Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed αDIV-L21C three-helical bundle peptide.

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 αDIV-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-αDIV-L21C when compared to the apo-αDIV-L21C.

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.

Ascorbyl and hydroxyl radical generation mediated by a copper complex adsorbed on gold.

This work presents the results obtained for a thioether derivative of bipyridine, (E,Z)-1-(4'-methyl-[2,2'-bipyridine]-4-yl)-N-(4(methylthio)phenyl)methanimine (4-mbpy-Bz-SMe), and its copper complex [Cu(4-mbpy-Bz-SMe)]. Electronic spectra acquired at 183 K of the cuprous complex [Cu(4-mbpy-Bz-SMe)] generated in situ indicated the formation of the peroxodicopper compound {[Cu(4-mbpy-Bz-SMe)](μ-O)}. A gold electrode modified with [Cu(4-mbpy-Bz-SMe)] (Au/[Cu]) was fully characterized by SERS spectroscopy, electrochemistry and impedance spectroscopy thus showing adsorption occurs through the sulfur atom of the 4-mbpy-Bz-SMe moieties.

A spectroelectrochemical investigation of the heme-based sensor DevS from Mycobacterium tuberculosis: a redox versus oxygen sensor.

Tuberculosis is one of the oldest known infectious diseases, responsible for millions of deaths annually around the world. The ability of Mycobacterium tuberculosis (Mtb) to enter into a dormant state has been considered integral to the success of this bacterium as a human pathogen. One of the key systems involved in regulating the entrance into dormancy is the differentially expressed in virulent strain sensor protein (DevS) [(dormancy survival sensor protein (DosS)].

Effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin.

In this work, we have studied the effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin (Mb), and by means of electrochemical techniques, we have shown that this reaction is undoubtedly affected following the interaction with the venom. Surface plasmon resonance, electrophoresis, UV-Vis, and circular dichroism showed that the interaction involves the attachment of some constituent of the venom to the protein, although not affecting its first and secondary structures. Mass spectra support this suggestion by showing the appearance of signals assigned to the Mb dimer and to a new species resulting from the interaction between Mb and the venom proteins.

Magnetic nanoparticles as a support for a copper (II) complex with nuclease activity.

Magnetic nanoparticles have been extensively explored for the development of platforms for drug delivery and imaging probes. In this work, we have used a modular capping strategy to produce magnetic gold-coated FeO (FeO@Au) nanoparticles, which have been decorated with a copper (II) complex containing a thioether derivative of clip-phen (FeO@Au@Cu), where the complex [Cu(2CP-Bz-SMe)] has affinity to bind DNA and proven nuclease activity (2CP-Bz-SMe=1,3-bis((1,10-phenanthrolin-2-yl)oxy)-N-(4-(methylthio)benzylidene)propan-2-imine). The functionalization of FeO@Au with the copper complex occurs through the sulfur atom of the thioether moiety, as indicated by Raman scattering on surface.

Small phospho-donors phosphorylate MorR without inducing protein conformational changes.

Phosphorylation is an essential mechanism of protein control and plays an important role in biology. The two-component system (TCS) is a bacterial regulation mechanism mediated by a response regulator (RR) protein and a kinase protein, which synchronize the regulatory circuit according to the environment. Phosphorylation is a key element in TCS function as it controls RR activity.

Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor.

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state.

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 (+19mV), the binding to ligands that switch off the kinase activity induces a negative shift, i.

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.

Hydroxyl Radical Generation and DNA Nuclease Activity: A Mechanistic Study Based on a Surface-Immobilized Copper Thioether Clip-Phen Derivative.

Coordination compounds of copper have been invoked as major actors in processes involving the reduction of molecular oxygen, mostly with the generation of radical species the assignment for which has, so far, not been fully addressed. In the present work, we have carried out studies in solution and on surfaces to gain insights into the nature of the radical oxygen species (ROS) generated by a copper(II) coordination compound containing a thioether clip-phen derivative, 1,3-bis(1,10-phenanthrolin-2-yloxy)-N-(4-(methylthio)benzylidene)propan-2-amine (2CP-Bz-SMe), enabling its adsorption/immobilization to gold surfaces. Whereas surface plasmon resonance (SPR) and electrochemistry of the adsorbed complex indicated the formation of a dimeric Cu(I) intermediate containing molecular oxygen as a bridging ligand, scanning electrochemical microscopy (SECM) and nuclease assays pointed to the generation of a ROS species.

Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis.

A 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.

Resonance assignment of DVU2108 that is part of the Orange Protein complex in Desulfovibrio vulgaris Hildenborough.

We report the 94 % assignment of DVU2108, a protein belonging to the Orange Protein family, that in Desulfovibrio vulgaris Hildenborough forms a protein complex named the Orange Protein complex. This complex has been shown to be implicated in the cell division of this organism. DVU2108 is a conserved protein in anaerobic microorganisms and in Desulfovibrio gigas the homologous protein was isolated with a novel Mo-Cu cluster non-covalently attached to the polypeptide chain.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 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.

Incorporation of molybdenum in rubredoxin: models for mononuclear molybdenum enzymes.

Molybdenum is found in the active site of enzymes usually coordinated by one or two pyranopterin molecules. Here, we mimic an enzyme with a mononuclear molybdenum-bis pyranopterin center by incorporating molybdenum in rubredoxin. In the molybdenum-substituted rubredoxin, the metal ion is coordinated by four sulfurs from conserved cysteine residues of the apo-rubredoxin and two other exogenous ligands, oxygen and thiol, forming a Mo((VI))-(S-Cys)4(O)(X) complex, where X represents -OH or -SR.

Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation.

Background: Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation.

ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase.

Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance.

Mo-Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas.

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2](3-) noncovalently bound to the polypeptide chain. The D.

Chromobacterium violaceum: important insights for virulence and biotechnological potential by exoproteomic studies.

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems.

Genome sequence of Exiguobacterium antarcticum B7, isolated from a biofilm in Ginger Lake, King George Island, Antarctica.

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e.g.

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 [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus.

Proteomics Analysis of the Effects of Cyanate on Chromobacterium violaceum Metabolism.

Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium.

Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254.

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.

Molybdenum induces the expression of a protein containing a new heterometallic Mo-Fe cluster in Desulfovibrio alaskensis.

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.