Theses and Dissertations at Montana State University (MSU)
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Item Versatility of cryo-electron microscopy as a structural technique informs iron mineral nucleation and growth in a mini-ferritin(Montana State University - Bozeman, College of Letters & Science, 2024) Gauvin, Colin Charles; Chairperson, Graduate Committee: C. Martin LawrenceIron is an enigmatic element. While necessary for life, it also contributes to the generation of reactive oxygen species via the Fenton reaction. To mitigate this, cellular life has evolved the ferritin family of proteins, including the 24 subunit ferritins and bacterioferritins, and the 12 subunit DPSL "mini-ferritins". Each of these catalyze the controlled oxidation and sequestration of iron as a hydrous ferric oxyhydroxide within their hollow protein cores. While there is a wealth of structural information on the unmineralized ferritins, little is known about the structures of the biomineralized forms, and the mechanism of ferric oxyhydroxide nucleation and growth. Here we report structural and biochemical characterization of a DPS-Like protein from Pyrococcus furiosus. This "thioferritin" utilizes a bacterioferritin-like ferroxidase center, but adopts the mini-ferritin quaternary structure, and is thus thought to sit at the evolutionary boundary between mini- and maxi-ferritins. In addition to the unmineralized structure, we report the 1.91 angstrom structure of P. furiosus thioferritin as it nucleates iron-oxyhydroxide distal to the ferroxidase site. In this very low iron form, a pair of conserved glutamate residues and unsaturated carbonyls at the 3-fold axis serve to template initial nucleation. We also determine structures of higher iron forms with a biomineralized ferrihydrite core, where C-terminal residues 170-176 interact directly with the initial mineral surface, which then grows towards the particle center. These studies provide important new insight into biological mechanisms for the controlled nucleation, growth and storage of ferric oxyhydroxide in this thioferritin specifically, and the ferritin superfamily as a whole.Item The influence of an iron deficient diet on the murine gut microbiome(Montana State University - Bozeman, College of Letters & Science, 2019) Coe, Genevieve Lea; Chairperson, Graduate Committee: Jennifer DuBoisIron is an essential nutrient for mammals 1. It is involved in multiple redox reactions that are essential for the survival of most organisms 2. There are two main types of iron that are absorbed from the diet: inorganic iron and heme 3. Dietary iron ingested by mammals is mostly absorbed in the small intestine; however, it is unclear whether the gut microbiome is involved in iron homeostasis or whether iron in the diet influences the microbiome. The goal of this project is to characterize the change in microbial composition in response to iron deficiency and iron repletion in conventional mice and define a baseline model for future studies involving the more complex human gut microbiome.Item Ultrafast photochemistry of aqueous iron(III) complexes(Montana State University - Bozeman, College of Letters & Science, 2017) Danforth, Rebecca Ann; Chairperson, Graduate Committee: Erik Grumstrup; Bern Kohler was a co-author of the article, 'Ultrafast photochemical dynamics of hexaaqua iron(III) ion' in the journal 'Chemical physics letters' which is contained within this thesis.The ultrafast photochemical dynamics of aqueous iron(III) solutions were measured utilizing ultrafast pump probe spectroscopy. Aqueous solutions of iron(III) were prepared at low pH (<4.5) and low iron(III) concentration (<5 mM) to allow for small aquairon(III) complexes and ferrihydrite to be studied. Small monomeric and dimeric aquairon(III) complexes were studied to elucidate the mechanisms involved in the formation of OH ° after UV excitation which were previously known to generate OH ° in vastly different quantities. Upon excitation of Fe 3+, a proton is released from a coordinated water molecule to generate FeOH 2+ in less than 200 fs. The newly generated FeOH 2+ can then undergo numerous recombination pathways to regenerate the Fe 3+. Approximately 10% of the excited Fe 3+ undergoes photoreduction and subsequent release of OH ° and Fe 2+ within 20 ps. Exciting FeOH 2+, results in homolysis to form Fe 2+ and OH ° with a wavelength dependent yield with a lifetime of 20 ps. Fe 2(OH) 2 4+ does not appear to generate significant quantities of OH ° however, the dimer is photostable in comparison to Fe 3+ and FeOH 2+. To further the understanding of the primary kinetics of iron(III) in aqueous solutions, ferrihydrite nanoparticles were studied. Ferrihydrite exhibits similar dynamics to hematite in which electrons are excited into the conduction band of ferrihydrite. The electrons can then relax to the bottom of the conduction band within 390 fs before undergoing various recombination process. This limits the amount of iron(III) converted into iron(II) in ferrihydrite. All iron(III) systems studied show unique kinetics after excitation that elucidate the mechanisms behind the generation of OH °.Item Role of the P-cluster and FeMo-cofactors in nitrogenase catalysis(Montana State University - Bozeman, College of Letters & Science, 2017) Keable, Stephen Michael Keable; Chairperson, Graduate Committee: John W. Peters; Andrew J. Rasmussen, Karamatullah Danyal, Brian J. Eilers, Gregory A. Prussia, Axl X. LeVan, Lance C. Seefeldt and John W. Peters were co-authors of the article, 'Three structural states of the nitrogenase P-cluster revealed in MOFE protein structures at poised potentials' submitted to the journal 'Biochemistry' which is contained within this thesis.; Jacopo Vertemara, Karamatullah Danyal, Andrew J. Rasmussen, Brian J. Eilers, Oleg A. Zadvornyy, Luca De Gioia, Giuseppe Zampella, Lance C. Seefeldt and John W. Peters were co-authors of the article, 'Acetylene interaction with the nitrogenase femo-cofactor investigated by structural and computational analysis' submitted to the journal 'Biochemistry' which is contained within this thesis.; Dissertation contains two articles of which Stephen Michael Keable is not the main author.Biological nitrogen fixation has been extensively researched for over four decades, yet due to the complex nature of this process, numerous questions still remain regarding the catalytic mechanism, and investigation of this system has relevance across a number of disciplines. Nitrogen is a fundamental element to all biological systems, primarily occurring in proteins and nucleic acids. However, most nitrogen on Earth is found in the form of nitrogen gas, a form that is biologically unavailable to most organisms owing to the strength of the triple bond between the two nitrogen atoms. The limited abundance of biologically accessible (or fixed) nitrogen has driven an anthropomorphic thrust to supplement the nitrogen cycle with nitrogenous fertilizers, thereby boosting agricultural output. The primary industrial method to produce these fertilizers, derived from the Haber-Bosch synthesis, is an energy intensive process that consumes approximately 1- 2% of the world's energy portfolio. This process utilizes metal iron catalysis, high temperatures and high pressures, along with hydrogen usually obtained from reformed fossil fuels, to reduce atmospheric nitrogen gas to ammonia. Aside from the environmental consequences that arise from the production of nitrogenous fertilizers, long-term agricultural application may also have disastrous ecological ramifications, such as eutrophication. Additionally, biological nitrogen fixation supports more than half the human population, and having a more complete understanding of this complex process has the potential to displace some of the demand for fertilizer production. The aforementioned reasons are clearly enough to warrant serious investigation into biological nitrogen fixation, however, the fascinating intricacies and comparative relevance to other biochemical systems further motivates the study of this system. The enzyme committed to this task, nitrogenase, orchestrates an elegant unidirectional multiple electron reduction and activation of the nitrogen triple bond. Historically, mechanistic characterization of this enzyme has been difficult for a number of reasons; however, studies to date have revealed many aspects of the process as biochemical techniques have improved. Nitrogenase is an oxygen sensitive, complex two-component enzyme that is mechanistically pertinent to many other biochemical processes. Presented here are studies revealing insight into substrate binding and the unique gated electron transfer mechanism of this fascinating enzyme.Item Biochemical characterization of the six-transmembrane epithelial antigen of the prostate family of metalloreductases(Montana State University - Bozeman, College of Letters & Science, 2015) Kleven, Mark Daniel; Chairperson, Graduate Committee: C. Martin Lawrence; George H. Gauss was the main author, and Mark D. Kleven, Anoop K. Sendamarai, Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site' in the journal 'Journal of biological chemistry' which is contained within this thesis.; Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'Characterization of a single B-type heme, FAD and metal binding sites in the transmembrane domain of six trans-membrane epithelial antigen of the prostate (Steap) family proteins' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.Iron and copper are the two most abundant transition metals in humans and are mediators of many essential cellular processes. The entry of these metals into cells require controlled processes, including their reduction prior to uptake. A group of integral membrane enzymes, the six-transmembrane epithelial antigen of the prostate (Steap) family, are able to perform this function. Steap3, in particular, functions as the primary ferric reductase in the transferrin cycle, the dominant mode of erythrocyte iron uptake. How these enzymes perform these functions has remained ill-defined. Here, the biochemical underpinnings of Steap metalloreductase activity have been investigated. To elucidate these mechanisms, expression systems for Steap3 and Steap4 have been developed in bacterial, insect, and human cell lines and purified to varying degrees. By analyzing the truncated cytoplasmic oxidoreductase domain of Steap4, it was found that NADPH is oxidized by transferring a pair of electrons to a flavin. With this truncation, however, flavin only binds weakly and the construct shows no ability to preferentially bind one type of flavin. In contrast, when the full length Steap3 was partially purified, it exhibits high-affinity FAD-binding, indicating that the transmembrane region of the protein contains the major structural features of the FAD binding site. Further, it was found that the cytoplasm-oriented loops between transmembrane helices formed the site. The next cofactor in the electron transport chain is a single b-type heme. Two strictly conserved histidines were identified that coordinate the heme and both are required for heme incorporation. The metal binding site at the extracellular face of the membrane was also characterized. Here, it was found that Steap3 and Steap4 share a conserved high-affinity iron binding site. Additionally, iron and copper both bind with similar affinities to Steap4. Two critical residues of the metal binding site were determined and their predicted proximity to the heme cofactor suggests that the electron is transfer is direct between cofactor and metal. Finally, it was found that Steap's are able to dimerize in the cells, forming homo- and heterodimers Together, the enzymatic mechanism has been characterized in-depth for the first time for these physiologically-significant enzymes.Item Nonaqueous organometallic electrochemistry : heterogeneous coupling of alkyl halides with reduced iron(Montana State University - Bozeman, College of Letters & Science, 1977) Hall, Jeffrey LouisItem Bis-cyclopentadienyl compounds of iron and vanadium(Montana State University - Bozeman, College of Letters & Science, 1957) Birkholz, Norman J.