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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Structure and function of hydrogenase from Azotobacter vinelandii

Sun, Jin-hua 03 September 1993 (has links)
Graduation date: 1994
42

The Role of Dimerization by Escherichia coli HypB in Hydrogenase Biosynthesis

Cai, Fang 15 December 2010 (has links)
Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.
43

The Role of Dimerization by Escherichia coli HypB in Hydrogenase Biosynthesis

Cai, Fang 15 December 2010 (has links)
Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.
44

A Biological Investigation of the Proteins Required for Nickel Insertion into Escherichia coli [NiFe] Hydrogenase

Chan Chung, Kim Cindy 05 January 2012 (has links)
[NiFe] hydrogenases are found in a variety of microorganisms and catalyze the reversible oxidation of hydrogen gas to protons and electrons. This enzyme has generated intense interest due to its contribution to pathogenicity in certain organisms as well as its application in bioremediation and the production of hydrogen as an alternative fuel source. The biosynthesis of the dinuclear active site requires a number of accessory proteins to chaperone and insert the metal cofactors to the awaiting large subunit of hydrogenase. The proteins responsible for nickel delivery to Escherichia coli hydrogenase 3 are HypA, HypB, and SlyD, however the mechanism by which this is accomplished is unclear. The goal of this work was to analyze the metal-binding abilities and protein interactions of these nickel insertion proteins to enhance our understanding of their roles. Isolated N-terminal peptide of HypB has similar high-affinity metal-binding to the full-length protein. This peptide binds nickel in a square planar site with three cysteinyl and a fourth N-terminal amine ligand. Additionally, studies with SlyD and HypA reveal protein interactions that occur during hydrogenase maturation. Pull-down experiments of a tagged variant of hydrogenase revealed multi-protein complexes with HypA, HypB, and SlyD. A complex between SlyD and hydrogenase forms prior to both nickel and iron insertion, supporting chaperone activity of SlyD during hydrogenase maturation. HypA can interact with hydrogenase in the absence of HypB and SlyD, and a possible role as the bridging protein during the nickel insertion event is proposed. In addition, fluorescent imaging of E. coli cells using a fluorescently labeled streptavidin conjugate revealed localization of both Strep-tagged II hydrogenase and HypA at or near the cell membrane, suggesting that enzyme maturation occurs proximal to metal transporters. This work provided a deeper understanding of the role that each of these proteins play in [NiFe] hydrogenase assembly and is helpful for any future applications of this enzyme.
45

A Biological Investigation of the Proteins Required for Nickel Insertion into Escherichia coli [NiFe] Hydrogenase

Chan Chung, Kim Cindy 05 January 2012 (has links)
[NiFe] hydrogenases are found in a variety of microorganisms and catalyze the reversible oxidation of hydrogen gas to protons and electrons. This enzyme has generated intense interest due to its contribution to pathogenicity in certain organisms as well as its application in bioremediation and the production of hydrogen as an alternative fuel source. The biosynthesis of the dinuclear active site requires a number of accessory proteins to chaperone and insert the metal cofactors to the awaiting large subunit of hydrogenase. The proteins responsible for nickel delivery to Escherichia coli hydrogenase 3 are HypA, HypB, and SlyD, however the mechanism by which this is accomplished is unclear. The goal of this work was to analyze the metal-binding abilities and protein interactions of these nickel insertion proteins to enhance our understanding of their roles. Isolated N-terminal peptide of HypB has similar high-affinity metal-binding to the full-length protein. This peptide binds nickel in a square planar site with three cysteinyl and a fourth N-terminal amine ligand. Additionally, studies with SlyD and HypA reveal protein interactions that occur during hydrogenase maturation. Pull-down experiments of a tagged variant of hydrogenase revealed multi-protein complexes with HypA, HypB, and SlyD. A complex between SlyD and hydrogenase forms prior to both nickel and iron insertion, supporting chaperone activity of SlyD during hydrogenase maturation. HypA can interact with hydrogenase in the absence of HypB and SlyD, and a possible role as the bridging protein during the nickel insertion event is proposed. In addition, fluorescent imaging of E. coli cells using a fluorescently labeled streptavidin conjugate revealed localization of both Strep-tagged II hydrogenase and HypA at or near the cell membrane, suggesting that enzyme maturation occurs proximal to metal transporters. This work provided a deeper understanding of the role that each of these proteins play in [NiFe] hydrogenase assembly and is helpful for any future applications of this enzyme.
46

Hydrogenases of Desulfovibrio desulfuricans G20 /

Ringbauer, Joseph A. January 2000 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 160-168). Also available on the Internet.
47

Hydrogenases of Desulfovibrio desulfuricans G20

Ringbauer, Joseph A. January 2000 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 160-168). Also available on the Internet.
48

Gas-Phase Photoelectron Spectroscopy and Computational Studies of [FeFe]-Hydrogenase Inspired-Catalysts for Hydrogen Production

Lockett, Lani Victoria January 2009 (has links)
The work presented in this dissertation focuses on the [FeFe]-hydrogenase active site as inspiration for the design and synthesis of complexes capable of the electrocatalytic generation of molecular hydrogen from protons and electrons. The majority of work discussed uses gas-phase photoelectron spectroscopy (PES) and density functional theory (DFT) to probe and analyze the bonding and electron distribution in potential catalysts. These two techniques are also used to explore the nature of cyanide as a ligand, due to its presence and unknown role in these enzymes. This dissertation begins with the study of (η⁵-C₅H₅)Fe(CO)₂X (FpX) and (η⁵- C₅Me₅)Fe(CO)₂X (Fp*X) complexes where X = H⁻, Cl⁻, and CN⁻ to assess and compare their π-accepting abilities, which is contradicted in the literature. The shifts in ionization energies measured by PES provide a measure of the relative bonding effects. The results indicate cyanide is, overall, a weak π-acceptor, and the σ- and π-donor interactions are important to understanding the chemistry. The molecule [(μ-ortho-C₆H₄S₂)][Fe(CO)₃]₂ was examined, in part due to the delocalized π-orbitals of the C₆H₄S₂ ligand, which could facilitate the redox chemistry necessary for catalysis. Computations show that upon ionization, the complex adopts a semi-bridging carbonyl; termed “rotated structure”. The reorganization energy of this geometry change was determined, which may provide understanding of how the active site in the enzyme enables electron transfer to achieve this catalysis. Next complexes of the form (μ-SCH₂XCH₂S)[Fe(CO)₃]₂, where X=CH₂, O, NH, ᵗBuN, MeN, were explored in order to provide insight to the unknown atom at the central bridging position of the alkyl chain in the [FeFe]-hydrogenase enzyme. The likelihood of a rotated cationic structure is also shown, with reorganization energy values similar to that seen for [(μ-ortho-C₆H₄S₂)][Fe(CO)₃]₂. The final chapter explores the replacement of selenium for sulfur in (μ- X(CH₂)₃X)[Fe(CO)₃]₂ and (μ-X(CH₂)₂CH(CH₃)X)[Fe(CO)₃]₂, where X is either sulfur or selenium. The PES data show destabilization of the selenium complex ionizations compared to the sulfur complexes and a lower reorganization energy was calculated. The computed HOMO-LUMO gap energy for the selenium-based complex is roughly 0.17 eV smaller than for the sulfur analogs, which may indicate a lower reduction potential is needed.
49

Preparation and Characterization of Hydrogenase Enzyme Active Site-inspired Catalysts: The Effects of Alkyl Bulk and Conformer Strain as Studied by Photoelectron Spectroscopy, Electrochemistry and Computational Methods

Petro, Benjamin J. January 2009 (has links)
A series of alkyldithiolatodiironhexacarbonyl complexes of the form &mu:-(RS2)Fe2(CO)6, where RS2 is: 1,2-ethanedithiolate (eth-cat), cis-1,2-cyclopentanedithiolate (pent-cat), cis-1,2-cyclohexanedithiolate (hex-cat), and 2-exo,3-exo-bicyclo[2.2.1]heptanedithiolate (norbor-cat), are reported. These complexes display structures and catalytic behavior toward production of molecular hydrogen with similarities to the active site of the diiron hydrogenase enzymes. Hydrogen production is desirable as an alternative fuel source and these catalysts are capable of producing H2 in the presence of weak acid under electrochemical conditions. Through understanding of the factors which control the catalytic activity of these catalysts it may be possible to contribute to the development of a hydrogen fuel economy.Significant scan-rate dependence under electrochemical conditions is observed, resulting in an initial 1-to-2 electron reduction depending on how quickly the singly reduced species can reorganize. The rate of this reorganization directly corresponds to the internal strain within the system and can be ranked in the following order of increasing rate of reorganization: pent-cat < norbor-cat < eth-cat < hex-cat. Additionally, these catalysts all successfully catalyze protons to molecular hydrogen under electrochemical conditions in the presence of acetic acid via an ECEC catalytic mechanism, where, E is an electrochemical step (reduction) and C is a chemical step (protonation).Density functional theory computations support the reported catalytic processes by calculating physically observable quantities, such as: pKa values, reduction potentials, adiabatic ionization energies and carbonyl stretching frequencies in the infrared (IR) region. These quantities were used to suggest reasonable reactive intermediates within the catalytic cycle. The electronic structure of each catalyst was examined using photoelectron spectroscopy and the global minimum cationic structure, in all cases, involves a structure with a bridging carbonyl ligand, akin to that of the enzyme active site.The most significant outcome of this work is the unprecedented diiron center rotation upon reduction. As conformational strain involving the dithiolate ligand increases, the rate of reorganization of the anion increases leading to cleavage of an iron-sulfur bond to provide an alternative protonation site, a key step toward molecular hydrogen formation. This site is less basic than the unrotated form and helps evolve H2 with thermodynamic favorability.
50

SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli

Kaluarachchi, Harini 10 January 2012 (has links)
SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric protein that can chelate up to 7 nickel ions with an affinity in the sub-nanomolar range. By truncation and mutagenesis studies we show that the unique C-terminal metal-binding domain of this protein is required for Ni(II) binding and that the protein coordinates this metal non-cooperatively. This activity of SlyD supports the proposed in vivo role of SlyD in nickel homeostasis. In addition to nickel, SlyD can bind a variety of other types of transition metals. Therefore it was feasible that the protein contributes to homeostasis of metals other than nickel. To test this hypothesis, the metal selectivity of the protein was examined. The preference of SlyD for the metals examined could be ordered as follows, Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) << Cu(I) indicating that the affinity of SlyD for the different metals follows the Irving-Williams series of metal-complex stabilities. Although the protein is unable to overcome the large thermodynamic preference in vitro for Cu(I) and exclude Zn(II) chelation, in vivo studies suggest a Ni(II)-specific function for the protein. To understand the function of SlyD as a metallochaperone, its interaction with HypB was investigated. This investigation revealed that SlyD plays a role in Ni(II) storage in E. coli and can function as a Ni(II)-donor to HypB. This study also revealed that SlyD can modulate the metal-binding as well as the GTPase activities of HypB. Based on the experimental data, a role for the HypB-SlyD complex in [NiFe]-hydrogenase biosynthesis is presented.

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