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|Title: ||A Biological Investigation of the Proteins Required for Nickel Insertion into Escherichia coli [NiFe] Hydrogenase|
|Authors: ||Chan Chung, Kim Cindy|
|Advisor: ||Zamble, Deborah|
Bacterial nickel homeostasis
|Issue Date: ||5-Jan-2012|
|Abstract: ||[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.|
|Appears in Collections:||Doctoral|
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