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Item The role of fixX in electron bifurcation(Montana State University - Bozeman, College of Letters & Science, 2016) Miller, Jacquelyn Marie; Chairperson, Graduate Committee: John W. PetersTwo known methods of physiological energy conservation are substrate level phosphorylation and electron transfer phosphorylation. Recently, electron bifurcation has been established as a third and key mechanism of energy conservation in biological processes. This coupling of endergonic and exergonic reactions allows for utilization of reducing potential to perform energetically expensive physiological reactions. A significant and energetically expensive physiological reaction is nitrogen fixation, which provides a substantial portion of the bioavailable nitrogen that life requires. Electron bifurcation is utilized by the FixABCX system that is up regulated during diazotrophic growth and is suggested to bifurcate electrons from NADH to quinone of the electron transport chain through high potential electron transfer proteins and to nitrogenase though low potential electron transfer proteins. The determination of how cellular mechanisms overcome the energy barriers of high potential electron transfers through electron bifurcation is crucial for our fundamental understanding of energy transfer and energy conservation. The work presented in this thesis aims to progress the present knowledge in this third mechanism of energy conservation and shows support for a protein in the FixABCX complex, FixX, as the low potential electron acceptor in the complex. Numerous organisms were investigated as potential model systems for FixABCX with varying degrees of success. The genome of the organism, Roseiflexus castenholzii, contains both the nitrogenase and fixABCX genes and has successfully been used to obtain FixX. This protein shows homology to ferredoxin, a physiological reductant of the nitrogenase Fe protein in some organisms. EPR spectroscopy and sequence analysis suggests FixX contains 2 [4Fe-4S] clusters, while a potentiometric titration shows the clusters to have highly negative mid-point potentials. The preliminary evidence supports FixX of the FixABCX system to be a low potential electron transfer protein.Item Purification and characterization of gene products of the nitrous oxide reductase gene cluster(Montana State University - Bozeman, College of Letters & Science, 2002) Henery, Shannon MichelleItem Characterization of NosX from Achromobacter cycloclastes and co-crystallization of N 2OR and PAZ(Montana State University - Bozeman, College of Letters & Science, 2013) Ijima, Fumihiro; Chairperson, Graduate Committee: David M. DooleyNitrous oxide (N 2O) is known as a greenhouse gas and an ozone-depleting substance. Denitrifying bacteria such as Achromobacter cycloclastes emit N 2O into the atmosphere during the denitrification process. Up to seventy percent of the total N 2O emission on the earth is derived from this process. N 2O is a very stable substance and its lifetime in the atmosphere is 114 years. Investigation of N 2O reductase (N 2OR) and its accessory proteins may ultimately be useful in the design of catalytic systems, either biological or chemical, for the control of N 2O level. The first aim of this research is to characterize NosX, which is one of the accessory proteins for N 2OR. Although NosX was shown to be involved in the activation of N 2OR, the structure and function of NosX and the mechanism of NosX on N 2OR activation are still unknown. Furthermore, NosX has never been isolated and probed. In this study, culture conditions for NosX expression in A. cycloclastes (AcNosX) were optimized to obtain adequate amounts of AcNosX (chapter 2), and AcNosX was characterized with UV/Vis, fluorescence, EPR, and MS spectrometry (chapter 3). The structure of AcNosX was investigated by computational and modeling methods (chapter 3). Crystallization trials were also undertaken. The mechanism of N 2OR activation by AcNosX was studied with UV/Vis and EPR spectrometry (chapter 4). We demonstrated that AcNosX was redox active and donated electrons to the Cu sites of AcN 2OR. The information revealed from this research will help to fully understand the activation mechanism of N 2OR with NosX. The second aim of the research is to investigate the interaction between AcN 2OR and its potential physiological electron donor, pseudoazurin (PAz) (chapter 5). X-ray crystallographic techniques were utilized to determine the structure of the AcN 2ORAcPAz complex and conditions were identified that grew crystals that included both AcN 2OR and AcPAz. Although the crystals showed diffraction patterns, further optimization is required to identify the complex structure. The information from this research will be important to elucidate the docking mechanism for electron transfer between N 2OR and PAz.