The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Ligia M Saraiva - One of the best experts on this subject based on the ideXlab platform.

  • the di iron ric protein ytfe of escherichia coli interacts with the dna binding protein from starved cells dps to diminish ric protein mediated redox stress
    Journal of Bacteriology, 2018
    Co-Authors: Liliana S O Silva, Joana M Baptista, Charlotte Batley, Simon C Andrews, Ligia M Saraiva
    Abstract:

    ABSTRACT The RIC (repair of iron clusters) protein of Escherichia coli is a di-iron Hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS. IMPORTANCE The mammalian immune system produces reactive oxygen and nitrogen species that kill bacterial pathogens by damaging key cellular components, such as lipids, DNA, and proteins. However, bacteria possess detoxifying and repair systems that mitigate these deleterious effects. The Escherichia coli RIC (repair of iron clusters) protein is a di-iron Hemerythrin-like protein that repairs stress-damaged iron-sulfur clusters. E. coli Dps is an iron storage protein of the ferritin superfamily with DNA-binding capacity that protects cells from oxidative stress. This work shows that the E. coli RIC and Dps proteins interact in a fashion that counters RIC protein-induced reactive oxygen species (ROS). Altogether, we provide evidence for the formation of a new bacterial protein complex and reveal a novel contribution for Dps in bacterial redox stress protection.

Radu Silaghidumitrescu - One of the best experts on this subject based on the ideXlab platform.

  • oxidative protection of hemoglobin and Hemerythrin by cross linking with a nonheme iron peroxidase potentially improved oxygen carriers for use in blood substitutes
    Biomacromolecules, 2014
    Co-Authors: Denisa Hathazi, Donald M. Kurtz, Eva Fischerfodor, Augustin C Mot, Florina Scurtu, Anetta Vaida, Iulia Lupan, Grigore Damian, Radu Silaghidumitrescu
    Abstract:

    The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and Hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

  • protein based blood substitutes recent attempts at controlling pro oxidant reactivity with and beyond hemoglobin
    Pharmaceuticals, 2013
    Co-Authors: Violetaflorina Scurtu, Augustin Cătălin Moţ, Radu Silaghidumitrescu
    Abstract:

    Reviewed here are recent attempts to produce protein-based artificial oxygen carriers (“blood substitutes”). Most of these involve chemical or physical modifications on hemoglobin, although a recent line of research using Hemerythrin instead of hemoglobin is also described. The focus is set on the extent to which these modifications alter the redox reactivity of the proteins, and on ways in which this can be done systematically and purposefully, within the framework of a working hypothesis where redox side-reactions hold an important role in the physiological outcome of experimental transfusions with artificial oxygen carriers.

  • towards Hemerythrin based blood substitutes comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells
    Journal of Biosciences, 2011
    Co-Authors: Eva Fischerfodor, Augustin C Mot, Florina Deac, Mariann Arkosi, Radu Silaghidumitrescu
    Abstract:

    Hemerythrin is a dioxygen-carrying protein whose oxidative/nitrosative stress-related reactivity is lower than that of hemoglobin, which may warrant investigation of Hemerythrin as raw material for artificial oxygen carriers (‘blood substitutes’). We report here the first biological tests for Hemerythrin and its chemical derivatives, comparing their performance with that of a representative competitor, glutaraldehyde-polymerized bovine hemoglobin. Hemerythrin (native or derivatized) exhibits a proliferative effect on human umbilical vein endothelial cell (HUVEC) cultures, as opposed to a slight inhibitory effect of hemoglobin. A similar positive effect is displayed on human lymphocytes by glutaraldehyde-polymerized Hemerythrin, but not by native or polyethylene glycol-derivatized Hemerythrin.

Sunney I Chan - One of the best experts on this subject based on the ideXlab platform.

  • the bacterioHemerythrin from methylococcus capsulatus bath crystal structures reveal that leu114 regulates a water tunnel
    Journal of Inorganic Biochemistry, 2015
    Co-Authors: Kelvin H C Chen, Phirnonphan Chuankhayan, Chunjung Chen, Mitsuhiro Fukuda, Sunney I Chan
    Abstract:

    The bacterioHemerythrin (McHr) from Methylococcus capsulatus (Bath) is an oxygen carrier that serves as a transporter to deliver O2 from the cytosol of the bacterial cell body to the particulate methane monooxygenase residing in the intracytoplasmic membranes for methane oxidation. Here we report X-ray protein crystal structures of the recombinant wild type (WT) McHr and its L114A, L114Y and L114F mutants. The structure of the WT reveals a possible water tunnel in the McHr that might be linked to its faster autoxidation relative to Hemerythrin in marine invertebrates. With Leu114 positioned at the end of this putative water tunnel, the hydrophobic side chain of this residue seems to play a prominent role in controlling the access of the water molecule required for autoxidation. This hypothesis is examined by comparing the autoxidation rates of the WT McHr with those of the L114A, L114Y and L114F mutants. The biochemical data are correlated with structural insights derived from the analysis of the putative water tunnels in the various McHr proteins provided by the X-ray structures.

  • isolation purification and characterization of Hemerythrin from methylococcus capsulatus bath
    Journal of Inorganic Biochemistry, 2008
    Co-Authors: Weichun Kao, Vincent C C Wang, Yiche Huang, Tachau Chang, Sunney I Chan
    Abstract:

    Earlier work from our laboratory has indicated that a Hemerythrin-like protein was over-produced together with the particulate methane monooxygenase (pMMO) when Methylococcus capsulatus (Bath) was grown under high copper concentrations. A homologue of Hemerythrin had not previously been found in any prokaryote. To confirm its identity as a Hemerythrin, we have isolated and purified this protein by ion-exchange, gel-filtration and hydrophobic interaction chromatography, and characterized it by mass spectrometry, UV–visible, CD, EPR and resonance Raman spectroscopy. On the basis of biophysical and multiple sequence alignment analysis, the protein isolated from M. capsulatus (Bath) is in accord with Hemerythrins previously reported from higher organisms. Determination of the Fe content in conjunction with molecular-weight estimation and mass analysis indicates that the native Hemerythrin in M. capsulatus (Bath) is a monomer with molecular mass 14.8 kDa, in contrast to Hemerythrins from other eukaryotic organisms, where they typically exist as a tetramer or higher oligomers.

Liliana S O Silva - One of the best experts on this subject based on the ideXlab platform.

  • the di iron ric protein ytfe of escherichia coli interacts with the dna binding protein from starved cells dps to diminish ric protein mediated redox stress
    Journal of Bacteriology, 2018
    Co-Authors: Liliana S O Silva, Joana M Baptista, Charlotte Batley, Simon C Andrews, Ligia M Saraiva
    Abstract:

    ABSTRACT The RIC (repair of iron clusters) protein of Escherichia coli is a di-iron Hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS. IMPORTANCE The mammalian immune system produces reactive oxygen and nitrogen species that kill bacterial pathogens by damaging key cellular components, such as lipids, DNA, and proteins. However, bacteria possess detoxifying and repair systems that mitigate these deleterious effects. The Escherichia coli RIC (repair of iron clusters) protein is a di-iron Hemerythrin-like protein that repairs stress-damaged iron-sulfur clusters. E. coli Dps is an iron storage protein of the ferritin superfamily with DNA-binding capacity that protects cells from oxidative stress. This work shows that the E. coli RIC and Dps proteins interact in a fashion that counters RIC protein-induced reactive oxygen species (ROS). Altogether, we provide evidence for the formation of a new bacterial protein complex and reveal a novel contribution for Dps in bacterial redox stress protection.

Donald M. Kurtz - One of the best experts on this subject based on the ideXlab platform.

  • Spectroscopy and DFT Calculations of Flavo–Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates
    2018
    Co-Authors: Andrew C. Weitz, Donald M. Kurtz, Nitai Giri, Rosanne E. Frederick, Emile L. Bominaar, Michael P. Hendrich
    Abstract:

    Flavo–diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in Hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N2O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N2O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS1·S2), were found to differ substantially, +17 cm–1 for the diiron mononitrosyl and +60 cm–1 for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mössbauer hyperfine parameters and DFT calculations confirmed a semibridging NO– ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO– ligands, which could initiate N–N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O

  • Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures
    2017
    Co-Authors: Andrew C. Weitz, Donald M. Kurtz, Jonathan D. Caranto, Nitai Giri, Emile L. Bominaar, Michael P. Hendrich
    Abstract:

    Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in Hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm–1 to +32 cm–1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-μ-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO

  • oxidative protection of hemoglobin and Hemerythrin by cross linking with a nonheme iron peroxidase potentially improved oxygen carriers for use in blood substitutes
    Biomacromolecules, 2014
    Co-Authors: Denisa Hathazi, Donald M. Kurtz, Eva Fischerfodor, Augustin C Mot, Florina Scurtu, Anetta Vaida, Iulia Lupan, Grigore Damian, Radu Silaghidumitrescu
    Abstract:

    The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and Hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

  • Oxidative Protection of Hemoglobin and Hemerythrin by Cross-Linking with a Nonheme Iron Peroxidase: Potentially Improved Oxygen Carriers for Use in Blood Substitutes
    2014
    Co-Authors: Denisa Hathazi, Donald M. Kurtz, Florina Scurtu, Anetta Vaida, Iulia Lupan, Grigore Damian, Augustin C. Mot, Eva Fischer-fodor, Radu Silaghi-dumitrescu
    Abstract:

    The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe­(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and Hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times

  • A Bacterial Hemerythrin Domain Regulates the Activity of a Vibrio cholerae Diguanylate Cyclase
    2012
    Co-Authors: Ruth A. Schaller, Syed Khalid Ali, Karl E. Klose, Donald M. Kurtz
    Abstract:

    The first demonstrated example of a regulatory function for a bacterial Hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O2 and undergo autoxidation. The amino acid sequence encoded by the VC1216 gene from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial diguanylate cyclases (DGCs) that catalyze formation of cyclic di-(3′,5′)-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon being exposed to air. Vc Bhr-DGC showed approximately 10 times higher DGC activity in the diferrous than in the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in V. cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low-dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity