The orange light emitting luciferase from the rare Euryopa clarindae adult railroadworm (Coleoptera:Phengodidae): structural/functional and evolutionary relationship with green and red emitting luciferases.

Railroadworms luciferases emit the widest range of bioluminescence colors among beetles, ranging from green to red, being model enzymes to investigate the structure and bioluminescence colors relationships. Only three active railroadworms luciferases from the larval stage have been cloned and investigated: the Phrixothrix hirtus head lanterns red-emitting luciferase (PhRE); the Phrixothrix vivianii lateral lanterns green emitting luciferases (PvGR) and the Phengodes sp. dorsal lanterns yellow-green emitting luciferase (Ph). No active luciferase emitting in the yellow-orange region, however, has been cloned yet. Here we report the cloning and characterization of the orange emitting luciferase from the adult males of a rare Brazilian Cerrado railroadworm, Euryopa clarindae, and the transcriptional identification of two isozymes from the Amazon forest Mastinomorphus sp. railroadworm. The luciferase of E. clarindae has 548 residues, emits orange bioluminescence (600 nm), and displays intermediate kinetic values [KM(luciferin) = 50 µM, KM(ATP) ~ 170 µM] between those reported for green-emitting lateral lanterns and red emitting head lanterns luciferases. It displays 74-78% identity with the lateral lanterns luciferases of other railroadworms and 70% with the head lantern PhRE luciferase, and 96% with the larval Mastinomorphus sp. Mast-1, suggesting that this larva could be from the Euryopa genus. The phylogenetic analysis and kinetic/functional properties, place this orange-emitting enzyme as an intermediate form between the green-emitting lateral lanterns and red-emitting head lanterns luciferases. Major structural differences that could be associated with bioluminescence color determination are a relatively larger cavity size, and substitutions in the loops 223-235 and 311-316, especially N/C/T311, and their interactions which may help to close the bottom of LBS.

Mining reactive triplet carbonyls in biological systems.

Aliphatic triplet carbonyls can be treated as short-lived radicals, since both species share similar reactions such as hydrogen atom abstraction, cyclization, addition, and isomerization. Importantly, enzyme-generated triplet carbonyls excite triplet molecular oxygen to the highly reactive, electrophilic singlet state by resonance energy transfer, which can react with proteins, lipids, and DNA. Carbonyl triplets, singlet oxygen, and radicals are endowed with the potential to trigger both normal and pathological responses. In this paper, we present a short review of easy, fast, and inexpensive preliminary tests for the detection of transient triplet carbonyls in chemical and biological systems. This paper covers direct and indirect methods to look for triplet carbonyls based on their spectral distribution of chemiluminescence, photoproduct analysis, quenching of light emission by conjugated dienes, and enhancement of light emission by the sensitizer 9,10-dibromoanthracence-2-sulfonate ion (DBAS).

Fungal bioassays for environmental monitoring.

Environmental pollutants are today a major concern and an intensely discussed topic on the global agenda for sustainable development. They include a wide range of organic compounds, such as pharmaceutical waste, pesticides, plastics, and volatile organic compounds that can be found in air, soil, water bodies, sewage, and industrial wastewater. In addition to impacting fauna, flora, and fungi, skin absorption, inhalation, and ingestion of some pollutants can also negatively affect human health. Fungi play a crucial role in the decomposition and cycle of natural and synthetic substances. They exhibit a variety of growth, metabolic, morphological, and reproductive strategies and can be found in association with animals, plants, algae, and cyanobacteria. There are fungal strains that occur naturally in soil, sediment, and water that have inherent abilities to survive with contaminants, making the organism important for bioassay applications. In this context, we reviewed the applications of fungal-based bioassays as a versatile tool for environmental monitoring.

Oxidative Modification of Proteins: From Damage to Catalysis, Signaling, and Beyond.

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption. Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance. Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions. Antioxid. Redox Signal. 35, 1016-1080.

Aerobic co-oxidation of hemoglobin and aminoacetone, a putative source of methylglyoxal.

Aminoacetone (1-aminopropan-2-one), a putative minor biological source of methylglyoxal, reacts like other α-aminoketones such as 6-aminolevulinic acid (first heme precursor) and 1,4-diaminobutanone (a microbicide) yielding electrophilic α-oxoaldehydes, ammonium ion and reactive oxygen species by metal- and hemeprotein-catalyzed aerobic oxidation. A plethora of recent reports implicates triose phosphate-generated methylglyoxal in protein crosslinking and DNA addition, leading to age-related disorders, including diabetes. Importantly, methylglyoxal-treated hemoglobin adds four water-exposed arginine residues, which may compromise its physiological role and potentially serve as biomarkers for diabetes. This paper reports on the co-oxidation of aminoacetone and oxyhemoglobin in normally aerated phosphate buffer, leading to structural changes in hemoglobin, which can be attributed to the addition of aminoacetone-produced methylglyoxal to the protein. Hydroxyl radical-promoted chemical damage to hemoglobin may also occur in parallel, which is suggested by EPR-spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide and ethanol. Concomitantly, oxyhemoglobin is oxidized to methemoglobin, as indicated by characteristic CD spectral changes in the Soret and visible regions. Overall, these findings may contribute to elucidate the molecular mechanisms underlying human diseases associated with hemoglobin dysfunctions and with aminoacetone in metabolic alterations related to excess glycine and threonine.

l-Tryptophan Interactions with the Horseradish Peroxidase-Catalyzed Generation of Triplet Acetone.

Triplet carbonyls generated by chemiexcitation are involved in typical photobiochemical processes in the absence of light. Due to their biradical nature, ultraweak light emission and long lifetime, electronically excited triplet species display typical radical reactions such as isomerization, fragmentation, cycloaddition and hydrogen abstraction. In this paper, we report chemical reactions in a set of amino acid residues induced by the isobutanal/horseradish peroxidase (IBAL/HRP) system, a well-known source of excited triplet acetone (Ac3* ). Accordingly, quenching of Ac3* by tryptophan (Trp) unveiled parallel enzyme damage and inactivation, likely explained by scavenging of IBAL tertiary radical reaction intermediate and Ac3* -derived 2-hydroxy-i-propyl radical. Quenching constants were calculated from Stern-Volmer plots, and the structure of radical adducts was revealed by mass spectrometry. As expected, a concurrent Schiff-type adduct was found to be one of the reaction by-products. These findings draw attention to potential structural and functional changes in enzymes involved in the electronic chemiexcitation of their products.

Oxidative modification of proteins: from damage to catalysis, signaling and beyond

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteotoxicity and cellular homeostasis disruption. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data has been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies and, the fate of the modified proteins is of clinical relevance. Future directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions.

Evaluation of Phenolic Compound Toxicity Using a Bioluminescent Assay with the Fungus Gerronema viridilucens.

Basidiomycetes (phylum Basidiomycota) are filamentous fungi characterized by the exogenous formation of spores on a club-shaped cell called a basidium that are often formed on complex fruiting bodies (mushrooms). Many basidiomycetes serve an important role in recycling lignocellulosic material to higher trophic levels, and some show symbiotic relationships with plants. All known bioluminescent fungi are mushroom-forming basidiomycetes in the order Agaricales. Hence, the disruption of the basidiomycete community can entirely compromise the carbon cycle in nature from fungi to higher trophic levels. The fungus Gerronema viridilucens was used in the present study to investigate the toxicity of a phenolic compound series based on the inhibition of its bioluminescence. The median effect concentration (EC50) obtained from curves of bioluminescence inhibition versus log [phenolic compound] showed that 2,4,6-trichlorophenol was the most toxic compound in the series. The log EC50 values of all phenolic compounds were then used for the prediction of their toxicity. The univariate correlation of log EC50 values obtained from 6 different phenolic compounds was stronger with the dissociation constant (pKa ) than with 1-octanol/water partition coefficient (KOW ). Nevertheless, the toxicity can be better predicted by using both parameters, suggesting that the phenol-driven uncoupling of fungus mitochondrial adenosine triphosphate synthesis is the origin of phenolic compound toxicity to the test fungus. Environ Toxicol Chem 2020;39:1558-1565. © 2020 SETAC.

Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,ß-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k2 = 6 × 103 M-1 s-1, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of •OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (O21Δg). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.

Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae) is the first record of a bioluminescent fungus-gnat in South America.

Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.

Chemiexcitation and Its Implications for Disease.

Quantum mechanics rarely extends to molecular medicine. Recently, the pigment melanin was found to be susceptible to chemiexcitation, in which an electron is chemically excited to a high-energy molecular orbital. In invertebrates, chemiexcitation causes bioluminescence; in mammals, a higher-energy process involving melanin transfers energy to DNA without photons, creating the lethal and mutagenic cyclobutane pyrimidine dimer that can cause melanoma. This process is initiated by NO and O2- radicals, the formation of which can be triggered by ultraviolet light or inflammation. Several chronic diseases share two properties: inflammation generates these radicals across the tissue, and the diseased cells lie near melanin. We propose that chemiexcitation may be an upstream event in numerous human diseases.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado.

Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model.

Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a "mitochondrial-targeted" action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl)ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado

ABSTRACT Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

Bioluminescence: a fungal nightlight with an internal timer.

A recent study shows that green light emission by Neonothopanus gardneri mushrooms, endemic to coconut forests of Northern Brazil, is controlled by a circadian clock. Furthermore, insects are attracted by the light, raising the possibility that bioluminescence functions in spore dispersal and fungal dissemination.

Photochemistry. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.

Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts that are typically created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. We found that in melanocytes, CPDs are generated for >3 hours after exposure to UVA, a major component of the radiation in sunlight and in tanning beds. These "dark CPDs" constitute the majority of CPDs and include the cytosine-containing CPDs that initiate UV-signature C→T mutations. Dark CPDs arise when UV-induced reactive oxygen and nitrogen species combine to excite an electron in fragments of the pigment melanin. This creates a quantum triplet state that has the energy of a UV photon but induces CPDs by energy transfer to DNA in a radiation-independent manner. Melanin may thus be carcinogenic as well as protective against cancer. These findings also validate the long-standing suggestion that chemically generated excited electronic states are relevant to mammalian biology.

Excited singlet molecular O2(¹Δg) is generated enzymatically from excited carbonyls in the dark.

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 ((1)Δg). Indirect analysis of O2 ((1)Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)Σg(-))], chemical trapping of (18)O2 ((1)Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

Ferricytochrome (c) directly oxidizes aminoacetone to methylglyoxal, a catabolite accumulated in carbonyl stress.

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH4(+) ion, and H2O2 coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O2(•-) and HO (•) radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E0 values = -0.51 and -1.0 V) to ferricytochrome c (E0 = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH4(+) ion. In the presence of oxygen, aminoacetone enoyl and O2(•-) radicals propagate aminoacetone oxidation to methylglyoxal and H2O2. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells.

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 µM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 µM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.