Theses and Dissertations at Montana State University (MSU)
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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 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 Development of density functional methods for iron-sulfur clusters : application to the elusive structure of the iron-molybdenum cofactor of nitrogenase(Montana State University - Bozeman, College of Letters & Science, 2011) Harris, Travis Victor; Chairperson, Graduate Committee: Robert K. SzilagyiIron-sulfur proteins are found in all forms of life, performing key functions in many of the most important biological processes, such as nitrogen fixation, respiration, DNA repair, and photosynthesis. The functional centers of these metalloproteins are iron-sulfur clusters, which are efficient electron-transfer agents, and provide a range of other functions, from catalysis to iron sensing. The main focus of this dissertation is to eliminate the structural uncertainties of one of the most complex iron-sulfur clusters, the iron-molybdenum cofactor of nitrogenase. The long-term goal of nitrogenase research is to understand all of the molecular-level details that enable biological nitrogen fixation, which may lead to alternatives to the fossil fuel-reliant, high temperature and pressure Haber-Bosch process for industrial nitrogen fixation. The research presented here is all based on computational chemistry, using primarily density functional theory with comprehensive experimental support from the literature. Computational chemistry can help explain experimental findings, and it can be used to probe structural and mechanistic details that cannot be observed through experiment; however, methods for theoretical modeling of iron-sulfur clusters must be developed before accurate results can be obtained. In this work, the best available methods (functionals, basis sets, and population analyses) are developed by comparing calculated properties of a series of model iron-sulfur complexes with experimental reference data. The newly recommended methods are then applied to a protein-embedded cluster, and the effects of the network of hydrogen bonds, dipoles, and electrostatic interactions are systematically probed. In addition to new insights regarding the role of the protein in tuning iron-sulfur cluster properties, an algorithm is derived for constructing computational models that capture all significant environmental effects. Future work will benefit from the foundations developed in these first two studies. The final work is a computational evaluation of the uncertain composition, charge, magnetic structure, and protonation state of the iron-molybdenum cofactor, based on a series of criteria defined by all available experimental data. The results show that the [Mo IV -2Fe II -5Fe III -9S ²- -C ⁴-] composition with hydroxyl-protonated homocitrate ligand is favored over the [Mo IV -4Fe II -3Fe III -9S ²- -N ³- ] composition preferred in the literature, adding new context to the many proposed mechanisms for biological nitrogen fixation.Item Investigations of nucleotide-dependent electron transfer and substrate binding in nitrogen fixation and chlorophyll biosynthesis(Montana State University - Bozeman, College of Letters & Science, 2009) Sarma, Ranjana; Chairperson, Graduate Committee: John W. PetersThe studies presented in this thesis include studies of nucleotide-dependent conformations of the electron donor protein in nitrogenase and dark-operative protochlorophyllide reductase (DPOR) characterized using small-angle x-ray scattering and x-ray diffraction methods. Nitrogen fixation and chlorophyll synthesis are involved in the reduction of high energy bonds under physiological conditions. Both make use of elegant reaction mechanisms made possible by complex enzyme systems which are evolutionarily related. Nitrogenase reduces nitrogen to ammonia and is a two-component metalloenzyme composed of Fe protein and MoFe protein. For nitrogen reduction, the Fe protein and MoFe protein associate and dissociate in a manner concomitant with hydrolysis of at least two MgATP molecules and enables the concomitant transfer of at least one electron from Fe protein to MoFe protein. During chlorophyll biosysnthesis, the rate limiting step is catalyzed by a two-component metalloenzyme called DPOR. The two components of DPOR are BchL and BchNB proteins and these share high level of sequence similarity with the Fe protein and the MoFe protein, respectively. Based on this sequence similarity and biochemical data available, it is proposed that the reaction mechanism is similar to nitrogenase mechanism in which the components of DPOR associate and dissociate in a nucleotide dependent manner, to enable intercomponent electron transfer. Fe protein and BchL present as unique examples of proteins that couple nucleotide dependent conformational change to enable electron transfer for high energy bond reduction. The present studies have been directed at studying the low resolution studies of MgATP-bound wild-type Fe protein and its comparison to the structure of the proposed mimic, i.e, L127 Delta Fe protein. The studies presented show evidence of the MgATP-bound wild-type Fe protein having a conformation very different from the L127 Delta Fe protein. The chapters also include detailed characterization of the structure of BchL in both MgADP bound and nucleotide-free states which offer detailed insights in the structure based mechanism of BchL, with primary focus on identifying key residues involved in componenet docking and in electron transfer. Together, the studies on the Fe protein and BchL have furthered our understanding of mechanism of electron transfer in these complex enzyme systems.Item Insights into microbial metabolism(Montana State University - Bozeman, College of Letters & Science, 2011) Burgess, Mary Catherine; Chairperson, Graduate Committee: John W. PetersNitrogen fixation (catalyzed by the enzyme nitrogenase), cellular respiration (completed through the Tricarboxylic Acid (TCA) cycle) and mercury detoxification (through mercury methylation) are three metabolic processes used by a wide variety of microorganisms, but that also have far reaching impacts on nutrient cycling in the environment. Roseiflexus castenholzii has been found to have a unique nitrogenase gene cluster encoding several nitrogenase homologs, including the structural proteins NifH and NifDK and the radical SAM protein, NifB, necessary for cofactor biosynthesis. However, the genome of R. castenholzii lacks the suite of nitrogenase accessory proteins necessary for nitrogen fixation. To investigate the metabolic role of these nitrogenase homologs, expression and purification protocols were developed that aid in the biochemical characterization of these proteins. Synechococcus sp. PCC 7002 encodes three novel TCA proteins, contrary to previous studies that indicated these phototrophs have incomplete TCA cycles. Expression, purification and preliminary crystallization trials were completed on the three novel TCA proteins in order to gain insight into the structure of the proteins which will elucidate the mechanism of each novel enzyme and provide evidence into the novel TCA cycle utilized by these cyanobacteria. The third project presented examines the role of microorganisms in metabolizing mercury, producing methylmercury and providing an entry point for methylmercury into the food chain in Yellowstone National Park (YNP). In this project, environmental samples were enriched for a sulfate reducing organism and a culture containing three sulfate reducing bacteria (SRB) has been established. The SRB that are present and active in the enrichment samples are known to reduce sulfate and may be responsible for the presence of methyl mercury in algal mats that bioaccumulates through the food chain in YNP. The enrichment of SRB in this culture will enable the identification and characterization of the organisms that are capable of methylating mercury in hydrothermal systems. Collectively, the results presented herein increase the knowledge base of three metabolic processes used by microorganisms: nitrogen fixation, cellular respiration through the TCA cycle and mercury detoxification; these results will contribute to a broader understanding of how these processes have evolved and their impacts on the environment.Item Nucleotide dependent conformational changes in the nitrogenase Fe protein(Montana State University - Bozeman, College of Letters & Science, 2005) Sen, Sanchayita; Chairperson, Graduate Committee: John W. PetersNitrogenase is a complex metal-containing enzyme that catalyzes the conversion of nitrogen gas to ammonia. During nitrogenase catalysis the Fe protein and the molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. The role of nucleotide binding and hydrolysis in nitrogenase catalysis is one of the most fascinating aspects of nitrogenase function. The Fe protein upon binding to MgATP undergoes a huge conformational change which is important for subsequent steps of nitrogenase reaction mechanism. Therefore structural characterization of the Fe protein bound to MgATP will provide a basis on how MgATP binding promotes the complex formation whereas hydrolysis to MgADP leads to the dissociation of the macromolecular complex structure. Towards these ends we have conducted structural studies on a site-directed variant of the Fe protein which is a close mimic of the MgATP conformational state. Structural characterization of this Leu127 deletion variant revealed a distinctly new conformation of the Fe protein which arises from the rigid body reorientation of the homodimeric Fe protein subunits with respect to each other. The structure not only provides the first basis on rationalizing the initial docking interactions between the component proteins but also helps us to dissect the conformational changes on the Fe protein which occur upon nucleotide binding from those conformational changes that are imposed on the Fe protein by the MoFe protein during complex formation. Having this structure in hand, we have developed several other experimental approaches like Mass spectrophotometry and Small Angle X-ray Scattering/Diffraction (SAXS) techniques to probe the relationship between the Leu127 deletion variant a close structural mimic of MgATP bound "on state" and the actual MgATP bound state which is more difficult to probe crystallographically. These studies will help us to compare the different nucleotide bound states (MgADP and MgATP) of the Fe protein in solution that will help to predict the level of conformational change that is induced in the Fe protein that makes it compatible for binding to the MoFe protein in the nitrogen catalysis cycle.Item Defining the ecological interactions that drove the evolution of biological nitrogen fixation(Montana State University - Bozeman, College of Letters & Science, 2012) Hamilton, Trinity Lynn; Chairperson, Graduate Committee: John W. Peters; Eric S. Boyd and John W. Peters were co-authors of the article, 'Environmental constraints underpin the distribution and phylogenetic diversity of NIFH in the Yellowstone geothermal complex' in the journal 'Microbial ecology' which is contained within this thesis.; Rachel K. Lange, Eric S. Boyd and John W. Peters were co-authors of the article, 'Biological nitrogen fixation in acidic high-temperature geothermal springs in Yellowstone National Park, Wyoming' in the journal 'Environmental microbiology' which is contained within this thesis.; Marcus Ludwig, Ray Dixon, Eric S. Boyd, Patricia C. Dos Santos, Joao C. Setubal, Donald A. Bryant, Dennis R. Dean and John W. Peters were co-authors of the article, 'Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii' in the journal 'Journal of bacteriology' which is contained within this thesis.; Marty Jacobson, Marcus Ludwig, Eric S. Boyd, Donald A. Bryant, Dennis R. Dean and John W. Peters were co-authors of the article, 'Differential accumulation of NIF structural gene MRNA in Azotobacter vinelandii' in the journal 'Journal of bacteriology' which is contained within this thesis.All life requires fixed forms of nitrogen (N). On early Earth, fixed N was supplied through abiotic mechanisms, which became limiting to an expanding biome, precipitating the emergence of biological nitrogen fixation. Today, most biological nitrogen fixation is catalyzed by molybdenum (Mo)-dependent nitrogenase (Nif). Alternative forms of the enzyme contain either vanadium (V) or only iron (Fe) instead of Mo, but are only found in taxa that encode Nif. Geochemical evidence suggests Mo bioavailability was limited on the early Earth, leading to the hypothesis that alternative forms of nitrogenase are ancestral. Evidence presented here suggests that in fact Nif emerged first in a methanogenic archaeon. Previous studies revealed a widespread distribution of nif along geochemical gradients but little is known about the environmental conditions that drove its evolution. An analytical approach allowed examination of the role environment played in shaping the evolution of Nif across geochemical gradients in Yellowstone National Park. The distribution of nifH was widespread and not constrained by temperature or pH alone, but exhibited evidence of niche conservatism imposed by salinity, and seemed dispersal limited. Activity measurements in sediments collected from high-temperature acidic springs confirmed the potential for N ₂ fixation in these environments. These data expand our understanding of the habitat range and environmental drivers of N ₂ fixing organisms. In organisms that encode alternative nitrogenases, Nif is preferred for nitrogen fixation. In addition, the alternative forms of the enzyme do not encode the full suite enzymes necessary for assembling the active site metal cofactors. Presumably, the selective pressure driving the evolution of alternative nitrogenase would have been provided by Mo limitation. Transcriptome studies of a model organism which encodes all three forms of nitrogenase reveals the genes associated with expression of each nitrogenase and the interplay between systems that enables nitrogen fixation in the absence of Mo and fixed N. These analyses suggest the alternative nitrogenases would not function in the absence of Nif biosynthetic machinery and expression of nitrogenase is regulated by fixed N limitation and metal availability. The results presented here help elucidate the environmental conditions that have driven nitrogenase evolution, resulting in the extant enzyme.