169,856 research outputs found
Cyclic voltammetry and spectroelectrochemistry of cytochrome c(8) from Rubrivivax gelatinosus. Implications in photosynthetic electron transfer
No full textWe present here the electrochemical characterization of cytochrome c(8) from light-grown cells of the purple phototroph Rubrivivax gelatinosus. At 25 degrees C, cytochrome c(8) exhibits a quasi-reversible, diffusion-controlled, electrochemical process as observed by cyclic voltammetry (CV) using a pyrolitic graphite microelectrode (10 mM Tris . HCl buffer, 250 mM NaCl). The pH dependence of the reduction potential, investigated in the range 4.9-8.2, allows the calculation of E-m,E-7 = +294 mV, and further yields values of pK(OX) = 6.24 and pK(red) = 6.87, probably associated to the ionization of a heme propionate group in the oxidized and reduced form of cytochrome c(8), respectively. The calculated reduction potentials of completely protonated and deprotonated cytochrome c(8) are +322 and +284 mV, respectively. The equilibrium reduction potential of cytochrome c(8) was also determined at pH 8 by recording its visible absorption spectrum as a function of the applied potential using an optically transparent electrode (OTE) cell (E-m = +276 mV). These observations indicate that cytochrome c(8) might play a significant role in photosynthetic electron transport and associated proton translocation in purple bacteria
High resolution crystal structure of Rubrivivax gelatinosus cytochrome c’
No full textThe structure of the cytochrome c' from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29 A and 1.50 A for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes
Modulation of Bacillus pasteurii cytochrome c(553) reduction potential by structural and solution parameters
No full textDirect cyclic voltammetry and H-1 NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c(553) isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c(553) at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell: revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275-308 K; Delta S degrees'= -162.7 +/- 1.2 J mol(-1) K-1, Delta H degrees' = -53.0 +/- 0.5 kJ mol(-1), Delta G degrees' = -4.5 +/- 0.1 kJ mol(-1), E degrees' = +47.0 +/- 0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c(553). Both effects are more accentuated in the high-temperature range (308-323 K; Delta S degrees'= -294.1 +/- 8.4 J mol(-1) K-1. Delta H degrees' = -93.4+\-3.1 kJ mol(-1), Delta G degrees'= -5.8 +/- 0.6 kJ mol(-1), E degrees' = +60.3 +/- 5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-ir conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Huckel theory, yields values of E-(I=0)degrees',= -25.4 +/- 1.4 mV, z(red)=-11.3, and z(ox)= -10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7-10 and an increase at more acidic pH, allowing the calculation of pK(O) = 5.5 and pK(R) = 5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c(553). H-1 NMR spectra of the oxidized paramagnetic cytochrome c(553) indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288-340 K at pH 7 and in the pH range 4.8-10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0-0.2 M causes a slight change of the H-1 NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5,2 +/- 1.1 M-1. The pH dependence of the isotropically shifted H-1 NMR signals of the oxidized cytochrome displays at least one ionization step with pK(O)=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c(553)
Identification of the bacteria associated to the phycosphere of the Chlorella-like strain SEC_LI_ChL_1
No full textThe associations and the co-evolution of bacteria and eukaryotic microalgae are raising great interest in the last years, especially in the ???phycosphere???, that is the area around the algal cells, where extracellular products of the algae are used by bacteria; here, different interactions between microalgae and bacteria might occur. The Chlorella-like strain SEC_LI_ChL_1 was previously characterized with a multidisciplinary integrated approach based on phylogenetic reconstructions, morphological-ultrastructural analysis and physiological characterization in presence of different trophic conditions. In this work, the isolation and characterization of fifteen cultivable and one uncultivable bacterial strains strictly associated to the phycosphere of strain SEC_LI_ChL_1 was carried out. The molecular identification followed by the taxonomic reconstruction allowed to highlight the presence of a ???primary??? culturable associated bacterium taxonomically related to Stenotrophomonas rhizophila, and other culturable bacterial strains taxonomically related to Actinobacteria (Microbacterium), Gammaproteobacteria (Pseudomonas, Stenotrophomonas and Pseudoxanthomonas), Alphaproteobacteria (Bosea, and Brevundimonas), Bacteroidetes and Firmicutes (Priestia and Bacillus). Since the microalgal strain was tolerant to different metal concentrations, the cultivable bacterial strains were exposed to the same metals at the same concentrations, highlighting similar tolerance patterns to the microalgae. The IAA production ability was tested as well in order to highlight this PGP trait in the isolated strains. An uncultivable bacterium taxonomically related to Shinella sp. was also molecularly characterized from the DNA of washed algal cells thus suggesting, in this case, an even more strict connection with the algae. A possible role in the microalgal growth promotion and defence against envi-ronmental pollutants is here discussed for all the bacterial strains associated to the SEC_LI_ChL_1 phycosphere
Screening of trace metal elements for pollution tolerance of freshwater and marine microalgal strains: Overview and perspectives
No full textMicroalgae represent a putative solution to decontaminate metal polluted aquatic sites (phycoremediation). Seven different freshwater and seawater microalgal strains (Nannochloropsis sp., Dunaliella sp., Phaeodactylum sp., Chlorella sp., Isochrysis sp., Euglena sp. and Chlorogonium sp.) were exposed to five metals (Cu, Zn, As (III), Fe and Ni) at three concentrations each, simulating highly polluted sites. The experiment was conducted for a week; the survival ability of each strain and the photosynthetic pigments content (chlorophyll a, b and carotenoids) were evaluated, together with the Optical Density of each culture, pH, growth rate and biomass. Results highlighted different resistance patterns towards metals characterizing each algal strain, and the tolerance of all the microalgal strains towards arsenite. For the first time, the metal resistance pattern of Chlorogonium sp. was evaluated. Finally, our Euglena sp. and Dunaliella sp. strains were considered among the most promising organisms for phycoremediation of freshwater and seawater polluted sites respectively
Redox activity of caffeic acid towards iron (III) complexed in a polygalacturonate network
No full textThe transfer of several metal ions from the soil to the plant absorbing cells is mediated principally by organic molecules of low molecular weight with complexing and reducing activity, among which caffeic acid (CAF) is particularly important. Here we report the results of a survey which deals with the oxidation of CAF by the Fe(III) ions bound to a polygalacturonate network (Fe(III)-PGA network). The interaction between Fe(III) and CAF was studied by using Fe(III)-PGA networks equilibrated in the 2.4-7.0 pH range by means of kinetic and spectroscopic methods. The reducing power was found to depend on the nature of the Fe(III)-PGA network complexes: when the ferric ion was complexed only by the PGA carboxylic groups, a high redox activity was observed, whereas the Fe(III) reduction was found to be lower when a hydroxylic group was inserted in the Fe(III) coordination sphere. The iron complexed in the network was protected from hydrolysis reactions, as shown by the high pH values at which its reduction occurred. Two different fractions of Fe(II) produced were identified, one diffusible and another exchangeable with CaCl2 6.0 mM. The existence of the exchangeable form was attributed to the electrostatic interaction of the Fe(II) ions with the carboxylate groups of the fibrils and with the degradation products of CAE The arrangement of the fibrils was altered following the substitution of Ca(II) by Fe(III) ions and was restored following the seduction of Fe(III) by CAF
Fluorescence spectroscopy structural studies of Bp-UreG in solution
No full textThe nickel-enzyme urease is a α3β3γ3 complex that catalyses the hydrolysis of urea in the last step of nitrogen mineralization. Full catalytic function requires the interaction of four accessory proteins: UreD, UreF, UreG and UreE. The monomer form of UreG (23kD) contains a fully conserved P-loop nucleotide-binding motif and has been proposed to be essential, as UreDFG complex, in coupling cellular metabolism and the assembly of urease. Here, the different intrinsic fluorescence properties of the single tryptophan residue (W192) of the UreG accessory protein from B. pasteurii have been analyzed to provide structural information. Under native conditions, at 20°C in 20 mM phosphate buffer, pH 7.5, BpUreG exists predominantly as a dimer with the emission maximum at 336 nm, an anisotropy decay correlation time, φ, of 32 ns and a KI collisional quenching rate constant, kq, of 0.35x109sec-1M-1. In the presence of 1.8 M Gdn-HCl, the emission maximum is shifted to 347 nm with an increase of 10% of the fluorescence intensity. Under the same condition an anisotropy decay correlation time, φ, of 11 ns and a KI collisional quenching rate constant, kq, of 0.99x109sec-1M-1 were recovered. In addition, fluorescence-detected denaturation curves revealed that the tryptophan peptide chain is fully unfolded at 2 M Gdn-HCl. Altogether our results indicate the existence of a relatively unstable region at the C-terminus of BpUreG with the tryptophan-containing segment that can move from a buried to an exposed configuration. Loss of local structure can be concomitant with dimer dissociation, but is apparently unrelated to the folding state of the residual protein core. This work was supported with a MIUR-PRIN 2003 to S.L.C
The intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy
No full textUreG is an essential protein for the in vivo activation of urease. In a previous study, UreG from Bacillus pasteurii was shown to behave as an intrinsically unstructured dimeric protein. Here, intrinsic and extrinsic fluorescence experiments were performed, in the absence and presence of denaturant, to provide information about the form (fully folded, molten globule, premolten globule, or random coil) that the native state of BpUreG assumes in solution. The features of the emission band of the unique tryptophan residue (W192) located on the C-terminal helix, as well as the rate of bimolecular quenching by potassium iodide, indicated that, in the native state, W192 is protected from the aqueous polar solvent, while upon
addition of denaturant, a conformational change occurs that causes solvent exposure of the indole side chain. This structural change, mainly affecting the C-terminal helix, is associated with the release of static quenching, as shown by resolution of the decay-associated spectra. The exposure of protein hydrophobic sites, monitored using the fluorescent probe bis-ANS, indicated that the native dimeric state of BpUreG is disordered even though it maintains a significant amount of tertiary structure. ANS fluorescence also indicated that, upon addition of a small amount of GuHCl, a transition to a molten globule state occurs, followed by formation of a pre-molten globule state at a higher denaturant concentration. The latter form is resistant to full unfolding, as also revealed by far-UV circular dichroism spectroscopy. The hydrodynamic parameters obtained by time-resolved fluorescence anisotropy at maximal denaturant concentrations (3 M GuHCl) confirmed the existence of a disordered but stable dimeric protein core. The nature of the forces holding together the two monomers of BpUreG was investigated. Determination of free thiols in native or denaturant conditions, as well as light scattering experiments in the absence and presence of dithiothreitol as a reducing agent, under native or denaturing conditions, indicates that a disulfide bond, involving the unique conserved cysteine C68, is present under native conditions and maintained upon addition of denaturant. This covalent bond is therefore important for the stabilization of the dimer under native conditions. The intrinsically disordered structure of UreG is discussed with respect to the role of this protein as a chaperone in the urease assembly system
The Ni(II)-Binding Activity of the Intrinsically Disordered Region of Human NDRG1, a Protein Involved in Cancer Development
No full textNickel exposure is associated with tumors of the respiratory tract such as lung and nasal cancers, acting through still-uncharacterized mechanisms. Understanding the molecular basis of nickel-induced carcinogenesis requires unraveling the mode and the effects of Ni(II) binding to its intracellular targets. A possible Ni(II)-binding protein and a potential focus for cancer treatment is hNDRG1, a protein induced by Ni(II) through the hypoxia response pathway, whose expression correlates with higher cancer aggressiveness and resistance to chemotherapy in lung tissue. The protein sequence contains a unique C-terminal sequence of 83 residues (hNDRG1*C), featuring a three-times-repeated decapeptide, involved in metal binding, lipid interaction and post-translational phosphorylation. In the present work, the biochemical and biophysical characterization of unmodified hNDRG1*C was performed. Bioinformatic analysis assigned it to the family of the intrinsically disordered regions and the absence of secondary and tertiary structure was experimentally proven by circular dichroism and NMR. Isothermal titration calorimetry revealed the occurrence of a Ni(II)-binding event with micromolar affinity. Detailed information on the Ni(II)-binding site and on the residues involved was obtained in an extensive NMR study, revealing an octahedral paramagnetic metal coordination that does not cause any major change of the protein backbone, which is coherent with CD analysis. hNDRG1*C was found in a monomeric form by light-scattering experiments, while the full-length hNDRG1 monomer was found in equilibrium between the dimer and tetramer, both in solution and in human cell lines. The results are the first essential step for understanding the cellular function of hNDRG1*C at the molecular level, with potential future applications to clarify its role and the role of Ni(II) in cancer development
Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease
No full textUrease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG
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