (Zn) and 18 (Pb). The degree of contamination was also evaluated by calculating a metal pollution index and the more polluted reaches were identified. Metal concentration variations in plants and sediments in two different surveys (1988 and 1989) were studied by factor analysis. Three factors are sufficient to characterize data variance. Factors corresponding to plants and sediments show different metal composition and provide evidence that industrial effluents and drainage waters from a mining area are the main causes of variations of element concentrations. The seasonal variations of metal concentrations both in plants and sediments are also discussed.

Computational Studies of the Luciferase Light-Emitting Product: Oxyluciferin.

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 inhibition.

Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector

The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl‐adenylate (L‐AMP), a by‐product that results from oxidation of luciferyl‐adenylate (LH2‐AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50 = 6 nm). We have shown that the CoA induced stabilization of light emission does not result from an allosteric effect but is due to the thiolytic reaction between CoA and L‐AMP, which gives rise to dehydroluciferyl‐CoA (L‐CoA), a much less powerful inhibitor (IC50 = 5 µm). Moreover, the Vmax for L‐CoA formation was determined as 160 min−1, which is one order of magnitude higher than the Vmax of the bioluminescence reaction. Results obtained with CoA analogues also support the thiolytic reaction mechanism: CoA analogues without the thiol group (dethio‐CoA and acetyl‐CoA) do not react with L‐AMP and do not antagonize its inhibitor effect; CoA and dephospho‐CoA have free thiol groups, both react with L‐AMP and both antagonize its effect. In the case of dephospho‐CoA, it was shown that it reacts with L‐AMP forming dehydroluciferyl‐dephospho‐CoA. Its slower reactivity towards L‐AMP explains its lower potency as antagonist of the inhibitory effect of L‐AMP on the light reaction. Moreover, our results support the conjecture that, in the bioluminescence reaction, the fraction of LH2‐AMP that is oxidized into L‐AMP, relative to other inhibitory products or intermediates, increases when the concentrations of the substrates ATP and luciferin increases.

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 (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).

Firefly chemiluminescence and bioluminescence: efficient generation of excited states.

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.