[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.

[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.

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.

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.

abstract: The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the 'large' catalytic subunit and usually have three iron-sulfur clusters within a 'small' electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.