Universiẗat, Diss., 2003--München.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1952. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: John W. Peters. Includes bibliographical references (leaves 164-187).

The enzyme nitrogenase, which catalyzes nitrogen fixation in Azotobacter vinelandii, consists of two components, the Fe protein (NifH) and the MoFe protein (NifDK). NifK contains several highly conserved residues implicated in its functions throughout the protein. However, the carboxyl terminus of NifK is not implicated in any of the known functions of this protein. Therefore, the present study explored the role of carboxyl terminal region of NifK. The results of growth analysis showed that when the media was adjusted to be slightly acidic, the strain that expresses the mutated NifK yielded a lower growth compared to the wild type. These observations implied that the carboxyl terminus of the NifK contributes to the formation of a stable nitrogenase complex when A. vinelandii is grown in acidic environment. The proper interaction between NifH and NifK is essential for the nitrogenase conformation. To determine how the interaction is influenced by the characteristics of the amino acids available at position 112 of NifH, we introduced residue mutations to the codon encoding for Glu112. Growth analyses indicated that mutant strains are capable of propagation under nitrogen-deficit conditions although the growth rate is lower than that of wild type strain. Therefore the charge carried by the amino acid at position 112 of NifH plays a minor role in the interaction whereas; a more important factor is the length of the side chains. The research on hydrogenase expressed by bacteria shed light on the possibilities of utilizing this novel energy source. We endeavored to take advantage of the nature of A. vinelandii and construct an A. vinelandii mutant strain expressing Fe-hydrogenase. This ongoing research involves molecular manipulation of the enzyme-encoding gene hydA. The synthetic hydA was incorporated and expressed in A. vinelandii strain DJ54. At the same time, we screened several biomass materials for their capabilities in sustaining diazotrophic growth of A. vinelandii. The result indicated that the HydA protein can be expressed in A. vinelandii under certain conditions and a number of biomass substances can be supportive ingredients for putative biohydrogen media.

No description available.

Based on what we have found, we proposed a model for Ni presenting by HypB involved in hydrogenase maturation. HypB binds two Ni ions in the apo- and GDP-bound form. Ni binding also induces dimerization of HypB. Upon GTP binding, HypB can bind an extra Ni ion at the dimeric interface. GTP hydrolysis will release the extra Ni ion, which may be subsequently inserted into hydrogenases during the maturation process. / Furthermore, two Ni binding sites were determined in a monomeric HypB. One is the cluster including C92, H93 and C122, the other is composed of H97 and H101. Upon GTP-dependent dimerization, HypB can bind an extra Ni ion. Our results have shown that the C92/H93/C122 is involved in binding the extra Ni ion, and such binding requires both cysteine residues in the reduced form. Since the GTP-induced dimerization of HypB is coupled to bind an extra Ni, so HypB could act as a GTP-mediated switch that regulate one Ni release from the GTP-bound form to the GDP-bound form. / In the future, we will attempt to crystallize AfHypB in complex with GDP, GTP analogue and AfHypA. Availability of good quality crystals will pave way for the structure determination of AfHypA and AfHypA/HypB complex. And the results obtained will provide a better understanding of the mechanism of functional interaction between HypA and HypB and how HypA and HypB play a role in Ni ion delivery for hydrogenase maturation. / The assembly of the [NiFe]-hydrogenases requires incorporation of Ni ions into the enzyme's metallocenter, which process requires the GTPase activity of HypB and HypA. Due to the essential role in assembly of the active site of hydrogenases, the two proteins were defined as hydrogenase maturation factors. To better understand the mechanism of GTP hydrolysis-dependent Ni delivery accomplished by HypB and HypA, our work focuses on the structure-function study of AfHypB from Archaeoglobus fulgidus and the functional interaction between AfHypA and AfHypB. / The intrinsic GTPase activity of AfHypB is very low, suggesting that AfHypB requires a G-protein activating protein (GAP) to activate its GTPase activity. Although AfHypB can interact with AfHypA to form 1:1 heterodimer, our data suggests that AfHypA is not a GAP for AfHypB. In addition, the FRET results showed that AfHypA could serve as a GEF (G-protein exchange factor) to activate the AfHypB from GDP-bound form to GTP-bound form and facilitate the dissociation of AfHypB dimer in the presence of GMPPNP. / Up to now, we have solved the structure of apo-AfHypB by X-ray crystallography. Crystals of AfHypB were grown using the hanging-drop-vapor-diffusion method and diffracted to ∼2.3 A. It belonged to space group P2(1)2(1)2, with unit cell dimensions a=72.49, b=82.33, c=68.66 A, alpha=beta=gamma=90°. Two molecules of AfHypB were found in an asymmetric unit. Structural comparison between the apo-AfHypB and GTP-bound HypB from M jannachii showed that the GTP binding broke the salt-bridge between K43 and D66, and induced conformational changes in the switch I loop and helix-3, which caused the HypB to form dimer and bind an extra Ni ion. The GTP-bound form of HypB was ready for Ni presenting. And GTP hydrolysis could induce the conformational changes of HypB in the switch I loop and helix-3, which dissociate the HypB dimer into the monomeric GDP-bound form. / Li, Ting. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 98-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Hydrogenases catalyse the reversible oxidation and production of H₂. They have been the subject of intense interest in recent years since these enzymes, or catalysts inspired by them, may greatly enhance our exploitation of H₂ as an energy carrier in a future ‘green’ H₂-based economy. However, a major challenge to the future use of these catalysts is their reactions with small molecule inhibitors, such as O₂ or CO. This thesis presents studies using Protein Film Electrochemistry, in which an enzyme is adsorbed onto an electrode to give an electroactive film. Although most hydrogenases are inhibited or irreversibly damaged by even trace O₂, certain O₂-tolerant hydrogenases are unusual in that they are able to sustain H₂ oxidation activity in the presence of O₂. Results outlined in this thesis suggest that the O₂ tolerance of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha relies upon O₂ attack generating exclusively the ‘Ready’ inactive state (formed by complete, four-electron reduction of O₂), which subsequently reactivates both rapidly and at high potential. The results contributed to a new explanation for how hydrogenases in certain microbes survive O₂. Electrochemical studies performed on a variant enzyme suggest that a modified proximal FeS cluster plays a role in conferring this O₂ tolerance. Studies of an enzymatic H₂/O₂ fuel cell employing the O₂-tolerant [NiFe]-hydrogenase Hyd1 from Escherichia coli as the anodic catalyst highlight the subtle influence of the reactions of the hydrogenase with O₂ on the power characteristics of the fuel cell under various operating conditions. This research also identifies straight-chain aldehydes as unprecedented inhibitors of H₂ production by the [FeFe]-hydrogenases. However, some of these results cannot currently be made freely available as they are to be published at a later date in academic journals.

Trichomonas vaginalis is a flagellated microaerophilic protozoan from the group Excavata that cause trichomoniasis, the most common nonviral sexually transmitted disease in the world. This thesis deals with the study of hydrogenases, enzymes catalyzing reversible conversion of protons and electrons to molecular hydrogen. In T. vaginalis, hydrogenases have been identified so far only in hydrogenosomes, modified anaerobic mitochondia that are involved in energy metabolism. We proved the presence of this enzyme also in the cytosol of T. vaginalis. Among several hydrogenase paralogues present in the genome, we selected an appropriate gene for the putative cytosolic hydrogenase (C-Hyd) and verified its cytosolic localization in the cells with overexpressed C-Hyd protein. Based on the determination of hydrogenase activities in different cell compartments and fractions obtained by affinity chromatography, we demonstrated the hydrogenase activity of C-Hyd protein, which means that C-Hyd is a functional hydrogenase. Identification of hydrogenase in T. vaginalis cytosol changes our understanding of trichomonad core metabolism and opens the door for the research of unexplored metabolic capabilities of this parasite.

No description available.