RESUMO
Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.
Assuntos
Neoplasias , Nitrosaminas , Tabaco sem Fumaça , Humanos , Carcinógenos/toxicidade , Mutagênicos , Neoplasias/induzido quimicamente , Nitratos , Nitritos , Nitrosaminas/toxicidade , Nitrosaminas/química , Nitrosaminas/metabolismo , Tabaco sem Fumaça/toxicidadeRESUMO
The development of new anticancer compounds is one of the challenges of bioinorganic and medicinal chemistry. Naringenin and its metal complexes have been recognized as promising inhibitors of cell proliferation, having enormous potential to act as an antioxidant and antitumorigenic agent. Lung cancer is the second most commonly diagnosed type of cancer. Therefore, this study is devoted to investigate the effects of Cu(II), naringenin (Nar), binary Cu(II)-naringenin complex (CuNar), and the Cu(II)-naringenin containing bathophenanthroline as an auxiliary ligand (CuNarBatho) on adenocarcinoma human alveolar basal epithelial cells (A549 cells) that are used as models for the study of drug therapies against lung cancer. The ternary complex shows selectivity being high cytotoxic against malignant cells. The cell death generated by CuNarBatho involves ROS production, loss of mitochondrial membrane potential, and depletion of GSH level and GSH/GSSG ratio. The structure-relationship activity was assessed by comparison with the reported Cu(II)-naringenin-phenanthroline complex. The CuNarBatho complex was synthesized and characterized by elemental analysis, molar conductivity, mass spectrometry, thermogravimetric measurements and UV-VIS, FT-IR, EPR, Raman and 1H-NMR spectroscopies. In addition, the binding to bovine serum albumin (BSA) was studied at the physiological conditions (pH = 7.4) by fluorescence spectroscopy.
Assuntos
Antineoplásicos , Complexos de Coordenação , Neoplasias Pulmonares , Antineoplásicos/química , Antioxidantes/farmacologia , Cátions , Complexos de Coordenação/química , Cobre/química , Flavanonas , Dissulfeto de Glutationa , Humanos , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Fenantrolinas/farmacologia , Espécies Reativas de Oxigênio , Soroalbumina Bovina/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s-1) lower than that of wt SmNir (kcat=240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.
Assuntos
Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Nitrito Redutases/metabolismo , Sinorhizobium meliloti/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cobre/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Nitrito Redutases/química , Nitrito Redutases/genética , Nitritos/metabolismo , Oxirredução , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes/metabolismo , Sinorhizobium meliloti/genética , Espectrofotometria UltravioletaRESUMO
The activity of sulfate-reducing bacteria (SRB) intensifies the problems associated to corrosion of metals and the solution entails significant economic costs. Although molybdate can be used to control the negative effects of these organisms, the mechanisms triggered in the cells exposed to Mo-excess are poorly understood. In this work, the effects of molybdate ions on the growth and morphology of the SRB Desulfovibrio alaskensis G20 (DaG20) were investigated. In addition, the cellular localization, ion uptake and regulation of protein expression were studied. We found that molybdate concentrations ranging between 50 and 150 µM produce a twofold increase in the doubling time with this effect being more significant at 200 µM molybdate (five times increase in the doubling time). It was also observed that 500 µM molybdate completely inhibits the cellular growth. On the context of protein regulation, we found that several enzymes involved in energy metabolism, cellular division and metal uptake processes were particularly influenced under the conditions tested. An overall description of some of the mechanisms involved in the DaG20 adaptation to molybdate-stress conditions is discussed.
Assuntos
Desulfovibrio/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Molibdênio/metabolismo , Proliferação de Células/efeitos dos fármacos , Desulfovibrio/efeitos dos fármacos , Desulfovibrio/crescimento & desenvolvimento , Íons/química , Íons/metabolismo , Íons/toxicidade , Molibdênio/toxicidadeRESUMO
Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.
Assuntos
Azurina/metabolismo , Elétrons , Nitrito Redutases/metabolismo , Sinorhizobium meliloti/química , Azurina/química , Azurina/genética , Oxirredução , Sinorhizobium meliloti/metabolismo , TermodinâmicaRESUMO
The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, ß = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Desulfovibrio/enzimologia , Compostos de Tungstênio/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Desulfovibrio/efeitos dos fármacos , Dados de Sequência Molecular , Molibdênio/farmacologia , Periplasma/metabolismo , Ligação ProteicaRESUMO
Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 µM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 µM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.
Assuntos
Desulfovibrio/enzimologia , Formiato Desidrogenases/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Molibdênio/farmacologia , Tungstênio/farmacologia , Desulfovibrio/metabolismo , Formiato Desidrogenases/genéticaRESUMO
Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.
Assuntos
Formiato Desidrogenases/química , Formiato Desidrogenases/isolamento & purificação , Formiatos/química , Modelos Moleculares , Molibdênio/química , Tungstênio/química , Dióxido de Carbono/química , Catálise , Simulação por Computador , Desulfovibrio/enzimologia , Desulfovibrio/metabolismo , Desulfovibrio desulfuricans/enzimologia , Desulfovibrio desulfuricans/metabolismo , Desulfovibrio gigas/enzimologia , Desulfovibrio gigas/metabolismo , Elétrons , Cinética , Conformação Molecular , Oxirredução , Conformação ProteicaRESUMO
The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 µM, V = 9.4 ± 0.5 µM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.
Assuntos
Membrana Celular/metabolismo , Nitrato Redutases/metabolismo , Nitratos/metabolismo , Sinorhizobium meliloti/metabolismo , Azidas/farmacologia , Cloratos/farmacologia , Cianetos/farmacologia , Cinética , Molibdênio/metabolismo , Nitrato Redutases/antagonistas & inibidores , Oxirredução , Sinorhizobium meliloti/enzimologia , Relação Estrutura-AtividadeRESUMO
Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ â Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 â T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π â Cu2+ LMCT transitions.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/metabolismo , Grupo dos Citocromos c/metabolismo , Nitrito Redutases/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Clonagem Molecular , Cobre/metabolismo , Grupo dos Citocromos c/genética , Nitrito Redutases/genética , Oxirredução , Regulação para CimaRESUMO
The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.
Assuntos
Proteínas de Bactérias/biossíntese , Desulfovibrio/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Metaloproteínas/biossíntese , Molibdênio/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Desulfovibrio/fisiologia , Metaloproteínas/genética , Dados de Sequência Molecular , Molibdênio/fisiologiaRESUMO
The EPR characterization of the molybdenum(V) forms obtained on formate reduction of both as-prepared and inhibited formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774, an enzyme that catalyzes the oxidation of formate to CO(2), is reported. The Mo(V) EPR signal of the as-prepared formate-reduced enzyme is rhombic (g(max)=2.012, g(mid)=1.996, g(min)=1.985) and shows hyperfine coupling with two nuclear species with I=1/2. One of them gives an anisotropic splitting and is not solvent exchangeable (A(max)=11.7, A(mid)=A(min)=non-detectable, A-values in cm(-1)x10(-4)). The second species is exchangeable with solvent and produces a splitting at the three principal g-values (A(max)=7.7, A(mid)=10.0, A(min)=9.3). The hyperfine couplings of the non-solvent and solvent exchangeable nuclei are assigned to the hydrogen atoms of the beta-methylene carbon of a selenocysteine and to a Mo ligand whose nature, sulfydryl or hydroxyl, is still in debate. The Mo(V) species obtained in the presence of inhibitors (azide or cyanide) yields a nearly axial EPR signal showing only one detectable splitting given by nuclear species with I=1/2 (g(max)=2.092, g(mid)=2.000, g(min)=1.989, A(max)=non-detectable, A(mid)=A(min)=7.0), which is originated from the alpha-proton donated by the formate to a proximal ligand of the molybdenum. The possible structures of both paramagnetic molybdenum species (observed upon formate reduction in presence and absence of inhibitors) are discussed in comparison with the available structural information of this enzyme and the structural and EPR properties of the closely related formate dehydrogenase-H from Escherichia coli.
Assuntos
Desulfovibrio desulfuricans/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Formiato Desidrogenases/química , Formiatos/química , Hidrogenase/química , Molibdênio/química , Complexos Multienzimáticos/química , Formiato Desidrogenases/metabolismo , Hidrogenase/metabolismo , Cinética , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , OxirreduçãoRESUMO
Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO42-, WO42- and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.
Assuntos
Desulfovibrio desulfuricans/enzimologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Compostos de Tungstênio/metabolismo , Substituição de Aminoácidos , Calorimetria , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
Aldehyde oxidoreductase (AOR) activity has been found in a number of sulfate-reducing bacteria. The enzyme that is responsible for the conversion of aldehydes to carboxylic acids is a mononuclear molybdenum enzyme belonging to the xanthine oxidase family. We report here the purification and characterization of AOR isolated from the sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254, an aminolytic strain performing thiosulfate dismutation. The enzyme is a homodimer (ca. 200 kDa), containing a molybdenum centre and two [2Fe-2S] clusters per monomer. UV/Visible and electron paramagnetic resonance (EPR) spectra of D. aminophilus AOR recorded in as-prepared and reduced states are similar to those obtained in AORs from Desulfovibrio gigas, Desulfovibrio desulfuricans and Desulfovibrio alaskensis. Despite AOR from D. aminophilus is closely related to other AORs, it presents lower activity towards aldehydes and no activity towards N-heterocyclic compounds, which suggests another possible role for this enzyme in vivo. A comparison of the molecular and EPR properties of AORs from different Desulfovibrio species is also included.
Assuntos
Aldeído Oxirredutases/química , Desulfovibrio/enzimologia , Coenzimas/química , Desulfovibrio/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas Ferro-Enxofre/química , Cinética , Metaloproteínas/química , Peso Molecular , Cofatores de Molibdênio , Oxirredução , Pteridinas/química , Especificidade da Espécie , Espectrofotometria UltravioletaRESUMO
Chlorite dismutase (Cld) is a b-type heme containing enzyme that catalyzes the reduction of chlorite into chloride plus dioxygen. This enzyme has gained attention because it can be used in the development of bioremediation processes, biosensors, and controlled dioxygen production. In the present work, Cld was purified from Magnetospirillum sp. cells cultured anaerobically with acetate/perchlorate until stationary phase. Biochemical, spectroscopic and X-ray crystallography methods showed that Cld from Magnetospirillum sp. is a ~140 kDa homopentamer comprising ~27.8 kDa monomers. Preliminary X-ray crystallography studies confirmed the quaternary structure and the presence of one b-type heme per monomer. The EPR spectroscopic signature of the as-purified Cld samples is affected by the buffer composition used during the purification. Potassium phosphate buffer is the only buffer that affected neither the spectral nor the kinetic properties of Cld. Kinetic studies in solution revealed that Cld from Magnetospirillum sp. decomposes chlorite at high turnover rates with optimal pH6.0. A temperature below 10 °C is required to avoid enzyme inactivation due to cofactor bleaching during turnover, and to achieve full substrate consumption. Cld kinetic parameters were not affected when kinetic assays were performed in the presence of air or under argon atmosphere, but chloride is a weak mixed inhibitor that modifies the EPR signal of as-prepared samples.
Assuntos
Magnetospirillum/enzimologia , Modelos Moleculares , Oxirredutases/química , Oxirredutases/metabolismo , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Heme/química , Concentração de Íons de Hidrogênio , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.
Assuntos
Azidas/química , Imidazóis/química , Magnetospirillum/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Tiocianatos/química , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Oxirredutases/isolamento & purificaçãoRESUMO
The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cobre , Desulfovibrio/química , Ferro , Metaloproteínas/química , Metaloproteínas/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desulfovibrio/genética , Desulfovibrio/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/fisiologia , Metaloproteínas/genética , Metaloproteínas/metabolismo , Molibdênio/química , Molibdênio/farmacologia , Estrutura Quaternária de Proteína/fisiologiaRESUMO
Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes.
Assuntos
Bactérias/enzimologia , Enzimas/química , Molibdênio/química , Sulfatos/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Enzimas/genética , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Modelos Moleculares , EstereoisomerismoRESUMO
Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe-4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to -500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe-4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.