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J A Cowan - One of the best experts on this subject based on the ideXlab platform.
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elucidation of a 4fe 4s cluster degradation pathway rapid kinetic studies of the degradation of chromatium vinosum hipip
Journal of Biological Inorganic Chemistry, 2001Co-Authors: Matthew W Foster, Shumin Bian, Kristene K Surerus, J A CowanAbstract:Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-potential Iron Protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened Mossbauer signals, but delta EQ (1.1 mm/s) and delta (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mossbauer experiments, occurs with a measured rate constant kobs approximately 0.72 +/- 0.35 s-1 that is consistent with results from time-resolved electronic absorption and fluorescence (kobs approximately 0.4 +/- 0.1 s-1) and EPR (kobs approximately 0.62 +/- 0.18 s-1) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the Protein, facilitating protonation of the cluster, but not degradation per se. Release of Iron ions occurs even more slowly with kobs approximately 0.07 +/- 0.02 s-1, as determined by the appearance of the g = 4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, Mossbauer and EPR measurements.
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factors influencing redox thermodynamics and electron self exchange for the fe4s4 cluster in chromatium vinosum high potential Iron Protein the role of core aromatic residues in defining cluster redox chemistry
Biochemistry, 1996Co-Authors: Aileen Soriano, Shumin Bian, And Anshu Agarwal, J A CowanAbstract:The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic [Fe4S4]3+/2+ cluster of Chromatium vinosum high potential Iron Protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements. Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66. Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster. These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's [Agarwal, A., Li, D., & Cowan, J. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9440−9444], in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized [Fe4S4]3+ cluster.
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synthesis cloning and expression of a synthetic gene for high potential Iron Protein from chromatium vinosum
Biochemical and Biophysical Research Communications, 1993Co-Authors: Anshu Agarwal, Jian Tan, Mesut Eren, A Tevelev, Siu Man Lui, J A CowanAbstract:Abstract A synthetic gene encoding the peptide sequence for the low molecular weight (Mr ∼ 9600 Da) high-potential Iron Protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonudeotides varying in size from 42-mers to 48-mers. After cloning the gene into a pET-21d(+) vector, expression of holoProtein in yields of 35 mg/liter of culture was obtained following induction with isopropyl-β-D-thiogalactoside (IPTG). The recombinant Protein was characterized by electronic absorption, 1H NMR, electrochemistry, N-terminal sequencing and amino acid analysis. This is the first example of the expression of a high potential ferredoxin containing a fully constituted [Fe4S4] cluster.
Douglas C Rees - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the all ferrous 4fe 4s 0 form of the nitrogenase Iron Protein from azotobacter vinelandii
Biochemistry, 2001Co-Authors: Pavel Strop, Patricia M Takahara, H J Chiu, Hayley C Angove, Barbara K Burgess, Douglas C ReesAbstract:The structure of the nitrogenase Iron Protein from Azotobacter vinelandii in the all-ferrous [4Fe-4S]0 form has been determined to 2.25 A resolution by using the multiwavelength anomalous diffraction (MAD) phasing technique. The structure demonstrates that major conformational changes are not necessary either in the Iron Protein or in the cluster to accommodate cluster reduction to the [4Fe-4S]0 oxidation state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands in Proteins of known structure reveals that the [4Fe-4S] cluster of the Iron Protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the ability of the Iron Protein to exist in three oxidation states.
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structures of the superoxide reductase from pyrococcus furiosus in the oxidized and reduced states
Biochemistry, 2000Co-Authors: Andrew P Yeh, Francis E. Jenney, Michael W. W. Adams, Douglas C ReesAbstract:Superoxide reductase (SOR) is a blue non-heme Iron Protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X., and Adams, M. W. W. (1999) Science 286, 306−309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like β-barrel fold that coordinates a mononuclear, non-heme Iron center. The Protein fold and metal center are similar to those observed previously for the homologous Protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fulop, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680−689]. Each Iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with...
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x ray crystal structure of the nitrogenase molybdenum Iron Protein from clostridium pasteurianum at 3 0 a resolution
Biochemistry, 1993Co-Authors: Jongsun Kim, Daniel Woo, Douglas C ReesAbstract:The crystal structure of the nitrogenase molybdenum-Iron (MoFe) Protein from Clostridium pasteurianum (Cp1) has been determined at 3.0-A resolution by a combination of isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging. The structure of Cp1, including the two types of metal centers associated with the Protein (the FeMo-cofactor and the P-cluster pair), is similar to that previously described for the MoFe-Protein from Azotobacter vinelandii (Av1). Unique features of the Cpl structure arise from the presence of an ~50-residue insertion in the α subunit and an ~50-residue deletion in the β subunit. As a consequence, the FeMo-cofactor is more buried in Cp1 than in Av1, since the insertion is located on the surface above the FeMo-cofactor. The location of this insertion near the putative nitrogenase Iron Protein binding site provides a structural basis for the observation that the nitrogenase Proteins from C. pasteurianum have low activity with complementary nitrogenase Proteins isolated from other organisms. Mechanistic implications of the Cp1 structure for substrate entry /product release, substrate binding to the FeMo-cofactor, and electron- and proton-transfer reactions of nitrogenase are discussed.
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crystallographic structure and functional implications of the nitrogenase molybdenum Iron Protein from azotobacter vinelandii
Nature, 1992Co-Authors: Douglas C ReesAbstract:The crystal structure of the nitrogenase molybdenum–Iron Protein from Azotobacter vinelandii has been determined at 2.7 A resolution. The α- and β-subunits in this α_2β_2 tetramer have similar polypeptide folds. The FeMo-cofactor is completely encompassed by the α-subunit, whereas the P-cluster pair occurs at the interface between α- and β-subunits. Structural similarities are apparent between nitrogenase and other electron transfer systems, including hydrogenases and the photosynthetic reaction centre.
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crystallographic structure of the nitrogenase Iron Protein from azotobacter vinelandii
Science, 1992Co-Authors: H Komiya, Millie M Georgiadis, Pinak Chakrabarti, J J Kornuc, Douglas C ReesAbstract:The nitrogenase enzyme system catalyzes the ATP (adenosine triphosphate)-dependent reduction of dinitrogen to ammonia during the process of nitrogen fixation. Nitrogenase consists of two Proteins: the Iron (Fe)-Protein, which couples hydrolysis of ATP to electron transfer, and the molybdenum-Iron (MoFe)-Protein, which contains the dinitrogen binding site. In order to address the role of ATP in nitrogen fixation, the crystal structure of the nitrogenase Fe-Protein from Azotobacter vinelandii has been determined at 2.9 angstrom (A) resolution. Fe-Protein is a dimer of two identical subunits that coordinate a single 4Fe:4S cluster. Each subunit folds as a single alpha/beta type domain, which together symmetrically ligate the surface exposed 4Fe:4S cluster through two cysteines from each subunit. A single bound ADP (adenosine diphosphate) molecule is located in the interface region between the two subunits. Because the phosphate groups of this nucleotide are approximately 20 A from the 4Fe:4S cluster, it is unlikely that ATP hydrolysis and electron transfer are directly coupled. Instead, it appears that interactions between the nucleotide and cluster sites must be indirectly coupled by allosteric changes occurring at the subunit interface. The coupling between Protein conformation and nucleotide hydrolysis in Fe-Protein exhibits general similarities to the H-Ras p21 and recA Proteins that have been recently characterized structurally. The Fe-Protein structure may be relevant to the functioning of other biochemical energy-transducing systems containing two nucleotide-binding sites, including membrane transport Proteins.
Mario Piccioli - One of the best experts on this subject based on the ideXlab platform.
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the Iron sulfur cluster in the oxidized high potential Iron Protein from ectothiorhodospira halophila
Journal of the American Chemical Society, 1993Co-Authors: Lucia Banci, Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Paolo Carloni, Stefano Ciurli, Mario PiccioliAbstract:In our efforts to characterize oxidized high-potential Iron-sulfur Proteins (HiPIP), we have investigated the oxidized HiPIP II from Ectothiorhodospira halophila through 1 H NMR and molecular dynamics (MD) calculations. This Protein has the most symmetric isotropic shift pattern of the β-CH 2 protons of the liganded cysteines, four signals being upfield and four downfield. 1 H NOE, NOESY, and TOCSY results have provided the necessary key connectivities to perform the assignment of the liganded cysteines, taking advantage of the structure of the HiPIP I isoProtein. It is found that the electronic distribution within the cluster is different with respect to the Chromatium vinosum and Rhodocyclus gelatinosus systems
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identification of the Iron ions of high potential Iron Protein from chromatium vinosum within the Protein frame through two dimensional nmr experiments
Journal of the American Chemical Society, 1992Co-Authors: Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Stefano Ciurli, Luigi Messori, Mario PiccioliAbstract:2D NMR experiments performed on both the oxidized and reduced form of the high potential Iron Protein (HiPIP) from Chromatium vinosum, a paramagnetic Iron sulfur Protein for which the crystal structure is known in both oxidation states, allowed us to detect a number of scalar and dipolar connectivities of the isotropically shifted signals. On this basis it was possible to firmly identify the signals of the β-CH 2 and α-CH protons of the cluster-liganded cysteines and perform their sequence-specific assignments
Francesco Capozzi - One of the best experts on this subject based on the ideXlab platform.
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ab initio solution and refinement of two high potential Iron Protein structures at atomic resolution
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Emilio Parisini, Claudio Luchinat, Francesco Capozzi, Paolo Lubini, Victor S Lamzin, George M SheldrickAbstract:The crystal structure of the reduced high-potential Iron Protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0.93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 Protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the Protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R_1=\sum\big||F_o|-|F_c|\big|\big/\sum|F_o| of 12.0 and 13.6%, respectively [data with I>2\sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe4S4]2+ cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe—S bonds along four approximately parallel cube edges, and eight long Fe—S bonds. The unique Protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the Protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the Protein rather than by structural differences in the cluster region.
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the Iron sulfur cluster in the oxidized high potential Iron Protein from ectothiorhodospira halophila
Journal of the American Chemical Society, 1993Co-Authors: Lucia Banci, Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Paolo Carloni, Stefano Ciurli, Mario PiccioliAbstract:In our efforts to characterize oxidized high-potential Iron-sulfur Proteins (HiPIP), we have investigated the oxidized HiPIP II from Ectothiorhodospira halophila through 1 H NMR and molecular dynamics (MD) calculations. This Protein has the most symmetric isotropic shift pattern of the β-CH 2 protons of the liganded cysteines, four signals being upfield and four downfield. 1 H NOE, NOESY, and TOCSY results have provided the necessary key connectivities to perform the assignment of the liganded cysteines, taking advantage of the structure of the HiPIP I isoProtein. It is found that the electronic distribution within the cluster is different with respect to the Chromatium vinosum and Rhodocyclus gelatinosus systems
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identification of the Iron ions of high potential Iron Protein from chromatium vinosum within the Protein frame through two dimensional nmr experiments
Journal of the American Chemical Society, 1992Co-Authors: Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Stefano Ciurli, Luigi Messori, Mario PiccioliAbstract:2D NMR experiments performed on both the oxidized and reduced form of the high potential Iron Protein (HiPIP) from Chromatium vinosum, a paramagnetic Iron sulfur Protein for which the crystal structure is known in both oxidation states, allowed us to detect a number of scalar and dipolar connectivities of the isotropically shifted signals. On this basis it was possible to firmly identify the signals of the β-CH 2 and α-CH protons of the cluster-liganded cysteines and perform their sequence-specific assignments
Claudio Luchinat - One of the best experts on this subject based on the ideXlab platform.
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ab initio solution and refinement of two high potential Iron Protein structures at atomic resolution
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Emilio Parisini, Claudio Luchinat, Francesco Capozzi, Paolo Lubini, Victor S Lamzin, George M SheldrickAbstract:The crystal structure of the reduced high-potential Iron Protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0.93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 Protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the Protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R_1=\sum\big||F_o|-|F_c|\big|\big/\sum|F_o| of 12.0 and 13.6%, respectively [data with I>2\sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe4S4]2+ cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe—S bonds along four approximately parallel cube edges, and eight long Fe—S bonds. The unique Protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the Protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the Protein rather than by structural differences in the cluster region.
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the Iron sulfur cluster in the oxidized high potential Iron Protein from ectothiorhodospira halophila
Journal of the American Chemical Society, 1993Co-Authors: Lucia Banci, Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Paolo Carloni, Stefano Ciurli, Mario PiccioliAbstract:In our efforts to characterize oxidized high-potential Iron-sulfur Proteins (HiPIP), we have investigated the oxidized HiPIP II from Ectothiorhodospira halophila through 1 H NMR and molecular dynamics (MD) calculations. This Protein has the most symmetric isotropic shift pattern of the β-CH 2 protons of the liganded cysteines, four signals being upfield and four downfield. 1 H NOE, NOESY, and TOCSY results have provided the necessary key connectivities to perform the assignment of the liganded cysteines, taking advantage of the structure of the HiPIP I isoProtein. It is found that the electronic distribution within the cluster is different with respect to the Chromatium vinosum and Rhodocyclus gelatinosus systems
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identification of the Iron ions of high potential Iron Protein from chromatium vinosum within the Protein frame through two dimensional nmr experiments
Journal of the American Chemical Society, 1992Co-Authors: Ivano Bertini, Claudio Luchinat, Francesco Capozzi, Stefano Ciurli, Luigi Messori, Mario PiccioliAbstract:2D NMR experiments performed on both the oxidized and reduced form of the high potential Iron Protein (HiPIP) from Chromatium vinosum, a paramagnetic Iron sulfur Protein for which the crystal structure is known in both oxidation states, allowed us to detect a number of scalar and dipolar connectivities of the isotropically shifted signals. On this basis it was possible to firmly identify the signals of the β-CH 2 and α-CH protons of the cluster-liganded cysteines and perform their sequence-specific assignments
