Theses and Dissertations at Montana State University (MSU)
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Item Quantitative 1 H NMR analyses of immunometabolic modulation in human macrophages(Montana State University - Bozeman, College of Letters & Science, 2019) Fuchs, Amanda Lee; Chairperson, Graduate Committee: Valerie Copie; Sage M. Schiller was an author and Wyatt J. Keegan, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Quantitative 1 H NMR metabolomics reveals distinct metabolic adaptations in human macrophages following differential activation' in the journal 'Metabolites' which is contained within this dissertation.; Sage M. Schiller was an author and Isaac R. Miller, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Pseudomonas aeruginosa planktonic- and biofilm-conditioned media elicit divergent responses in human macrophages' submitted to the journal 'PLoS pathogens' which is contained within this dissertation.Macrophages are innate immune cells that are found ubiquitously in nearly all human tissues, where they support host innate and adaptive immune responses in an effort to maintain systemic homeostasis. They are inherently plastic in nature and can dramatically modulate their functional phenotype according to pathogen and microenvironmental stimuli. Previous studies have shown that macrophages are particularly important for the resolution of inflammation in acute wound healing, which is marked by a phenotypic transition of wound macrophages from pro-inflammatory to anti-inflammatory. Chronic, or non-healing, wounds, such as diabetic, pressure, and venous leg ulcers, feature a prolonged host inflammatory response due in part to aberrant wound macrophage behavior. Non-healing in chronic wounds has also been shown to be dependent upon the establishment of pathogenic biofilms, which are more resistant to host defense mechanisms than planktonic, or free-floating, bacteria. Therefore, investigating macrophage dysregulation in the presence of bacterial biofilms has gained considerable interest. Here, 1D 1 H NMR-based metabolomics was utilized to identify metabolic pathways that are differentially modulated following primary human monocyte-derived macrophage activation with pro-inflammatory or anti-inflammatory stimuli relative to resting macrophages. Metabolic profiling of inflammatory macrophages indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provided evidence that inflammatory macrophages divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. In addition, we investigated which metabolic pathways are differentially modulated following primary human monocyte-derived macrophage exposure to Pseudomonas aeruginosa planktonic- and biofilm-conditioned media. Metabolic profiling of PCM- and BCM-exposed macrophages indicated a significant depletion of intracellular glucose without elevation of downstream glycolytic products. These metabolic patterns suggest that PCM- and BCM-exposed macrophages potentially divert glycolytic intermediates towards inositol phosphate metabolism. Overall, our studies provide additional support to previous findings, generate novel results regarding metabolic modulation of human macrophages following activation and exposure to planktonic- vs. biofilm-conditioned media, and contribute new insight to the field of immunometabolism.Item Unusual isomerization behavior of organic solutes at the aqueous-silica interface(Montana State University - Bozeman, College of Letters & Science, 2019) Purnell, Grace Elizabeth; Chairperson, Graduate Committee: Robert Walker; Robert A. Walker was a co-author of the article, 'Hindered isomerization at the silica/aqueous interface: surface polarity or restricted solvation?' in the journal 'Langmuir' which is contained within this dissertation.; Robert A. Walker was a co-author of the article, 'Surface solvation and hindered isomerization at the water/silica interface explored with second harmonic generation' in the journal 'The journal of chemical physics' which is contained within this dissertation.; Marshall T. McNally, Patrik R. Callis and Robert A. Walker were co-authors of the article, 'Buried liquid interfaces as a form of chemistry in confinement: the case of 4-dimethylaminobenzonitrile at the silica-aqueous interface' submitted to the journal 'The journal of the American Chemical Society' which is contained within this dissertation.; Marshall T. McNally, and Robert A. Walker were co-authors of the article, 'Isomerization at aqueous-silica interfaces and the role of solute structure' submitted to the journal 'Chemical physics letters' which is contained within this dissertation.Experiments described in this thesis address the question of how strong association between water molecules and the silica surface alter the solvation and isomerization behavior of adsorbed organic molecules from bulk solution limits. The work was motivated by the hypothesis that the ice-like structure induced by strong hydrogen bonding with the surface silanol groups would restrict solute isomerization. This hypothesis was tested using 2 surface-specific spectroscopic techniques: second harmonic generation (SHG) and time-correlated single photon counting in a total internal reflection geometry (TIR-TCSPC). This work examined two different 7-aminocoumarin dyes (Coumarin 151 and Coumarin 152) and dimethylaminobenzonitrile (DMABN). Coumarin 152 and DMABN both isomerize to form a twisted intramolecular charge transfer (TICT) state upon photoexcitation, whereas Coumarin 151 forms a simple (planar) intramolecular charge transfer state. SHG studies characterized the local solvation environment surrounding adsorbed molecules by providing electronic excitation energies that were compared to bulk excitation energies in different representative solvents. TIR-TCSPC measured the time-resolved emission of adsorbed molecules and quantified a solute's tendency to form TICT (or ICT) isomers at the aqueous-silica interface. Together, SHG and TIR-TCSPC provide a cohesive description of the local polarity across an aqueous-silica interface and how restricted solvent dynamics change a solute's photophysical chemistry. TIR-TCSPC studies reported that both C152 and DMABN are unable to isomerize to TICT states at the aqueous-silica interface, acting as if they were solvated in a nonpolar solvent or in a confined geometry. SHG studies confirm that the aqueous-silica interface is, in fact, more polar than the bulk aqueous limit, strongly implying that the observed effects are dynamic in origin rather than polarity driven. In contrast, studies of C151 show that this solute is largely insensitive to anisotropic, restrictive surface effects. Together results from these three molecules lead us to conclude that adsorption to the strongly associating aqueous-silica interface restricts large amplitude isomerization in organic molecules. Adsorption to less strongly associating interfaces does not cause this restriction. In the event that photo-induced isomerization does not require large amplitude motion, interfacial solvation has little effect on adsorbed solute behavior.Item Biochemical and biophysical characterization of plastic degrading aromatic polyesterases(Montana State University - Bozeman, College of Letters & Science, 2019) Topuzlu, Ece; Chairperson, Graduate Committee: Valerie Copie; Brandon C. Knott and Mark D. Allen were authors and Japheth Gado, Harry P. Austin, Erika Erickson, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Graham Dominick, Christopher W. Johnson, Valerie Copie, Christina M. Payne, H. Lee Woodcock, Gregg T. Beckham and John E. McGeehan were co-authors of the article, 'Structural and biochemical characterization of MHETASE' submitted to the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.As the world is producing more plastics than it can recycle, accumulation of manmade polymers in the environment is becoming one of the greatest global threats humanity is facing today. One of the major contributors to the plastics pollution problem is polyethylene terephthalate (PET), an aromatic polyester widely used in the packaging, beverage, garment and carpeting industries. As a response to the onslaught of plastics in the environment, fungi and bacteria are evolving metabolic pathways to convert plastics into useable energy sources. One of these organisms, a bacterium, Ideonella sakaiensis 201-F6, has recently been identified to convert PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), and to use these compounds for energy and growth. I. sakaiensis' ability to convert PET is made possible by two enzymes, named PETase and MHETase. As a first step, PETase breaks down the insoluble substrate PET into a soluble major hydrolysis product - mono-(2- hydroxyethyl) terephthalate (MHET), which is then further hydrolyzed by MHETase into TPA and EG. Crystal structure of PETase, as well as some of its biochemical features, have been reported several times to date, but MHETase has remained largely uncharacterized. This work focuses on further discovery-driven biophysical and biochemical characterization of PETase, visualization of PETase activity on various polyester surfaces, as well as the structural and biochemical characterizations of the MHETase enzyme. We have found that several aspects of PETase-mediated substrate surface modification hydrolysis mechanisms differ depending on the specific mechanical and material characteristics of the substrate. We have also found that PETase is inhibited by BHET. Additionally, we have solved the crystal structure of MHETase. MHETase consists of an alpha/beta hydrolase domain, and a 'lid' domain, commonly seen in lipases. Molecular dynamics simulations revealed the mechanism of MHETase action. Through bioinformatics approaches, we have also identified mutants of interest for improved MHETase activity. Coincubation of MHETase with PETase affects PET turnover in a synergistic fashion. Taken together, this work provides additional insights into the mechanisms of action of the PETase and MHETase enzymes, which may open new avenue for bioremediation and removing plastics from the environment in a sustainable manner.Item Proteomics analysis of the metabolic transition between aerobic and anaerobic conditions in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) Refai, Mohammed Yahya; Chairperson, Graduate Committee: Brian Bothner; Nina Paris, Hunter Fausset, Monika Tokmina Lukaszewska were co-authors of the article, 'Proteomics analysis of the transition between aerobic and anaerobic growth conditions in Escherichia coli' submitted to the journal 'Biochimica et biophysica acta' which is contained within this dissertation.As a facultative anaerobe, Escherichia coli has the ability to grow in anaerobic and aerobic environments. Despite detailed characterizations of this model organism in the presence and absence of oxygen, an in-depth understanding of changes to the proteome during transitions from aerobic to anaerobic growth is lacking. This thesis work focuses on elucidating how protein thiol oxidation and reduction change during a facultative anaerobe's transition from aerobic to anaerobic growth conditions, and pathways of thiol-mediated cell signaling. Redox driven changes in cysteine oxidation involved in signaling are referred to as 'thiol switches'. These modulate diverse biological activities ranging from gene expression and protein synthesis to environmental stress response. Surprisingly, little is known about the role of thiol switches during microbial transitions from aerobic and anaerobic growth conditions. To explore this uncharted territory, a mass-spectrometry (MS)-based proteomics workflow was developed and refined. Following extensive protocol optimization for high-throughput MS data processing, normalization, and pattern matching, the analytical pipeline was fine-tuned for the specific proteome-wide analysis of cysteine chemical modifications in E. coli. The approach was based on open-source software and publicly accessible databases, creating a transparent, reproducible, and easily sharable proteomics approach. Herein, the redox state and chemical forms of protein-based thiol switches in E. coli were characterized over time as the bacterium reversibly transitioned between aerobic and anaerobic growth conditions. Unexpectedly, differential alkylation analysis of cysteine-containing E. coli proteins revealed a higher degree of protein thiol oxidation under anaerobic growth conditions, a result not reported for E. coli or any other facultative anaerobe. Our proteome-wide analysis also revealed that cysteine redox potentials vary widely, and several specific E. coli proteins contain highly reactive thiols. These findings provide strong evidence for thiol-based signaling in E. coli in response to environmental changes such as aerobic to anaerobic growth transitions. Characterization of specific redox switches underlying metabolic changes associated with oxygen availability has uncovered a previously unknown E. coli cell signaling mechanism. Since transitioning between aerobic and anaerobic environments is associated with bacterial virulence, this work opens new avenues to target pathogenic facultative anaerobes and to develop novel thiol-based antibacterial therapies.Item Morphological adaptations facilitating attachment for archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2019) Hartman, Ross Alan; Chairperson, Graduate Committee: Mark J. YoungLittle is known regarding the attachment and entry process for any archaeal virus. The virus capsid serves multiple biological functions including: to protect the viral genome during transit between host cells, and to facilitate attachment and entry of the viral genome to a new host cell. Virus attachment is conducted without expenditure of stored chemical energy i.e. ATP hydrolysis. Instead, virus particles depend on diffusion for transportation and attachment from one host cell to another. This thesis examines the attachment process for two archaeal viruses. Sulfolobus turreted icosahedral virus (STIV) is well characterized for an archaeal virus. Still, no information is available concerning STIV attachment or entry. The research presented here shows that STIV attaches to a host cell pilus. Furthermore, combining the previously determined atomic model for the virus, with cryo-electron tomography, a pseudo-atomic model of the interaction between the host pilus and virus was determined. Based on this data, a model is proposed for the maturation of the virus capsid from a noninfectious to an infectious form, by dissociation of accessory proteins. Finally, a locus of genes is identified in the host cell, encoding proteins essential for viral infection, that are likely components of the pili structure recognized by STIV. The isolation of a new archaeal virus, Thermoproteus Piliferous Virus 1 (TSPV1), is also presented here. The TSPV1 virion has numerous fibrous extensions from the capsid, of varying length, that are the first observed for any virus. The capsid 2-3nm fibers likely serve to extend the effective surface area of the virus, facilitating attachment to host cells. Characterization of this new virus was conducted, including genome sequencing and determination of the protein identity for the capsid fibers. The research presented here provides a substantial advancement in our knowledge of the attachment process for archaeal viruses. Attachment to host pili is now emerging as a common theme for archaeal viruses. Furthermore, the isolation of the new archaeal virus TSPV1 demonstrates a novel strategy to increase the probability of interaction between a virus and host cell.Item When a lectin binds a sugar, and other sweet tales(Montana State University - Bozeman, College of Letters & Science, 2019) Bernhard, Samuel Pruitt; Chairperson, Graduate Committee: Mary J. Cloninger; Mackenzie S. Fricke was an author and Katharina Achazi, Paul Hillman, Willy Totten, Rainer Haag and Mary J. Cloningerwere co-authors of the article, 'The toxicity, uptake, and impact on galectin-3 mediated apoptosis of lactose functionalized dendrimers' submitted to the journal 'Biomolecules Special Issue: Moving Forward with Dendrimers' which is contained within this dissertation.The current state of chemotherapy and cancer treatment leaves much to be desired. Treatment is generally non-specific and relies on high dosage to achieve therapeutically relevant concentrations at target sites. Glycopolymer-drug conjugates, featuring targeting molecules and therapeutic prodrug on a water-soluble polymeric scaffold, offer a solution to these contemporary problems. Here, the complexity of glycopolymer design is explored through the lens of a biologically significant carbohydrate-binding receptor. In particular, galectin-3 is a complex Beta-galactoside binding lectin that experiences altered expression in many cancer pathologies and is implicated in metastasis, angiogenesis and poor overall prognosis. Galectin-3 mediates undesired cancer promoting processes through carbohydrate binding and oligomerization. A more complete understanding of the role galectin-3 plays in cancer progression will guide development of methods in the therapeutic intervention of these processes. In the interest of understanding galectin-3 and using it as a targeted receptor, its binding characteristics have been assessed through fluorescence lifetime and dynamic light scattering measurements. Employment of carbohydrates and glycopolymers including mannose, lactose, and lactose functionalized poly(amidoamine) (PAMAM) dendrimers, dendritic polyglycerols (dPG), and linear polymers (LP) provided insight into the carbohydrate binding avidity of galectin-3 and its propensity to oligomerize or form micron scale aggregates. A relationship between scaffold size and receptor recruitment was observed, which sheds light into multivalent binding motifs initiated by these glycopolymers and establishes a threshold for minimum requisite lactose functionality on lactose functionalized dendritic polyglycerols. In vitro cell based glycopolymer studies with AlexaFluor 647 and lactose functionalized PAMAM dendrimers revealed size-dependent uptake and demonstrated that accumulation occurs within the lysosome. Cellular aggregation experiments revealed that lactose functionalized LPs and dPGs influence galectin-3 mediated homotypic cellular aggregation and, in fact, augment this aggregation through receptor recruitment and cross-linking. The results reported here have provided a more fundamental understanding of galectin-3 binding interactions and have laid the groundwork for optimized glycopolymer-drug conjugate design.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 Reactive-atom scattering dynamics and liquid-vacuum interfacial structure(Montana State University - Bozeman, College of Letters & Science, 2019) Smoll, Eric James, Jr.; Chairperson, Graduate Committee: Timothy Minton; Maria Tesa-Serrate, Timothy K. Minton and Kenneth G. McKendrick were also authors of the article, 'Review of atomic and molecular collisions at liquid surfaces' in the journal 'Annual review of physical chemistry' which is contained within this dissertation.; Simon M. Purcell, Lucia D'Andrea, John M. Slattery, Duncan W. Bruce, Matthew L. Costen, Kenneth G. McKendrick and Timothy K. Minton were co-authors of the article, 'Probing conformational heterogeneity at the ionic liquid-vacuum interface by reactive atom scattering' in the journal 'The Journal of Physical Chemistry Letters' which is contained within this dissertation.; Timothy K. Minton was an author of the article, 'Scattering-angle randomization in nonthermal gas-liquid collisions' submitted to the journal 'Journal of physical chemistry C' which is contained within this dissertation.; John M. Slattery, Timothy K. Minton were also authors of the article, 'Probing a ruthenium coordination complex at the ionic liquid-vacuum interface with reactive atom scattering, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry' submitted to the journal 'Journal of physical chemistry C' which is contained within this dissertation.Experiments to characterize reactive and nonreactive gas-liquid scattering dynamics were carried out with the use of a crossed molecular beams apparatus configured for beam-surface scattering. In each experiment, the identity of the gas and liquid was strategically selected to reveal fundamental insights on the relationship between scattering observables and liquid-vacuum interfacial structure. This work is crucially important for the experimental advancement of liquid surface science and has the potential to impact our understanding of the chemical role of gas-liquid interfaces in the environment. An extensive literature review suggests that the inherent chemical specificity of reactive scattering is a promising probe of composition at the liquid-vacuum interface. We expand on what has been demonstrated in the literature by exploring F-atom scattering from the liquid-vacuum interface of deuterium labeled variants of the common ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C 4 mim][Tf 2 N]). The experimental data and new molecular dynamics simulations provide evidence for the extreme surface specificity of reactive scattering and help quantify the relative populations of [C 4mim] + conformations at the liquid-vacuum interface. Also, at a fixed incident angle, the site-specific IS flux angular distributions from [C 4mim] + were discovered to be related by the addition or subtraction of a line-shape proportional to a cos(θf) function. To investigate this phenomenon, a separate study of noble gas scattering from the liquid-vacuum interface of other low vapor pressure liquids was carried out. Our results support the generality of the relative cos(θf) character trend and demonstrate that the relative cos(θf) character between total flux angular distributions from squalane and a perfluoropolyether is independent of gas identity and incident angle suggesting that this metric is an intrinsic property of the liquid pair. The existing evidence suggests that the relative cos(θf) character between flux angular distributions is a result of angle-randomization from multiple collision scattering trajectories induced by atomic-scale corrugation at the liquid-vacuum interface. A study on the liquid-vacuum interface structure of a solution of [RuCl 2(p-cymene)P(C 8H 17) 3] in perdeuterated 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (d 11-[C 2mim][Tf 2N]) is also discussed. The experimental data suggest that [RuCl(p-cymene)P(C 8H 17) 3] + is enriched at the liquid-vacuum interface at the expense of d 11-[C 2mim] + and the hydrocarbon chains of the Ru-complex protrude into the vacuum.Item Complexation of lipids with cyclodextrin carriers for fully defined supplementation of cell culture(Montana State University - Bozeman, College of Letters & Science, 2019) Corbin, Elizabeth Dale; Chairperson, Graduate Committee: Edward Dratz and Renee Reijo Pera (co-chair)Induced Pluripotent Stem Cells (iPSCs) hold great promise for revolutionizing medicine and research. Scientists are currently able to reprogram adult cells of almost any type to a genetically 'open' state, pluripotency, wherein they lose the characteristics of their original cell type, and revert to a state that can reproduce indefinitely, and can be differentiated to many different cell types. IPSCs are currently grown in 'chemically defined' media that contains no animal derived components. This media eliminates animal and human sera because these tend to be quite variable and confound the reprogramming process, but the chemically defined media in use has almost no lipid content. The central goal of this project was to develop methods for chemically defined addition of lipids to cell culture media. The methods developed promise to be an advance in stem cell and general cell culture methodology, providing more reproducible experimental results, and supporting cells in culture with optimized lipid contents. In order to facilitate the addition of lipids to cell culture media without animal serum or serum albumin, complexation of individual lipids with a methyl beta-cyclodextrin starch was accomplished without addition of other molecules or oxidation of delicate lipids at a 1:1 stoichiometry. The lipid/MBCD complexes are soluble in aqueous media, and can be added individually or as a mixture to cell cultures. Application of complexed lipids to stem cells and fibroblasts revealed significant differences in lipid responses. Supplementation of human fibroblasts with a mixture of complexed lipids and other elements caused a 60% increase in proliferation over a 10 day period. Supplementation of stem cells with complexed lipids significantly increased proliferation, without reduction of pluripotency. Differences in lipid responses were also found between iPSC and embryonic stem cells, that may help elucidate differences between genetic or metabolic states which may point the way for more effective reprogramming of adult cells to pluripotency.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.