21 research outputs found
Source and reduction of nitrous oxide
PTDC/BBB-BQB/0129/2014.
UID/QUI/50006/2019.
UlD/Multi/04378/2019.
MSPC acknowledges FCT/MCTES for funding her "Research Position" (signed with FCT NOVA in accordance with DL.57/2016 and Lei 57/2017).
Sem PDF conforme despacho.Nitrous oxide is a potent greenhouse gas with a global warming impact 300-fold higher than carbon dioxide. Due to its exponential increase in the atmosphere and its implications in climate change there is the need to develop strategies to mitigate its emissions and to reduce it to the inert dinitrogen gas. Only three enzymes have been reported to be able to reduce nitrous oxide, namely nitrogenase, one multicopper oxidase and nitrous oxide reductase, with the latter being the only one with a relevant physiological activity. In this enzyme, reduction of nitrous oxide occurs in a unique catalytic tetranuclear sulfide center, named âCuZâ center, a complex center required to overcome the high activation barrier of this reaction. Nitrous oxide reductase can be isolated with âCuZâ center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), that differ in their catalytic and spectroscopic properties. Recently, another step towards a better understanding of the catalytic and activation mechanism of this enzyme was taken by identifying and spectroscopically characterizing an intermediate species of its catalytic cycle, CuZ 0 . A different approach for N 2 O reduction can be attained using model compounds. The unique structural motif present in âCuZâ center, a Cu 4 (” 4 -S), has been a challenge for inorganic synthesis but several synthetic clusters that mimic different forms of âCuZâ center have been reported. Model compounds for the oxidation states involved in N 2 O reduction are also available. The advances in this area will be discussed in light of the recent data, with structural and functional model compounds of N 2 OR active site.authorsversionpublishe
Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide
PB would thank the PTNMRPhD (PD/00065/2013). VLP thanks the NIH for support (GM141086).The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ⌠â408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UVâvisible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.publishersversionpublishe
The heme-based oxygen sensor Rhizobium etli FixL: influence of auxiliary ligands on heme redox potential and implications on the enzyme activity
Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the Fe redox couple of the heme domain. In this work, midpoint potentials (E) assigned to the Fe redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+ 19 mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. E = â 51, â 57 and â 156 mV for O, imidazole and CN, respectively. Upon binding to CO, which does not affect the kinase active, E was observed at + 21 mV. The potential values observed for Fe of the heme domain of ReFixL upon binding to CO and O do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the Fe state. Our results suggest that the conformational changes that switch off kinase activity upon O binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the Fe state and counterbalances the Fe-stabilizing influence of the O ligand
Molybdenum Induces the expression of a protein containing a new heterometallic Mo-Fe cluster in desulfoVibrio alaskensis
Biochemistry. 2009 Feb 10;48(5):873-82. doi: 10.1021/bi801773t.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
Synergy of DNA intercalation and catalytic activity of a copper complex towards improved polymerase inhibition and cancer cell cytotoxicity
Improving the binding of metal complexes to DNA to boost cancer cell cytotoxicity requires fine tuning of their structural and chemical properties. Copper has been used as a metal center in compounds containing intercalating ligands due to its ability to catalytically generate reactive oxygen species (ROS), such as hydroxyl radicals (OHË). We envision the synergy of DNA binding and ROS generation in proximity to target DNA as a powerful chemotherapy treatment. Here, we explore the use of [Cu(2CP-Bz-SMe)]2+(2CP-Bz-SMe = 1,3-bis(1,10-phenanthrolin-2-yloxy)-N-(4-(methylthio)benzylidene)propan-2-amine) for this purpose by characterizing its cytotoxicity, DNA binding, and ability to affect DNA replication through the polymerase chain reaction - PCR and nuclease assays. We determined the binding (Kb) and Stern-Volmer constants (KSV) for complex-DNA association of 5.8 ± 0.14 Ă 104and 1.64 (±0.08), respectively, through absorption titration and competitive fluorescence experiments. These values were superior to those of other Cu-complex intercalators. We hypothesize that the distorted trigonal bipyramidal geometry of [Cu(2CP-Bz-SMe)]2+allows the phenanthroline fragments to be better accommodated into the DNA double helix. Moreover, the aromaticity of these fragments increases the local hydrophobicity thus increasing the affinity for the hydrophobic domains of DNA. Nuclease assays in the presence of common reducing agents ascorbic acid, nicotinamide adenine dinucleotide, and glutathione showed the effective degradation of DNA due to thein situgeneration of OHË. The [Cu(2CP-Bz-SMe)]2+complex showed cytotoxicity against the following human cancer cells lines A549, MCF-7, MDA-MB-231 and MG-63 with half maximal inhibitory concentration (IC50) values of 4.62 ± 0.48, 5.20 ± 0.76, 5.70 ± 0.42 and 2.88 ± 0.66 ÎŒM, respectively. These low values of IC50, which are promising if compared to that of cisplatin, are ascribed to the synergistic effect of ROS generation with the intercalation ability into the DNA minor grooves and blocking DNA replication. This study introduces new principles for synergizing the chemical and structural properties of intercalation compounds for improved drug-DNA interactions targeting cancer.Fil: Romo, Adolfo I. B.. University of Illinois. Urbana - Champaign; Estados Unidos. Universidade Federal do Ceara; BrasilFil: Carepo, Marta P.. Universidade Nova de Lisboa; PortugalFil: Levin, Pedro. Universidad de Santiago de Chile; ChileFil: Nascimento, Otaciro R.. Universidade Federal do SĂŁo Carlos; BrasilFil: DĂaz, Daniel E.. Universidad de Santiago de Chile; ChileFil: Rodriguez Lopez, Joaquin. University of Illinois. Urbana - Champaign; Estados UnidosFil: Leon, Ignacio Esteban. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - La Plata. Centro de QuĂmica InorgĂĄnica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de QuĂmica InorgĂĄnica "Dr. Pedro J. Aymonino"; ArgentinaFil: Bezerra, Lucas F.. Universidade Federal do Ceara; BrasilFil: Lemus, Luis A.. Universidad de Santiago de Chile; ChileFil: DiĂłgenes, Izaura C. N.. Universidade Federal do Ceara; Brasi
The Tetranuclear Copper-Sulfide Center of Nitrous Oxide Reductase
Nitrous oxide reductase catalyzes the reduction of nitrous oxide (N2O) to dinitrogen (N2) and water at a catalytic tetranuclear copper sulfide center, named CuZ, overcoming the high activation energy of this reaction. In this center each Cu atom is coordinated by two imidazole rings of histidine side-chains, with the exception of one named CuIV. This enzyme has been isolated with CuZ in two forms CuZ(4Cu1S) and CuZ(4Cu2S), which differ in the CuI-CuIV bridging ligand, leading to considerable differences in their spectroscopic and catalytic properties. The Cu atoms in CuZ(4Cu1S) can be reduced to the [4Cu1+] oxidation state, and its catalytic properties are compatible with the nitrous oxide reduction rates of whole cells, while in CuZ(4Cu2S) they can only be reduced to the [1Cu2C-3Cu1C] oxidation state, which has a very low turnover number. The catalytic cycle of this enzyme has been explored and one of the intermediates, CuZ0, has recently been identified and shown to be in the [1Cu2+-3Cu1+] oxidation state. Contrary to CuZ(4Cu2S), CuZ0 is rapidly reduced intramolecularly by the electron transferring center of the enzyme, CuA, to [4Cu1+] by a physiologically relevant redox partner. The three-dimensional structure of nitrous oxide reductase with the CuZ center either as CuZ(4Cu1S) or as CuZ(4Cu2S) shows that it is a unique functional dimer, with the CuZ of one subunit receiving electrons from CuA of the other subunit. The complex nature of this center has posed some questions relative to its assembly, which are only partially answered, as well as to which is the active form of CuZ in vivo. The structural, spectroscopic, and catalytic features of the two forms of CuZ will be addressed here, as well as its assembly. The understanding of its catalytic features, activation, and assembly is essential to develop strategies to decrease the release of nitrous oxide to the atmosphere and to reduce its concentration in the stratosphere, as well as to serve as inspiration to synthetic inorganic chemists to develop new models of this peculiar and challenging copper sulfide center.publishersversionpublishe
Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis
The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Feâ4S] and one [4Feâ4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus.
From differential pulse voltammetry the following signals were identified: [3Fe-4S]+ 1/0 (E0âČ = â 158 ± 5 mV); [4Feâ4S]+ 2/+1 (E0âČ = â 474 ± 5 mV) and [3Feâ4S]0/â 2 (E0âČ = â 660 ± 5 mV). The effect of pH on these signals showed that the reduced [3Feâ4S]0 cluster has a pKÊčredâČ = 5.1 ± 0.1, the [4Feâ4S]+ 2/+1 centre is pH independent, and the [3Feâ4S]0/â2 reduction is accompanied by the binding of two protons. The ability of the [3Feâ4S]0 cluster to be converted into a new [4Feâ4S] cluster was proven. The redox potential of the original [4Feâ4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70 mV) of the redox potential of the original centre.
Being most [FeâS] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.Fil: Grazina, Raquel. Universidade Nova de Lisboa. Faculdade de CiĂȘncias e Tecnologia. Departamento de QuĂmica. REQUIMTE/CQFB; PortugalFil: Sousa, PatrĂcia M. Paes de. Universidade Nova de Lisboa. Faculdade de CiĂȘncias e Tecnologia. Departamento de QuĂmica. REQUIMTE/CQFB; PortugalFil: Brondino, Carlos Dante. Universidad Nacional del Litoral. Facultad de BioquĂmica y Ciencias BiolĂłgicas. Departamento de FĂsica; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Santa Fe; ArgentinaFil: Carepo, Marta S. P.. Universidade Nova de Lisboa. Faculdade de CiĂȘncias e Tecnologia. Departamento de QuĂmica. REQUIMTE/CQFB; PortugalFil: Moura, Isabel. Universidade Nova de Lisboa. Faculdade de CiĂȘncias e Tecnologia. Departamento de QuĂmica. REQUIMTE/CQFB; PortugalFil: Moura, JosĂ© J. G.. Universidade Nova de Lisboa. Faculdade de CiĂȘncias e Tecnologia. Departamento de QuĂmica. REQUIMTE/CQFB; Portuga
Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data
Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2âĄFC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments
Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis
International audienceA novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 +/- A 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde\â198) increased 6.6 +/- A 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria
Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceum ATCC 12472
Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field