Cassius V. Stevani

Studies on the Mechanism of the Excitation Step in Peroxyoxalate Chemiluminescence

Studies on the mechanism of excited state formation in the peroxyoxalate system have been performed, to corroborate the involvement of the well-known Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism in the chemi-excitation step of this complex sequence. The singlet quantum yields, extrapolated to infinite activator concentrations (ΦS∞), and relative rate constants (kCAT/kD) of the excitation step have been determined in the presence of several activators for two systems: (i) the complete peroxyoxalate reaction with bis(2,4,6-trichlorophenyl) oxalate; and (ii) the base-catalyzed reaction of 4-chlorophenyl O,O-hydrogen monoperoxyoxalate, an isolated key intermediate. For five activators commonly used in CIEEL studies (anthracene, 9,10-diphenylanthracene, 2,5-diphenyloxazole, perylene, and rubrene), a linear correlation of ln(kCAT/kD) with the voltammetric half-peak oxidation potential (Ep/2) of the activator was obtained for both systems. The values obtained with 9,10-dicyanoanthracene and 9,10-dimethoxyanthracene did not fit this correlation. A reasonable linear correlation between ln(ΦS∞) and Ep/2 was obtained for all activators. For the commonly used activators, this quantum yield (ΦS∞) dependence can be rationalized in terms of the free energy balance of the back electron transfer leading to the formation of the excited state of the activator. However, the ΦS∞ values obtained with 9,10-dimethoxyanthracene and 9,10-dicyanoanthracene cannot be explained on the basis of these considerations alone. Thus, although this work presents clear-cut evidence of the operation of the CIEEL mechanism in the peroxyoxalate reaction, the results obtained with less commonly used activators show that several mechanistic details of the CIEEL hypothesis remain to be elucidated.

Fungi bioluminescence revisited

A review of the research conducted during the past 30 years on the distribution, taxonomy, phylogeny, ecology, physiology and bioluminescence mechanisms of luminescent fungi is presented. We recognize 64 species of bioluminescent fungi belonging to at least three distinct evolutionary lineages, termed Omphalotus, Armillaria and mycenoid. An accounting of their currently accepted names, distributions, citations reporting luminescence and whether their mycelium and/or basidiomes emit light are provided. We address the physiological and ecological aspects of fungal bioluminescence and provide data on the mechanisms responsible for bioluminescence in the fungi.

The enzymatic nature of fungal bioluminescence

The uncertainty about the possible involvement of a luciferase in fungal bioluminescence has not only hindered the understanding of its biochemistry but also delayed the characterization of its constituents. The present report describes how in vitro light emission can be obtained enzymatically from the cold and hot extracts assay using different species of fungi, which also indicates a common mechanism for all these organisms. Kinetic data suggest a consecutive two-step enzymatic mechanism and corroborate the enzymatic proposal of Airth and Foerster. Finally, overlapping of light emission spectra from the fungal bioluminescence and the in vitro assay confirm that this reaction is the same one that occurs in live fungi.

Evidence that a single bioluminescent system is shared by all known bioluminescent fungal lineages

Since the early 20th century, many researchers have attempted to determine how fungi are able to emit light. The first successful experiment was obtained using the classical luciferin-luciferase test that consists of mixing under controlled conditions hot (substrate/luciferin) and cold (enzyme/luciferase) water extracts prepared from bioluminescent fungi. Failures by other researchers to reproduce those experiments using different species of fungi lead to the hypothesis of a non-enzymatic luminescent pathway. Only recently, the involvement of a luciferase in this system was proven, thus confirming its enzymatic nature. Of the 100,000 described species in Kingdom Fungi, only 71 species are known to be luminescent and they are distributed unevenly amongst four distantly related lineages. The question we address is whether the mechanism of bioluminescence is the same in all four evolutionary lineages suggesting a single origin of luminescence in the Fungi, or whether each lineage has a unique mechanism for light emission implying independent origins. We prepared hot and cold extracts of numerous species representing the four bioluminescent fungal lineages and performed cross-reactions (luciferin × luciferase) in all possible combinations using closely related non-luminescent species as controls. All cross-reactions with extracts from luminescent species yielded positive results, independent of lineage, whereas no light was emitted in cross-reactions with extracts from non-luminescent species. These results support the hypothesis that all four lineages of luminescent fungi share the same type of luciferin and luciferase, that there is a single luminescent mechanism in the Fungi, and that fungal luciferin is not a ubiquitous molecule in fungal metabolism.

Metal cation toxicity in the alga Gracilaria domingensis as evaluated by the daily growth rates in synthetic seawater

Macroalgae of the genus Gracilaria have considerable economic importance as raw material for agar production and belong to an important group of organisms that are tolerant of high concentrations of metal. The median inhibitory concentration (IC50) values obtained by measuring the ratio of fresh mass variation (i.e., daily growth rates) of the red macroalga Gracilaria domingensis during a 48-h aquatic toxicity assay are reported here. The alga was exposed to 14 different metal cations as well as the molybdate anion in synthetic seawater. The actual concentrations of these ionic species (at IC50 values) and the proportion of free ions (aqueous complexes) were determined by inductively coupled plasma atomic emission spectroscopy and the Environmental Protection Agency-recommended software, MINTEQA2, respectively. Based on the free IC50 values (IC50F), the ions were ranked in terms of toxicity: Cd2+ ≫ Cu2+ ≫ Pb2+ ≫ Zn2+ ≫ Ni2+ > Co2+ > La3+ ≫ Mn2+ > Ca2+ ~ Li+ ≫ MoO42− ≫ Sr2+ > Mg2+ ≫ K+ > Na+. As a member of the first trophic level in the marine food chain, G. domingensis is an appropriate target organism both for the development of toxicological assays and as a bioindicator of marine degradation.

Luminescent Mycena: new and noteworthy species

Seven species of Mycena are reported as luminescent, representing specimens collected in Belize, Brazil, Dominican Republic, Jamaica, Japan (Bonin Islands), Malaysia (Borneo) and Puerto Rico. Four of them represent new species (Mycena luxaeterna, M. luxarboricola, M. luxperpetua, M. silvaelucens) and three represent new reports of luminescence in previously described species (M. aff. abieticola, M. aspratilis, M. margarita). Mycena subepipterygia is synonymized with M. margarita, and M. chlorinosma is proposed as a possible synonym. Comprehensive descriptions, illustrations, photographs and comparisons with phenetically similar species are provided. A redescription of M. chlorophos, based on analyses of type specimens and recently collected topotypical material, is provided. The addition of these seven new or newly reported luminescent species of Mycena brings the total to 71 known bioluminescent species of fungi.

Mechanism of automotive clearcoat damage by dragonfly eggs investigated by surface enhanced Raman scattering

Dragonflies are attracted by the reflection of sunlight on car surfaces and lay their eggs on the clearcoat resin. Considering that the surface can reach up to 93°C and that during the egg hardening process (sclerotization) H2O2 is released, cysteine and cystine residues present in the egg protein can be oxidized to sulfinic and sulfonic acids. These are strong acids which, like acid rain, can hydrolyze the acrylic/melamine resin causing damage where the eggs were laid. Confocal Raman spectroscopy revealed that the spectra obtained from damaged and intact portions of the clearcoat were similar, in agreement with infrared absorption spectroscopy data. These data demonstrate that the attack by eggs, H2SO4 and cysteine/H2O2 only promotes solubilization of resin through acid hydrolysis of the resin ester and amide moieties. Furthermore, surface enhanced Raman scattering (SERS) spectra obtained from dragonfly eggs and cysteine/H2O2 reaction products treated with a silver colloid were very similar, thus confirming the presence of sulfinic and sulfonic acids.

Bioluminescent Mycena species from São Paulo, Brazil

Six species of bioluminescent agarics are described and illustrated from a single site in primary Atlantic Forest habitat in the Parque Estadual Turístico do Alto Ribeira, São Paulo State, Brazil. These include two new taxa of Mycena, viz. M. asterina and M. lucentipes. Luminescence in Mycena fera, M. singeri and M. discobasis is reported for the first time. In addition an undeterminable luminescent Mycena species is described and additional specimens of Gerronema viridilucens are documented. An accounting of known bioluminescent species of Mycena and a discussion of why they luminesce are presented.

Kinetic studies on the chemiluminescent decomposition of an isolated intermediate in the peroxyoxalate reaction

The peroxyoxalate system undergoes one of the most efficient chemiluminescence reactions and is the only one considered to involve an intermolecular chemically initiated electron exchange luminescence mechanism, with proven high efficiency. Several reactive intermediates have been proposed, which, upon interaction with a fluorescent activator, lead to excited-state generation. The synthesis and spectral characterization of 4-chlorophenyl O,O-hydrogen monoperoxyoxalate (1), a compound analogous to one of the proposed reactive intermediates, was recently reported. Here the results of a kinetic study on the chemiluminescent decomposition of this peracid 1, catalyzed by oxygen bases (potassium hydroxide, potassium tert-butoxide and potassium p-chlorophenolate) or nitrogen bases [pyridine, imidazole and 1,8-bis(dimethylamino)naphthalene] are presented. Based on the dependence of the observed rate constants on the base concentration, kinetic schemes are proposed for the catalyzed decomposition of 1 and rate constants are assigned to specific reaction steps. The results obtained with the nitrogen bases give further support in favor of 1,2-dioxetane dione as the reactive intermediate in the peroxyoxalate reaction.

Evaluation of metal toxicity by a modified method based on the fungus Gerronema viridilucens bioluminescence in agar medium

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+),Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables us to propose a metal toxicity series to Gerronema viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximately Ni(2+) approximately Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximately Na(+) > Ca(2+), and to shed some light on the mechanism of toxic action of metal cations to basidiomycete fungi.