Theses and Dissertations at Montana State University (MSU)
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Item New insights into radical initiation by radical S-adenosylmethionine enzymes and activation of [FeFe]-hydrogenase(Montana State University - Bozeman, College of Letters & Science, 2020) Impano, Stella; Chairperson, Graduate Committee: Joan B. Broderick; Hao Yang, Adrien Pagnier, Richard Jodts, Ryan Swimley, Eric M. Shepard, Sarah M. Hill, Christopher D. James, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Photolytic cleavage of S-adenosylmethionine' which is contained within this dissertation.; Eric M. Shepard, Hao Yang, Adrien Pagnier, Ryan Swimley, Emma Dolen, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Generation of an ethyl radical trapped in active sites of [FeFe]-hydrogenase maturase enzymes HydE AND HydG' which is contained within this dissertation.; Eric M. Shepard, Hao Yang, Jeremiah N. Betz, Adrien Pagnier, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'EPR and ENDOR spectroscopic evidence of an ammonium binding site in HydE' which is contained within this dissertation.; Adrien Pagnier, Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Investigation into all the necessary components required for [FeFe]-hydrogenase H-cluster maturation' which is contained within this dissertation.; Dissertations contains two articles of which Stella Impano is not the main author.Radical S-adenosylmethionine (SAM) enzymes harbor a [4Fe-4S] cluster in their active sites that coordinates a catalytically relevant small molecule SAM. During catalysis the S-5'C bond of SAM is reductively cleaved to generate a 5'-deoxyadenosyl radical that subsequently abstracts an H atom from substrate, allowing functionally diverse reactions to be achieved. Trapping of the 5'-deoxyadenosyl radical intermediate during turnover had proven difficult likely due to the formation of omega intermediate resulting from the oxidative addition of the 5'-deoxyadenosyl radical to the unique iron of the cluster. Recently, our laboratory showed that this elusive 5'-deoxyadenosyl can be liberated, captured, and characterized, in the absence of substrate, via photoinduced electron transfer (ET)-mediated reductive cleavage of SAM. Further, photolysis of [4Fe-4S] +-SAM complexes in different radical SAM enzymes revealed that the regioselective bond cleavage of SAM is dependent on the active site environment where either a 5'-deoxyadenosyl or a *CH 3, depending on the enzyme. When Sadenosyl- ethionine is used in place of SAM in the [4Fe-4S] +-SAM complex of HydE or HydG an ethyl radical is trapped. In either case, annealing of the methyl and ethyl radicals yields corresponding omega-like species, omega M and omega E, respectively. Functionally, HydE and HydG work together with a third protein HydF, to synthesize the H-cluster of [FeFe]-hydrogenase enzymes. HydG lyses tyrosine to generate CO and CN - ligands of the diiron core of the H-cluster, while the role and substrate of HydE are yet to be elucidated; however, it is hypothesized that this enzyme is responsible for dithiomethylamine (DTMA) bridge assembly. Our hypothesis is that HydE uses ammonium as a co-substrate and we propose that this polyatomic ion condenses with two CH 2S- like species to assemble the DTMA. We demonstrate for the first time via EPR and ENDOR spectroscopic techniques that HydE harbors an ammonium binding site; this NH 4 + would be stored in the active site of HydE prior to DTMA synthesis. Additionally, through in vitro [FeFe]-hydrogenase assays, we investigate what component of the essential E. coli lysate is required for H-cluster assembly. Results from this work suggest that the Hyd maturases are not the only proteins needed for H-cluster biosynthesis.Item The biofilm matrix in sulfate-reducing bacterial biofilms: potential roles for electron mediators and large proteins(Montana State University - Bozeman, College of Letters & Science, 2019) Krantz, Gregory Peter; Chairperson, Graduate Committee: Matthew Fields; Kilean Lucas, Erica L.-Wunderlich, Linh T. Hoang, Recep Avci, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm' in the journal 'Biofouling' which is contained within this dissertation.; Peter J. Walian, Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Large extracellular proteins sense hydrodynamic force and drive biofilm formation in Desulfovibrio vulgaris' which is contained within this dissertation.; Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Characterization of extracellular biofilm mutants cultivated on 1018 carbon steel in Desulfovibrio vulgaris Hildenborough' which is contained within this dissertation.Sulfate-reducing bacteria grow and form biofilms in soil and benthic environments across much of the Earth's surface. Formation of these prevalent biofilms requires the secretion of an extracellular polymeric substance (EPS) to allow the cells to stick together, as well as adhere to a surface. The specific interactions that occur between EPS components of an SRB biofilm are poorly understood. The data presented in this dissertation suggest the presence of two extracellular mechanisms utilized in these communities. The first mechanism was observed in a study altering the lactate (electron donor) and sulfate (electron acceptor) ratios to create limiting nutrient conditions in Desulfovibrio alaskensis G20 (G20) biofilms. G20 was grown under two conditions: electron donor limited (EDL) and electron acceptor limited (EAL) conditions. When grown on a 1018 carbon steel substrate, the G20 consumes all of the available lactate, and once limited, it turns to the high energy electrons in the Fe 0 for growth. Corrosion rates in the steel increased two fold compared to the EAL condition. Global metabolomic analysis revealed increased lumichrome levels under the EDL condition, which suggested higher flux through the riboflavin/FAD biosynthetic pathway. Previous research showed that synthetically adding riboflavin and FAD increases the corrosion rate of a SRB biofilm on 1018 carbon steel, and paired with these results, suggest G20 produces a flavin-based extracellular electron transfer molecule endogenously, and uses it to harvest high energy electrons from Fe 0 when limited for electron donor. The second mechanism was observed in Desulfovibrio vulgaris Hildenborough (DvH) biofilms grown on glass. Two proteins, DVU1012 and DVU1545 were found to be the most abundant extracellular peptides in a DvH biofilm. Single deletion strains for these proteins grew biofilm similar to the wild type strain, but a double deletion strain had decreased ability to form biofilm, demonstrating that at least one of the peptides must be present in order to form a biofilm. Exposure to increased shear force caused an large increase in wild-type biofilm biomass, yet eliminated the double mutant biofilm. These proteins are required for a DvH biofilm to respond to shear force.Item Relating protein structure to function: how protein dynamics maximizes energy gained by electron transfer in an anaerobic energy conservation mechanism(Montana State University - Bozeman, College of Letters & Science, 2019) Berry, Luke Montgomery; Chairperson, Graduate Committee: Brian Bothner; Angela Patterson, Natasha Pence, John Peters and Brian Bothner were co-authors of the article, 'Hydrogen deuterium exchange mass spectrometry of oxygen sensitive proteins' in the journal 'Bio-protocols' which is contained within this dissertation.; Saroj Poudel, Monika Tokmina-Lukaszewska, Daniel R. Colman, Diep M.N. Nguyen, Gerrit J. Schut, Micheal W.W. Adams, John W. Peters, Eric S. Boyd and Brian Bothner were co-authors of the article, 'H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle' in the journal 'Biochimica et biophysica acta (BBA) - general subjects' which is contained within this dissertation.; Monika Tokmina-Lukaszewska, Derek F. Harris, Oleg A. Zadvornyy, Simone Raugei, John W. Peters, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Combining in-solution and computational methods to characterize the structure-function relationship of the nitrogengase systems' which is contained within this dissertation.; Hayden Kallas, Derek F. Harris, Monika Tokmina-Lukaszewska, Simone Raugei, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Iron protein docking effects on MOFE protein dynamics: function of negative cooperativity and the regulation of electron trasfer' which is contained within this dissertation.Reduced ferredoxin (Fd) plays a critical role in anaerobic metabolism by acting as an alternative source of energy to adenosine triphosphate (ATP). The reduction potential of Fd is low (-450 mV) making it difficult to reduce individually. However, it has recently been discovered that a unique mechanism known as electron bifurcation allows anaerobic organisms to reduce Fd without suffering a loss of energy. Electron bifurcation was originally discovered in complex III of the electron transport chain, and increased the efficiency of the proton motive force without an overall change in the electron flow, minimizing energy loss. EB accomplishes this is by coupling a favorable (exergonic) and unfavorable (endergonic) reduction reaction. The exergonic reaction produces a singly reduced cofactor with a sufficiently negative reduction potential to allow the endergonic process to proceed. This allows anaerobic organisms to couple the formation of NADH, with the reduction of Fd. A detail of interest in the bifurcating mechanism is how these enzymes regulate the flow of electrons down the exergonic and endergonic branches to prevent multiple electrons from traveling down the exergonic branch. It is hypothesized that changes in the protein conformation alter the distance between cofactors altering the rate of electron transfer. To fully understand how changes in a protein's conformation regulates electron transfer in electron bifurcation we used a suite of in-solution techniques, such as H/D exchange and chemical cross-linking coupled to mass spectrometry to characterize the structure and dynamics of the model bifurcating enzyme, NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn), during the different steps of electron bifurcation. Additionally we also set out to use these techniques to characterize the structure and dynamics of the nitrogenase systems in order to obtain biophysical evidence of negative cooperativity in the various nitrogenase systems.Item Mechanisms of gating nucleotide-driven electron transfer in nitrogenase(Montana State University - Bozeman, College of Letters & Science, 2020) Pence, Natasha Kathrine; Chairperson, Graduate Committee: John W. Peters; Monika Tokmina-Lukaszewska, Zhi-Yong Yang, Rhesa N. Ledbetter, Lance C. Seefeldt, Brian B. Bothner and John W. Peters were co-authors of the article, 'Unraveling the interactions of the physiogical reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle' in the journal 'The Journal of Biological Chemistry' which is contained within this dissertation.The Mo-nitrogenase from Azotobacter vinelandii reduces N 2 to ammonia in an ATP-dependent process. It has two-components, the MoFe protein (MoFe) with the active site for N 2 reduction, and the Fe protein (FeP) that delivers electrons to MoFe. The less efficient alternative nitrogenases (Fe- and V-nitrogenases) have FeFe and VFe proteins with an additional subunit, termed gamma, whose role is unknown. Electron delivery to MoFe occurs through the Fe protein cycle (FeP cycle). This involves association between the FeP(MgATP 2) and MoFe, followed by electron transfer, ATP hydrolysis, release of P i, and dissociation of the FeP(MgADP 2) from MoFe. A study of the Fe protein cycle with the physiological electron donor flavodoxin (Fld), changed the rate-limiting step for nitrogenase catalysis, highlighting the important role of physiological protein donors in nitrogenase catalysis. However, it is unknown if Fld interacts with the MgADP or MgATP-bound state of the FeP. Insights from ClusPro 2.0 in silico docking models, time-resolved limited proteolysis and chemical cross-linking coupled with LC-MS and MALDI-TOF MS analysis show that the FeP(MgADP 2) forms a more productive complex with Fld, reducing competition between Fld and MoFe for the FeP(MgATP 2) to drive catalysis. To confirm our model, MicroScale Thermophoresis (MST) was developed to measure binding affinity between the FeP and nucleotides which agreed with previous measurements from isothermal calorimetry, confirming its application for nitrogenase. In silico docking models with ClusPro 2.0 and HADDOCK 2.2 identified structural differences between the Mo-nitrogenase and the alternative V- and Fe-nitrogenases that allow discrimination of protein-protein interactions that enable complex formation. The gamma subunit of the V- and Fe-nitrogenases mediates interactions between the nitrogenases, preventing competition between the least efficient Fe-nitrogenase and the Mo-nitrogenase. Finally, a pipeline was developed for homology modeling of potential physiological donor ferredoxin proteins (VnfF, FdxN, FixFd) associated with expression of the Mo-, V- or Fe-nitrogenases. Insights from in silico docking and assessment with the PRODIGY server were used to identify structural features that differentiate how these ferredoxins interact with the FePs of the three nitrogenases. Ultimately, nucleotide-dependent control of protein-protein interactions is necessary to support N 2 reduction and funnel electrons to the most efficient Mo-nitrogenase.Item Characterizing excited state transport and charge carrier dynamics in lead halide perovskites(Montana State University - Bozeman, College of Letters & Science, 2020) Hickey, Casey Lynn; Chairperson, Graduate Committee: Erik Grumstrup; Andrew H. Hill, Eric S. Massaro and Erik M. Grumstrup were co-authors of the article, 'Ultrafast excited-state transport and decay dynamics in cesium lead mixed halide perovskites' in the journal 'ACS energy letters' which is contained within this dissertation.; Andrew H. Hill and Erik M. Grumstrup were co-authors of the article, 'Screening links transport and recombination mechanisms in lead halide perovskites' in the journal 'The journal of physical chemistry C' which is contained within this dissertation.; Erik M. Grumstrup was a co-author of the article, 'Direct correlation of charge carrier transport to local crystal quality in lead halide perovskites' submitted to the journal 'Nano letters' which is contained within this dissertation.; Erik M. Grumstrup was a co-author of the article, 'A reduced artifact approach for determining diffusion coefficients in time-resolved microscopy' submitted to the journal 'The journal of physical chemistry C' which is contained within this dissertation.Understanding fundamental processes which drive the behavior of photoexcited charge carriers is essential to the development of novel semiconducting materials. The studies presented in this work combine ultrafast microscopy with a novel data analysis technique to provide an in-depth characterization of the excited state transport and recombination dynamics which occur in a series of lead halide perovskites. An investigation of the impact halide composition has on recombination dynamics in CsPbI 2Br revealed that trap-mediated recombination dominates at low fluences, with Auger recombination becoming increasingly important as the excitation density increases. Additionally, the average diffusivity measured for CsPbI 2Br (DA = 0.27 cm2/s) is nearly 10x lower than that observed in MAPbI 3. Further, it was determined that the dielectric constants relevant to photoexcited charge carriers in CsPbBr3 and MAPbBr3 perovskites (11.5 and 13, respectively) are intermediate between the high and low frequency limits, and that halide chemistry plays an integral role in determining the screening ability of lead halide perovskites. By correlating charge carrier diffusivities to locally measured crystal quality, it was found that solution processing methods can cause subtle lattice defects which act to impede transport and risk going undetected by bulk measurement techniques. Finally, to improve upon the traditional method for extracting diffusivities from transport measurements, which relies on perfectly Gaussian point spread functions, a new method was developed which instead relies on a numerical convolution of the actual point spread function with the diffusion equation. Compared to the traditional Gaussian method, the numerical convolution method proved to more accurately determine the diffusion coefficient, especially in the case of an anomalous point spread function.Item Partitioning of reactive oxygen species via the re-oxidation of electron transfer flavoprotein(Montana State University - Bozeman, College of Letters & Science, 2019) Austvold, Chase Kennor; Chairperson, Graduate Committee: Edward DratzThe biology of Reactive Oxygen Species are poorly understood. Within a healthy cell, Reactive Oxygen Species behave as signaling molecules, although overproduction leads to oxidative damage. In order to understand when the overproduction of Reactive Oxygen Species takes place, or leads to oxidative damage, the elementary step of quantification becomes necessary. Electron Transfer Flavoprotein is a known Reactive Oxygen Species producing enzyme and was studied. Electron Transfer Flavoprotein is a key-player within the production of energy within the eukaryotic mitochondria. The redox nature of Electron Transfer Flavoprotein's catalytic cofactor, flavin adenine dinucleotide produced two types of ROS; the superoxide anion (O 2 °-) and hydrogen peroxide (H 2 O 2). Electron Transfer Flavoprotein produced roughly five-fold more O 2 °-compared to H 2 O 2 as the enzyme became oxidized. It has been put forward that the production of these two Reactive Oxygen Species is dictated by the formation of a radical pair between the flavin adenine dinucleotide of Electron Transfer Flavoprotein and molecular oxygen. Two types of radical pairs can be formed, either in a triplet or singlet state, and the rate in which these states occur can be influenced by a static magnetic field. Therefore, the effect of a magnetic field on these products was also studied. Upon the suppression of magnetic field strength, the production of H 2 O 2 decreased and a proportional increase of O 2 °-was observed.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 Photo induced absorption study of model organic charge transfer systems(Montana State University - Bozeman, College of Letters & Science, 2000) Hyfield, Amy Alta ElizabethItem Optical properties and electron transfer in novel bis salicylimine diamino metallo-Schiff-base compounds(Montana State University - Bozeman, College of Letters & Science, 2002) Wilcox, Jeffrey RockefellerItem Interactions and electron transfer involved in pyruvate formate-lyase activation(Montana State University - Bozeman, College of Letters & Science, 2013) Crain, Adam Vernon; Chairperson, Graduate Committee: Joan B. Broderick; Kaitlin S. Duschene, John W. Peters and Joan B. Broderick were co-authors of the article, 'Iron sulfur clusters, S-adenosylmetionine enzymes, and their role in hydrogenase maturation' which is contained within this thesis.; Joan B. Broderick was a co-author of the article, 'Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP+ oxidoreductase and pyruvate formate-lyase activating enzyme' which is contained within this thesis.; Joan B. Broderick was a co-author of the article, 'Pyruvate formate-lyase: protein-protein interactions and activation by pyruvate formate-lyase activating enzyme' which is contained within this thesis.; Stephanie J. Maiocco, Sean J. Elliot and Joan B. Broderick were co-authors of the article, 'Elucidating the role of cation binding in PFL-AE' which is contained within this thesis.; Martina D. Van Hoy and Joan B. Broderick were co-authors of the article, 'Pyruvate:flavodoxin oxidoreductase is the electron donor for pyruvate formate-lyase activating enzyme' which is contained within this thesis.Pyruvate formate-lyase activating enzyme (PFL-AE) is one of the best-characterized members of the radical S-adenosyl-L-methionine (SAM) superfamily. The radical SAM enzymes utilize an iron-sulfur cluster and SAM to catalyze a diverse set of reactions such as vitamin synthesis, enzyme activation, DNA repair, and sulfur insertion to name a few. PFL-AE contains one [4Fe-4S] cluster coordinated by cysteine residues from a canonical CX3CX2C radical SAM motif. Iron-sulfur cluster-initiated reductive cleavage of S-adenosylmethionine results in a highly reactive 5'-deoxyadenosyl radical that abstracts a pro-S hydrogen from glycine 734 on PFL creating a stable glycyl radical. PFL utilizes this glycyl radical to catalyze the reaction pyruvate + CoA [] formate + acetyl-CoA, thereby providing the main source of acetyl-CoA for the citric acid cycle under anaerobic conditions. We have undertaken experiments using circular dichroism and isothermal titration calorimetry to characterize interactions between flavodoxin (Fld) and its cofactor (FMN). These experiments show that cofactor binding significantly increases flavodoxin stability and structure, which is required for protein-protein interactions. Anaerobic surface plasmon resonance experiments were used to provide insight into protein-protein interactions between the enzymes involved in PFL activation and in all cases, the proteins interact with low micromolar affinity. SAM binding experiments with PFL-AE were performed in the presence and absence of PFL, which demonstrate that PFL binding to PFL-AE does not alter SAM binding affinity for PFL-AE. PFL activation studies using PFL-AE in the presence of PFL substrates/analogues show that they are not required for PFL activation, however they do play a large role in activation and their inclusion resulted in 3.7 fold higher glycyl radical concentrations. In vivo concentrations were calculated for proteins and small molecules involved in PFL activation and activity in E. coli to provide a context for our measured equilibrium constants and to determine the amount of bound protein in vivo. Activity assays and UV-vis electron transfer assays show that pyruvate:flavodoxin oxidoreductase (PFOR) is capable of activating the PFL system. The aggregate data suggests that electron transfer from Fld to PFL-AE only occurs when SAM and PFL are bound to PFL-AE.