RESUMO
Cystathionine ß-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5'-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO(â¢) binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (â¼5-fold) and faster association (â¼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO(â¢) binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (â¼2-fold higher affinity) as compared with CO. Importantly, CO and NO(â¢) binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO(â¢). This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.
Assuntos
Monóxido de Carbono/metabolismo , Cistationina beta-Sintase/metabolismo , Óxido Nítrico/metabolismo , S-Adenosilmetionina/farmacologia , Heme/metabolismo , Humanos , Cinética , Metionina/metabolismo , OxirreduçãoRESUMO
Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H(2)S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H(2)S-producing human enzyme cystathionine ß-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Neoplasias do Colo/metabolismo , Cistationina beta-Sintase/metabolismo , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Monóxido de Carbono/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Cistationina beta-Sintase/química , Cistationina beta-Sintase/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Glutationa/metabolismo , Humanos , Cinética , Mitocôndrias/patologia , Modelos Moleculares , Óxido Nítrico/metabolismo , Fosforilação Oxidativa , S-Adenosilmetionina/metabolismo , Transdução de SinaisRESUMO
Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage.
Assuntos
Biocatálise , Citocromos/fisiologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Oxirredutases/fisiologia , Ácido Peroxinitroso/metabolismo , Grupo dos Citocromos b , Consumo de OxigênioRESUMO
The hexa-coordinate heme in the H2S-generating human enzyme cystathionine ß-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(â¢) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(â¢) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 µm). This discrepancy was herein reconciled by investigating the NO(â¢) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(â¢) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 µm. In line with this result, at 25 °C, NO(â¢) binds quickly to CBS (k on â¼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off â¼ 0.003 s(-1)). The observed rate constants for NO(â¢) binding were found to be linearly dependent on [NO(â¢)] up to â¼ 800 µm NO(â¢), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(â¢) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(â¢). The novel findings reported here shed new light on CBS regulation by NO(â¢) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(â¢), CO, and H2S in cell signaling.
Assuntos
Cistationina beta-Sintase/metabolismo , Óxido Nítrico/metabolismo , Monóxido de Carbono/metabolismo , Cistationina beta-Sintase/química , Heme/química , Heme/metabolismo , Humanos , Cinética , Oxirredução , Ligação ProteicaRESUMO
Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity.
Assuntos
Proteínas de Bactérias/química , Entamoeba histolytica/química , Ferro/química , Lisina/química , Oxirredutases/química , Tirosina/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Entamoeba histolytica/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ferro/metabolismo , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismoRESUMO
Cytochrome bd is a prokaryotic respiratory quinol:O2 oxidoreductase, phylogenetically unrelated to the extensively studied heme-copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O2, NO and H2O2 is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
Assuntos
Proteínas de Bactérias/metabolismo , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Oxirredutases/metabolismo , Bactérias/enzimologia , Bactérias/metabolismoRESUMO
Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.
Assuntos
Giardia lamblia/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/efeitos da radiação , Cristalografia por Raios X , Giardia lamblia/química , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Alinhamento de Sequência , Raios XRESUMO
Giardia intestinalis is the most frequent protozoan agent of intestinal diseases worldwide. Though commonly regarded as an anaerobic pathogen, it preferentially colonizes the fairly oxygen-rich mucosa of the proximal small intestine. Therefore, when testing new potential antigiardial drugs, O2 should be taken into account, since it also reduces the efficacy of metronidazole, the gold standard drug against giardiasis. In this study, 46 novel chalcones were synthesized by microwave-assisted Claisen-Schmidt condensation, purified, characterized by high-resolution mass spectrometry, (1)H and (13)C nuclear magnetic resonance, and infrared spectroscopy, and tested for their toxicity against G. intestinalis under standard anaerobic conditions. As a novel approach, compounds showing antigiardial activity under anaerobiosis were also assayed under microaerobic conditions, and their selectivity against parasitic cells was assessed in a counterscreen on human epithelial colorectal adenocarcinoma cells. Among the tested compounds, three [30(a), 31(e), and 33] were more effective in the presence of O2 than under anaerobic conditions and killed the parasite 2 to 4 times more efficiently than metronidazole under anaerobiosis. Two of them [30(a) and 31(e)] proved to be selective against parasitic cells, thus representing potential candidates for the design of novel antigiardial drugs. This study highlights the importance of testing new potential antigiardial agents not only under anaerobic conditions but also at low, more physiological O2 concentrations.
Assuntos
Antiprotozoários/efeitos adversos , Antiprotozoários/farmacologia , Chalconas/química , Chalconas/farmacologia , Giardia lamblia/efeitos dos fármacos , Piperazinas/química , Piperidinas/química , Antiprotozoários/química , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Chalconas/efeitos adversos , Humanos , PiperazinaRESUMO
BACKGROUND: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. SCOPE OF REVIEW: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO(2)(-)) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O(2). All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Respiração Celular/fisiologia , Citocromos c/metabolismo , Humanos , Modelos Biológicos , OxirreduçãoRESUMO
Extending our previous observations, we have shown on HaCat cells that melatonin, at ~10-9 M concentration, transiently raises not only the expression of the neuronal nitric oxide synthase (nNOS) mRNA, but also the nNOS protein synthesis and the nitric oxide oxidation products, nitrite and nitrate. Interestingly, from the cell bioenergetic point of view, the activated NO-related chemistry induces a mild decrease of the oxidative phosphorylation (OXPHOS) efficiency, paralleled by a depression of the mitochondrial membrane potential. The OXPHOS depression is apparently balanced by glycolysis. The mitochondrial effects described have been detected only at nanomolar concentration of melatonin and within a time window of a few hours' incubation; both findings compatible with the melatonin circadian cycle.
Assuntos
Ritmo Circadiano , Melatonina/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Trifosfato de Adenosina/biossíntese , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lactatos/metabolismo , Melatonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Óxido Nítrico Sintase Tipo I/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Fatores de TempoRESUMO
The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.
Assuntos
Biocatálise , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Grupo dos Citocromos b , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Compostos Ferrosos/metabolismoRESUMO
A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, â¼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide-induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.
Assuntos
Antioxidantes/farmacologia , Metabolismo Energético/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Melatonina/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Células Cultivadas , Humanos , Queratinócitos/citologia , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Nitritos/metabolismo , Oxirredução , Fosforilação Oxidativa , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cell respiration is controlled by nitric oxide (NO) reacting with respiratory chain complexes, particularly with Complex I and IV. The functional implication of these reactions is different owing to involvement of different mechanisms. Inhibition of complex IV is rapid (milliseconds) and reversible, and occurs at nanomolar NO concentrations, whereas inhibition of complex I occurs after a prolonged exposure to higher NO concentrations. The inhibition of Complex I involves the reversible S-nitrosation of a key cysteine residue on the ND3 subunit. The reaction of NO with cytochrome c oxidase (CcOX) directly involves the active site of the enzyme: two mechanisms have been described leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) or a more labile nitrite-derivative (CcOX-NO (2) (-) ). Both adducts are inhibited, though with different K(I); one mechanism prevails on the other depending on the turnover conditions and availability of substrates, cytochrome c and O(2). SH-SY5Y neuroblastoma cells or lymphoid cells, cultured under standard O(2) tension, proved to follow the mechanism leading to degradation of NO to nitrite. Formation of CcOX-NO occurred upon rising the electron flux level at this site, artificially or in the presence of higher amounts of endogenous reduced cytochrome c. Taken together, the observations suggest that the expression level of mitochondrial cytochrome c may be crucial to determine the respiratory chain NO inhibition pathway prevailing in vivo under nitrosative stress conditions. The putative patho-physiological relevance of the interaction between NO and the respiratory complexes is addressed.
Assuntos
Mitocôndrias/fisiologia , Óxido Nítrico/fisiologia , Animais , HumanosRESUMO
BACKGROUND: The shortage of essential surgical care in sub-Saharan Africa is a worldwide problem. Lack of healthcare workers, surgeons and very limited resources are the reason for this critical health crisis. Furthermore in Africa many physicians as soon as they get a degree emigrate in more attractive countries. METHODS: "Multidisciplinary teaching support to a new Faculty of Medicine in N'Djamena - Chad" is a teaching cooperation program between «Sapienza¼ University of Rome and the University Hospital "Le Bon Samaritain" in N'Djamena, Chad. The project started in 2010, with the aim of cooperating in teaching medical students of African origin and in training Italian residents in sub-Saharan surgical and internal pathologies. In Chad the greatest surgical burden (about 11% of the total global diseases) is created by injuries, cancers, congenital anomalies, appendicitis, bowel obstructions, hernias, abscesses (by amebiasis or others) and obstetric emergencies. CONCLUSIONS: Up till now healthcare in Africa especially in rural areas has been provided by international organisations,we believe that academic collaborations between high-income and low-income Nations is necessary to meet the real needs of the african surgical workforce; at the same time it is very useful for store of knowledge of our residents.
Assuntos
Especialidades Cirúrgicas/educação , Chade , Hospitais Universitários , Cooperação Internacional , Cidade de RomaRESUMO
Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c550 acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c552 to the terminal ba3- and caa3-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite â SOR(HB8) â cytochrome c550 â cytochrome c552 â ba3 oxidase/caa3 oxidase â O2. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus.
Assuntos
Grupo dos Citocromos c/metabolismo , Redes e Vias Metabólicas , Sulfitos/metabolismo , Thermus thermophilus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O(2) -scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O(2) -detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O(2) to H(2) O rapidly (â¼3 µM O(2) × min × 10(6) cells at 37 °C) and with high affinity (C(50) = 3.4 ± 0.7 µM O(2)). Following a short-term (minutes) exposure to H(2) O(2) ≥ 100 µM, the O(2) consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome-inhibitor MG132. Instead, H(2) O(2) does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.
Assuntos
Giardia lamblia/fisiologia , Intestino Delgado/parasitologia , Estresse Oxidativo , Animais , Peróxido de Hidrogênio/metabolismo , Consumo de OxigênioRESUMO
Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Potenciais da Membrana/fisiologia , Consumo de Oxigênio , Linhagem Celular , Cianetos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ionóforos/farmacologia , Modelos Estatísticos , Nigericina/farmacologia , Fosforilação , Valinomicina/farmacologiaRESUMO
Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.
Assuntos
Giardia lamblia/metabolismo , Hemoglobinas/metabolismo , Óxido Nítrico/metabolismo , Aerobiose , Giardia lamblia/efeitos dos fármacos , Hemoglobinas/genética , Humanos , NAD/metabolismo , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Nitrosoglutationa/farmacologiaRESUMO
Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.
Assuntos
Ataxia Telangiectasia/metabolismo , Respiração Celular , Linfócitos/metabolismo , Óxido Nítrico/fisiologia , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Glicólise , Humanos , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
Flavodiiron proteins (FDPs) are enzymes identified in prokaryotes and a few pathogenic protozoa, which protect microorganisms by reducing O(2) to H(2)O and/or NO to N(2)O. Unlike most prokaryotic FDPs, the protozoan enzymes from the human pathogens Giardia intestinalis and Trichomonas vaginalis are selective towards O(2). UV/vis and EPR spectroscopy showed that, differently from the NO-consuming bacterial FDPs, the Giardia FDP contains an FMN with reduction potentials for the formation of the single and the two-electron reduced forms very close to each other (E(1)=-66+/-15mV and E(2)=-83+/-15mV), a condition favoring destabilization of the semiquinone radical. Giardia FDP contains also a non-heme diiron site with significantly up-shifted reduction potentials (E(1)=+163+/-20mV and E(2)=+2+/-20mV). These properties are common to the Trichomonas hydrogenosomal FDP, and likely reflect yet undetermined subtle structural differences in the protozoan FDPs, accounting for their marked O(2) specificity.