RESUMO
Building stimuli-responsive supramolecular systems is a way for chemists to achieve spatio-temporal control over complex systems as well as a promising strategy for applications ranging from sensing to drug-delivery. For its large spectrum of biological and biomedical implications, adenosine 5'-triphosphate (ATP) is a particularly interesting target for such a purpose but photoresponsive ATP-based systems have mainly been relying on covalent modification of ATP. Here, we show that simply mixing ATP with AzoDiGua, an azobenzene-guanidium compound with photodependent nucleotide binding affinity, results in the spontaneous self-assembly of the two non-fluorescent compounds into photoreversible, micrometer-sized and fluorescent aggregates. Obtained in water at room temperature and physiological pH, these supramolecular structures are dynamic and respond to several chemical, physical and biological stimuli. The presence of azobenzene allows a fast and photoreversible control of their assembly. ATP chelating properties to metal dications enable ion-triggered disassembly and fluorescence control with valence-selectivity. Finally, the supramolecular aggregates are disassembled by alkaline phosphatase in a few minutes at room temperature, resulting in enzymatic control of fluorescence. These results highlight the interest of using a photoswitchable nucleotide binding partner as a self-assembly brick to build highly responsive supramolecular entities involving biological targets without the need to covalently modify them.
RESUMO
Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.
Assuntos
Proteínas Ferro-Enxofre , Mathanococcus , Humanos , Mathanococcus/genética , Uridina/metabolismo , Cisteína/metabolismo , Biossíntese de Proteínas , RNA de Transferência/genética , Enxofre/metabolismo , Proteínas Ferro-Enxofre/metabolismoRESUMO
Control of the transmetalation degree of organoiron(II) species is a critical parameter in numerous Fe-catalyzed cross-couplings to ensure the success of the process. In this report, we however demonstrate that the selective formation of a monotransmetalated FeII species during the catalytic regime counterintuitively does not alone ensure an efficient suppression of the nucleophile homocoupling side reaction. It is conversely shown that a fine control of the transmetalation degree of the transient FeIII intermediates obtained after the activation of alkyl electrophiles by a single-electron transfer (SET), achievable using σ-donating additives, accounts for the selectivity of the cross-coupling pathway. This report shows for the first time that both coordination spheres of FeII resting states and FeIII short-lived intermediates must be efficiently tuned during the catalytic regime to ensure high coupling selectivities.
RESUMO
Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli , Ferro/metabolismo , RNA de Transferência/metabolismo , Enxofre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Copper acetate and copper resinate pigments are bimetallic CuII complexes in which metal atoms are bridged by four carboxylate ligands (acetate or abietate). Prepared with lindseed oil as binder, these green pigments were particularly used in easel paintings between the 15th and 17th centuries. Unfortunately, they had the tendency to darken in an irreversible way, explaining why they fell into disuse. The darkening mechanism of films of copper pigments in linseed oil is studied by electron paramagnetic resonance (EPR) and by optical absorption spectroscopy (OAS). EPR and OAS reveal different chemical and photochemical behaviors depending on the type of copper complex and on the binding oil. The effect of light is investigated by illuminating the films at â¼410 nm in the bridging ligand-to-metal charge transfer (LMCT) transition. The photodarkening manifests itself as the appearance of an optical absorption band around 22â¯000 cm-1 and a decrease of the EPR intensity of bimetallic copper complexes. These effects are explained by the photoinduced substitution of acetate (or abietate) bridging ligands by dioxygen molecules from ambient atmosphere. The resulting peroxo-CuII dimer is characterized by a red shift of the LMCT and an increase of the exchange interaction in the ground state, which is responsible for the decrease of the EPR intensity due to the depletion of the paramagnetic S = 1 state. This mechanism explains the differences in darkening intensity observed with different pigment compositions (resinate versus acetate, raw linseed oil versus boiled linseed oil).
RESUMO
It is generally believed that CO2 electroreduction to multi-carbon products such as ethanol or ethylene may be catalyzed with significant yield only on metallic copper surfaces, implying large ensembles of copper atoms. Here, we report on an inexpensive Cu-N-C material prepared via a simple pyrolytic route that exclusively feature single copper atoms with a CuN4 coordination environment, atomically dispersed in a nitrogen-doped conductive carbon matrix. This material achieves aqueous CO2 electroreduction to ethanol at a Faradaic yield of 55 % under optimized conditions (electrolyte: 0.1 m CsHCO3 , potential: -1.2â V vs. RHE and gas-phase recycling set up), as well as CO electroreduction to C2 -products (ethanol and ethylene) with a Faradaic yield of 80 %. During electrolysis the isolated sites transiently convert into metallic copper nanoparticles, as shown by operando XAS analysis, which are likely to be the catalytically active species. Remarkably, this process is reversible and the initial material is recovered intact after electrolysis.
RESUMO
Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O2 Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O2 range.IMPORTANCE In order to colonize environments with large O2 gradients or fluctuating O2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O2 In contrast, the long-described pathway for ubiquinone biosynthesis requires O2 as a substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2 range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O2 levels.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxigênio/metabolismo , Ubiquinona/biossíntese , Anaerobiose , Escherichia coli/genéticaRESUMO
Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds. The recombinant purified protein produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro. A shorter protein version devoid of its N-terminal putative chloroplast transit peptide, Δ46AtDGAT3, was more stable in vitro, allowing biochemical and spectroscopic characterization. The results obtained demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Diacilglicerol O-Aciltransferase/química , Diacilglicerol O-Aciltransferase/metabolismo , Escherichia coli/crescimento & desenvolvimento , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/química , Cloroplastos/metabolismo , Diacilglicerol O-Aciltransferase/genética , Escherichia coli/genética , Germinação , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Sementes/fisiologia , Tiorredoxinas/metabolismoRESUMO
The contribution of copper complexes of salen-based Schiff bases N, N'-bis(salicylidene)ethylenediamine (C1), N, N'-bis(4-hydroxysalicylidene)ethylenediamine (C2), and N, N'-bis(5-hydroxysalicylidene)ethylenediamine (C3) to the flame retardancy of thermoplastic polyurethane (TPU) is investigated in the context of minimizing the inherent flammability of TPU. Thermal and fire properties of TPU are evaluated. It is observed that fire performances vary depending upon the substitution of the salen framework. Cone calorimetry [mass loss calorimetry (MLC)] results show that, in TPU at 10 wt % loading, C2 and C3 reduce the peak of heat release rate by 46 and 50%, respectively. At high temperature, these copper complexes undergo polycondensation leading to resorcinol-type resin in the condensed phase and thus acting as intumescence reinforcing agents. C3 in TPU is particularly interesting because it delays significantly the time to ignition (MLC experiment). In addition, pyrolysis combustion flow calorimetry shows reduction in the heat release rate curve, suggesting its involvement in gas-phase action. Structural changes of copper complexes and radical formation during thermal treatment as well as their influence on fire retardancy of TPU in the condensed phase are investigated by spectroscopic studies supported by microscopic and powder diffraction studies. Electron paramagnetic resonance (EPR) spectroscopy was fully used to follow the redox changes of Cu(II) ions as well as radical formation of copper complexes/TPU formulations in their degradation pathways. Pulsed EPR technique of hyperfine sublevel correlation spectroscopy reveals evolution of the local surrounding of copper and radicals with a strong contribution of nitrogen fragments in the degradation products. Further, the spin state of radicals was investigated by the two-dimensional technique of phase-inverted echo-amplitude detected nutation experiment. Two different radicals were detected, that is, one monocarbon radical and an oxygen biradical. Thus, the EPR study permits to deeply investigate the mode of action of copper salen complexes in TPU.
RESUMO
Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a protein-bound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.
Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência/química , Compostos de Sulfidrila/química , Enxofre/química , Sítios de Ligação , Catálise , Clonagem Molecular , Genoma Bacteriano , Proteínas Ferro-Enxofre/química , Modelos Biológicos , Família Multigênica , Oxirredução , RNA de Transferência/genética , Espectrofotometria Ultravioleta , Sulfurtransferases/genética , Thermotoga maritima/genéticaRESUMO
Electron paramagnetic resonance (EPR) spectra of mixtures are often difficult to interpret due to the superposition of spectral contribution of various species present in the complex materials. It is challenging to accurately identify the number of pure compounds present and to extract their pure spectra. In this study, the powerful chemometric method, multivariate curve resolution-alternating least squares (MCR-ALS), is applied to identify different paramagnetic centers. This method is used to simultaneously extract, with no prior knowledge, the pure spectra and the corresponding concentration profiles of all the compounds in the unknown and unresolved mixtures. The goal of our work is to apply, for the first time, this new chemometrics methodology, MCR-ALS, on EPR spectroscopic data in order to characterize a series of distinct but strongly overlapping spectra of various paramagnetic species.
Assuntos
Algoritmos , Artefatos , Técnicas de Química Combinatória/métodos , Misturas Complexas/análise , Misturas Complexas/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
FhaC is an integral outer membrane protein of the whooping cough agent Bordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/FhaC pair is a prototypic TpsA/TpsB system of the widespread 'Two-Partner Secretion' pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram-negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X-ray structure of FhaC shows that the transmembrane ß-barrel channel hypothesized to serve as the FHA-conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N-terminal α helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N-terminal 'plug' helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas de Secreção Bacterianos , Bordetella pertussis/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Conformação ProteicaRESUMO
The quest for new oxides with cations containing active lone-pair electrons (E) covers a broad field of targeted specificities owing to asymmetric electronic distribution and their particular band structure. Herein, we show that the novel compound BaCoAs2 O5, with lone-pair As(3+) ions, is built from rare square-planar Co(2+) O4 involved in direct bonding between As(3+) E and Co(2+) dz2 orbitals (Co-As=2.51â Å). By means of DFT and Hückel calculations, we show that this σ-type overlapping is stabilized by a two-orbital three-electron interaction allowed by the high-spin character of the Co(2+) ions. The negligible experimental spin-orbit coupling is expected from the resulting molecular orbital scheme in O3 AsE-CoO4 clusters.
RESUMO
Green and blue ultramarine pigments are characterized by the sodalite structure Na(6)(Al(6)Si(6)O(24)) and colored inserted species. These chromophores are sulfur species: S(3)(-) (blue) and S(2)(-) (yellow). Both radicals are encapsulated inside the ß-cages. They contribute to the EPR spectrum of ultramarine pigments. The well-known strong EPR signal observed in all ultramarine pigments Continuous-Wave (CW) spectra has long been assigned to S(3)(-) (g = 2.029). In contrast, the S(2)(-) contribution is still subject to controversy because its signal in ultramarine pigments was not resolved even at low temperature in CW-EPR experiments. In this study, we identify unambiguously for the first time by CW-EPR and field sweep-echo detected (FS-ED) EPR the signal of S(2)(-) in ultramarine pigments and we determine its tensor components: g(1) = 2.69(6), g(2) = 2.03(4) and g(3) = 1.86(4).
RESUMO
2-Hydroxyisoquinoline-1,3(2H,4H)-dione was recently discovered as a scaffold for the inhibition of HIV-1 integrase and the ribonuclease H function of HIV-1 reverse transcriptase. First, we investigate its interaction with Mg(2+) and Mn(2+) using different spectroscopic techniques and report that 2-hydroxyisoquinoline-1,3(2H,4H)-dione forms a 1:1 complex with Mg(2+) but a 1:2 complex with Mn(2+). The complex formation requires enolization of the ligand. ESR spectroscopy shows a redox reaction between the ligand and Mn(2+) producing superoxide anions. Second, 2-hydroxyisoquinoline-1,3(2H,4H)-dione, its magnesium complex, and its 4-methyl and 2-hydroxy-4-methoxycarbonylisoquinoline-1,3(2H,4H)-diones were tested as inhibitors of HIV-1 integrase, reverse transcriptase ribonuclease H, and DNA polymerase functions. Their antiviral activities were evaluated and 2-hydroxy-4-methoxycarbonyl-isoquinoline-1,3(2H,4H)-dione was found to inhibit the viral replication of HIV-1 in MT-4 cells. Cross-resistance was measured for this compound on three different viral strains. Experimental data suggest that the antiviral activity of 2-hydroxy-4-methoxycarbonylisoquinoline-1,3(2H,4H)-dione is probably due to the RNase H inhibition.
Assuntos
Quelantes/síntese química , Complexos de Coordenação/síntese química , Inibidores de Integrase de HIV/síntese química , HIV-1/efeitos dos fármacos , Isoquinolinas/síntese química , Magnésio , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Domínio Catalítico , Linhagem Celular , Quelantes/química , Quelantes/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Farmacorresistência Viral , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/enzimologia , Humanos , Isomerismo , Isoquinolinas/química , Isoquinolinas/farmacologia , Espectroscopia de Ressonância Magnética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC50's within the 1-10 microM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b-c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Naftalenos/química , Naftalenos/farmacologia , Fármacos Anti-HIV/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Integrase de HIV/síntese química , HIV-1/enzimologia , Humanos , Naftalenos/síntese químicaRESUMO
The design of profen hybrids containing a NO donor moiety connected to an aliphatic spacer led to compounds with a similar cyclooxygenase inhibition compared to their parent profen and with significant antiproliferative activities on PC3 cells. However, inhibition of COX-2 pathway alone did not seem sufficient to inhibit cancer cell proliferation, and NO-release in a time-dependent manner strongly contributes to this activity.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Química Farmacêutica/métodos , Óxido Nítrico/química , Neoplasias da Próstata/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Masculino , Modelos QuímicosRESUMO
Ferroquine (FQ or SR97193) is a unique ferrocene antimalarial drug candidate which just entered phase IIb clinical trials in autumn 2007. FQ is able to overcome the chloroquine (CQ) resistance problem, an important limit to the control of Plasmodium falciparum, the principal causative agent of malaria. However, as for other therapeutic agents such as chloroquine (CQ) and artemisin, its mechanism of action remains partially unknown. Most investigations have so far focused on comparing the activity of FQ to that of CQ in order to understand how the ferrocene core contributes to a stronger antiplasmodial activity. Studies have already shown that the ferrocene altered the shape, volume, lipophilicity, basicity and also electronic profile of the parent molecule and, hence, its pharmacodynamic behavior. However, few investigations have been undertaken to probe the real contribution of redox properties of the ferrocene (iron(II))/ferricinium (iron(III)) system in FQ as reported in this article. In our experimental and theoretical approach, we considered the redox profile of the ferrocene core of FQ in the specific conditions (acidic and oxidizing) of the parasitic digestive vacuole as a possible discriminating property from CQ in the antimalarial activity.