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1.
FEBS J ; 291(6): 1275-1294, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38129989

RESUMO

Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O2 /H2 O2 and/or NO detoxification through the reduction of these species to H2 O or N2 O, respectively. All FDPs contain a minimal catalytic unit of two identical subunits, each one having a metallo-ß-lactamase-like domain harboring the catalytic diiron site, and a flavodoxin-like domain. However, more complex and diverse arrangements in terms of domains are found in this family, of which the class H enzymes are among the most complex. One of such FDPs is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp. wolfei str. Goettingen G311. Besides the core domains, this protein is predicted to have three additional ones after the flavodoxin core domain: two short-chain rubredoxins and a NAD(P)H:rubredoxin oxidoreductase-like domain. This enzyme, FDP_H, was produced and characterized and the presence of the predicted cofactors was investigated by a set of biochemical and spectroscopic methodologies. Syntrophomonas wolfei FDP_H exhibited a remarkable O2 reduction activity with a kcat = 52.0 ± 1.2 s-1 and a negligible NO reduction activity (~ 100 times lower than with O2 ), with NADH as an electron donor, that is, it is an oxygen-selective FDP. In addition, this enzyme showed the highest turnover value for H2 O2 reduction (kcat = 19.1 ± 2.2 s-1 ) ever observed among FDPs. Kinetic studies of site-directed mutants of iron-binding cysteines at the two rubredoxin domains demonstrated the essential role of these centers since their absence leads to a significant decrease or even abolishment of O2 and H2 O2 reduction activities.


Assuntos
Clostridiales , NAD , Oxirredutases , Oxirredutases/metabolismo , NAD/metabolismo , Flavodoxina/metabolismo , Cinética , Composição de Bases , Filogenia , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Oxigênio/metabolismo , Oxirredução
2.
Biophys J ; 120(23): 5395-5407, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34688593

RESUMO

Geobacter sulfurreducens possesses over 100 cytochromes that assure an effective electron transfer to the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA family, composed of five periplasmic triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome synthetized by G. sulfurreducens in Fe(III)-reducing conditions but is not present in cultures grown on fumarate. This cytochrome has a low sequence identity with the PpcA family cytochromes and a different heme coordination, based on the analysis of its amino acid sequence. In this work, amino acid sequence analysis, site-directed mutagenesis, and complementary biophysical techniques, including ultraviolet-visible, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopies, were used to characterize GSU0105. The cytochrome has a low percentage of secondary structural elements, with features of α-helices and ß-sheets. Nuclear magnetic resonance shows that the protein contains three low-spin hemes (Fe(II), S = 0) in the reduced state. Electron paramagnetic resonance shows that, in the oxidized state, one of the hemes becomes high-spin (Fe(III), S = 5/2), whereas the two others remain low-spin (Fe(III), S = 1/2). The data obtained also indicate that the heme groups have distinct axial coordination. The apparent midpoint reduction potential of GSU0105 (-154 mV) is pH independent in the physiological range. However, the pH modulates the reduction potential of the heme that undergoes the low- to high-spin interconversion. The reduction potential values of cytochrome GSU0105 are more distinct compared to those of the PpcA family members, providing the protein with a larger functional working redox potential range. Overall, the results obtained, together with an amino acid sequence analysis of different multiheme cytochrome families, indicate that GSU0105 is a member of a new group of triheme cytochromes.


Assuntos
Proteínas de Bactérias , Citocromos , Compostos Férricos , Geobacter/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocromos/química , Citocromos/genética , Heme/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução
3.
Environ Microbiol ; 23(8): 4112-4125, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245087

RESUMO

Clostridia comprise bacteria of environmental, biotechnological and medical interest and many commensals of the gut microbiota. Because of their strictly anaerobic lifestyle, oxygen is a major stress for Clostridia. However, recent data showed that these bacteria can cope with O2 better than expected for obligate anaerobes through their ability to scavenge, detoxify and consume O2 . Upon O2 exposure, Clostridia redirect their central metabolism onto pathways less O2 -sensitive and induce the expression of genes encoding enzymes involved in O2 -reduction and in the repair of oxidized damaged molecules. While Faecalibacterium prausnitzii efficiently consumes O2 through a specific extracellular electron shuttling system requiring riboflavin, enzymes such as rubrerythrins and flavodiiron proteins with NAD(P)H-dependent O2 - and/or H2 O2 -reductase activities are usually encoded in other Clostridia. These two classes of enzymes play indeed a pivotal role in O2 tolerance in Clostridioides difficile and Clostridium acetobutylicum. Two main signalling pathways triggering O2 -induced responses have been described so far in Clostridia. PerR acts as a key regulator of the O2 - and/or reactive oxygen species-defence machinery while in C. difficile, σB , the sigma factor of the general stress response also plays a crucial role in O2 tolerance by controlling the expression of genes involved in O2 scavenging and repair systems.


Assuntos
Clostridioides difficile , Clostridium acetobutylicum , Clostridium/genética , Oxigênio , Fator sigma
5.
mBio ; 11(5)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900801

RESUMO

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2 O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Trato Gastrointestinal/fisiologia , Oxigênio/metabolismo , Anaerobiose , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/patogenicidade , Trato Gastrointestinal/microbiologia , Técnicas de Inativação de Genes , Hemeritrina/genética , Hemeritrina/metabolismo , Peróxido de Hidrogênio/metabolismo , Rubredoxinas/genética , Rubredoxinas/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo
6.
Free Radic Biol Med ; 140: 36-60, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30735841

RESUMO

Microbial anaerobes are exposed in the natural environment and in their hosts, even if transiently, to fluctuating concentrations of oxygen and its derived reactive species, which pose a considerable threat to their anoxygenic lifestyle. To counteract these stressful conditions, they contain a multifaceted array of detoxifying systems that, in conjugation with cellular repairing mechanisms and in close crosstalk with metal homeostasis, allow them to survive in the presence of O2 and reactive oxygen species. Some of these systems are shared with aerobes, but two families of enzymes emerged more recently that, although not restricted to anaerobes, are predominant in anaerobic microbes. These are the iron-containing superoxide reductases, and the flavodiiron proteins, endowed with O2 and/or NO reductase activities, which are the subject of this Review. A detailed account of their physicochemical, physiological and molecular mechanisms will be presented, highlighting their unique properties in allowing survival of anaerobes in oxidative stress conditions, and comparing their properties with the most well-known detoxifying systems.


Assuntos
Ferro/metabolismo , Estresse Oxidativo , Oxirredutases/metabolismo , Proteínas/metabolismo , Anaerobiose/genética , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Sci Rep ; 8(1): 10164, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29977056

RESUMO

Flavodiiron proteins (FDPs) are enzymes with a minimal core of two domains: a metallo-ß-lactamase-like, harbouring a diiron center, and a flavodoxin, FMN containing, domains. FDPs are O2 or NO reducing enzymes; for many pathogens, they help mitigate the NO produced by the immune system of the host, and aid survival during fluctuating concentrations concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. Clostridium difficile, a threatening human pathogen, encodes two FDPs: one with the two canonical domains, and another with a larger polypeptide chain of 843 amino acids, CD1623, with two extra domains, predicted to be a short-rubredoxin-like and an NAD(P)H:rubredoxin oxidoreductase. This multi-domain protein is the most complex FDP characterized thus far. Each of the predicted domains was characterized and the presence of the predicted cofactors confirmed by biochemical and spectroscopic analysis. Results show that this protein operates as a standalone FDP, receiving electrons directly from NADH, and reducing oxygen to water, precluding the need for extra partners. CD1623 displayed negligible NO reductase activity, and is thus considered an oxygen selective FDP, that may contribute to the survival of C. difficile in the human gut and in the environment.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/metabolismo , Flavoproteínas/metabolismo , Ferro/metabolismo , NAD/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Sequência de Aminoácidos , Anaerobiose , Proteínas de Bactérias/química , Biocatálise , Flavoproteínas/química , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Oxirredutases/química , Domínios Proteicos , Espectrofotometria Ultravioleta
8.
FEMS Microbiol Lett ; 365(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29240952

RESUMO

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-ß-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.


Assuntos
Flavoproteínas Transferidoras de Elétrons/química , Variação Genética , Óxido Nítrico/metabolismo , Oxirredutases/química , Oxigênio/metabolismo , Motivos de Aminoácidos , Catálise , Flavoproteínas Transferidoras de Elétrons/classificação , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Flavinas/metabolismo , Ferro/metabolismo , Modelos Moleculares , Oxirredutases/classificação , Oxirredutases/genética , Oxirredutases/metabolismo , Domínios Proteicos
9.
Appl Microbiol Biotechnol ; 99(20): 8329-36, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26278535

RESUMO

The increasing dependency on fossil fuels has driven researchers to seek for alternative energy sources. Renewable energy sources such as sunlight, wind, or water are the most common. However, since the 1990s, other sources for energy production have been studied. The use of microorganisms such as bacteria or archaea to produce energy is currently in great progress. These present several advantages even when compared with other renewable energy sources. Besides the energy production, they are also involved in bioremediation such as the removal of heavy metal contaminants from soils or wastewaters. Several research groups have demonstrated that these organisms are able to interact with electrodes via heme and non-heme iron proteins. Therefore, the role of iron as well as iron metabolism in these species must be of enormous relevance. Recently, the influence of cellular iron regulation by Fur in the Geobacter sulfurreducens growth and ability to produce energy was demonstrated. In this review, we aim to briefly describe the most relevant proteins involved in the iron metabolism of bacteria and archaea and relate them and their biological function with the ability of selected organisms to produce energy.


Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Fontes de Energia Bioelétrica , Eletricidade , Eletrodos/microbiologia , Ferro/metabolismo , Archaea/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento
10.
Radiat Environ Biophys ; 54(1): 111-121, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25249071

RESUMO

Organisms are often exposed to different types of ionizing radiation that, directly or not, will promote damage to DNA molecules and/or other cellular structures. Because of that, organisms developed a wide range of response mechanisms to deal with these threats. Endonuclease III is one of the enzymes responsible to detect and repair oxidized pyrimidine base lesions. However, the effect of radiation on the structure/function of these enzymes is not clear yet. Here, we demonstrate the effect of UV-C radiation on E. coli endonuclease III through several techniques, namely UV-visible, fluorescence and Mössbauer spectroscopies, as well as SDS-PAGE and electrophoretic mobility shift assay. We demonstrate that irradiation with a UV-C source has dramatic consequences on the absorption, fluorescence, structure and functionality of the protein, affecting its [4Fe-4S] cluster and its DNA-binding ability, which results in its inactivation. An UV-C radiation-induced conversion of the [4Fe-4S](2+) into a [2Fe-2S](2+) was observed for the first time and proven by Mössbauer and UV-visible analysis. This work also shows that the DNA-binding capability of endonuclease III is highly dependent of the nuclearity of the endogenous iron-sulfur cluster. Thus, from our point of view, in a cellular context, these results strengthen the argument that cellular sensitivity to radiation can also be due to loss of radiation-induced damage repair ability.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/efeitos da radiação , Proteínas de Escherichia coli/efeitos da radiação , Proteínas Ferro-Enxofre/efeitos da radiação , Raios Ultravioleta , DNA/metabolismo , Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Análise Espectral
11.
Radiat Environ Biophys ; 52(2): 211-20, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23412012

RESUMO

Several studies have sought to understand the health effects of occupational exposure to cosmic radiation. However, only few biologic markers or associations with disease outcomes have so far been identified. In the present study, 22 long- and 26 medium-haul male Portuguese airline pilots and 36 factory workers who did not fly regularly were investigated. The two groups were comparable in age and diet, were non-smokers, never treated with ionizing radiation and other factors. Cosmic radiation exposure in pilots was quantified based on direct monitoring of 51 flights within Europe, and from Europe to North and South America, and to Africa. Indirect dose estimates in pilots were performed based on the SIEVERT (Système informatisé d'évaluation par vol de l'exposition au rayonnement cosmique dans les transports aériens) software for 6,039 medium- and 1,366 long-haul flights. Medium-haul pilots had a higher cosmic radiation dose rate than long-haul pilots, that is, 3.3 ± 0.2 µSv/h and 2.7 ± 0.3 µSv/h, respectively. Biological tests for oxidative stress on blood and urine, as appropriate, at two time periods separated by 1 year, included measurements of antioxidant capacity, total protein, ferritin, hemoglobin, creatinine and 8-hydroxy-2-deoxyguanosine (8OHdG). Principal components analysis was used to discriminate between the exposed and unexposed groups based on all the biological tests. According to this analysis, creatinine and 8OHdG levels were different for the pilots and the unexposed group, but no distinctions could be made among the medium- and the long-haul pilots. While hemoglobin levels seem to be comparable between the studied groups, they were directly correlated with ferritin values, which were lower for the airline pilots.


Assuntos
Aeronaves , Radiação Cósmica , Exposição Ocupacional/análise , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Creatinina/urina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Ferritinas/sangue , Hemoglobinas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Análise de Componente Principal , Doses de Radiação
12.
J Am Chem Soc ; 134(26): 10822-32, 2012 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-22681596

RESUMO

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a µ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.


Assuntos
Ceruloplasmina/metabolismo , Ferritinas/metabolismo , Compostos Ferrosos/metabolismo , Proteínas de Bactérias/química , Ceruloplasmina/química , Desulfovibrio vulgaris/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Ferritinas/química , Compostos Ferrosos/química
13.
Biochem J ; 446(1): 125-33, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22642556

RESUMO

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H(2)O(2) oxidizes Fe(2+) ions at much higher reaction rates than O(2). The H(2)O(2) oxidation of two Fe(2+) ions was proven by Mössbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mössbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H(2)O(2) as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , DNA Bacteriano/metabolismo , Desulfovibrio vulgaris/metabolismo , Ferritinas/metabolismo , Ferro/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Clonagem Molecular , Grupo dos Citocromos b/efeitos dos fármacos , Grupo dos Citocromos b/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Ferritinas/efeitos dos fármacos , Ferritinas/genética , Peróxido de Hidrogênio/farmacologia , Oxirredução , Estresse Oxidativo , Espectrofotometria Ultravioleta , Espectroscopia de Mossbauer
14.
Biochem J ; 438(3): 485-94, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21682694

RESUMO

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 × 107 M⁻¹ · s⁻¹ and 1.3 × 106 M⁻¹ · s⁻¹ for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 × 106 M⁻¹ · s⁻¹ for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.


Assuntos
Oxirredutases/química , Desulfovibrio vulgaris/enzimologia , Desulfovibrio vulgaris/metabolismo , Eletroquímica , Transporte de Elétrons , Ferro/química , Ferro/metabolismo , Cinética , Oxirredução , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio , Rubredoxinas/química , Rubredoxinas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Superóxidos/química , Superóxidos/metabolismo
15.
J Inorg Biochem ; 103(10): 1314-22, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19699535

RESUMO

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/fisiologia
16.
Org Biomol Chem ; 5(1): 69-77, 2007 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-17164909

RESUMO

The synthetic flavylium salt 6-hydroxy-4'-(dimethylamino)flavylium hexafluorophosphate displays a set of pH-driven chemical reactions in aqueous solutions, involving the formation of hemiketal species and chalcones with cis and trans configurations. Such reactions were studied by steady-state and transient UV-Vis spectroscopy and by stopped-flow techniques. A novel and more generalized kinetic scheme is developed, in order to take account of possible acid/base pairs that occur in the network of chemical reactions as the pH is changed. It is found that the formation of the hemiketal species by hydration of the flavylium is slow, and it is not possible to isolate each process that leads to the formation of the cis-chalcone (hydration and tautomerization reactions). The cis/trans isomerization reaction of cis-chalcone is slow, and the system takes several hours to reach equilibrium after a pH jump at room temperature. In basic conditions, negatively charged trans-chalcones are dominant. Comparison with other flavylium compounds shows that the hydration process is affected mainly by the amino group, while the hydroxyl group influences the tautomerization and isomerization reactions.

17.
Org Biomol Chem ; 2(19): 2802-8, 2004 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-15455153

RESUMO

The synthetic flavylium salt 7-(N,N-diethylamino)-4'-hydroxyflavylium tetrafluoroborate gives rise in aqueous solution to a complex network of chemical reactions driven by pH. The system was studied by 1H NMR, single crystal X-ray diffraction, steady state and transient UV-Vis spectrophotometry as well as stopped flow. The crystal structure shows a high degree of coplanarity between the pyrylium system and the phenyl group in position 2. Thermodynamic and kinetic constants for the pH dependent network of chemical reactions were obtained. The introduction of an amino group in position 7 allows formation of protonated species leading, in particular, to a tautomeric form of the protonated cis-chalcone, H+, whose absorption spectra is rather red shifted, in comparison with the correspondent protonated trans-chalcone, H+. The H+ species can be rapidly converted into the flavylium cation through a first order process with lifetime of 0.2 s at pH = 2.35. This new reaction channel confers this compound a peculiar behaviour in acidic media, allowing to define an unidirectional pH driven reaction cycle.


Assuntos
Etilaminas/química , Etilaminas/síntese química , Flavonoides/química , Cristalografia por Raios X , Flavonoides/síntese química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Termodinâmica
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