Theses and Dissertations at Montana State University (MSU)
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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 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 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.