Browsing by Subject "electron transfer"

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    Exploring properties of ferredoxins that control electron transfer to nitrogen fixation and anaerobic aromatic compound degradation in Rhodopseudomonas palustris
    (2024-05) Lewis, Nathan
    All of life requires the movement of electrons, from donor to acceptor, to keep metabolism turning, to synthesize special metabolites, and to generate cellular energy. Ferredoxins (Fds) are small electron carrying proteins, roughly 6 to 12 kDa, which are involved in some of the most important biochemical processes including biological nitrogen fixation, photosynthesis, hydrogen generation, and anaerobic aromatic compound degradation (AACD). Fds emerged relatively early on an evolutionary timescale, which enabled their duplication and specialization as a product of selective pressure for efficient electron flow. As a product of their diversification, Fds are known to have different reactivities with various electron transfer pathways. It has been demonstrated that the regulation of gene expression, structural differentiation of Fds, and electrochemical compatibility of Fds and their redox partners can all play into the ability of a Fd to react with one pathway over another. What remains unknown, however, is how these properties need to change to get one pathway to support another. In this work, I use the purple non-sulfur bacterium Rhodopseudomonas palustris to study the determinants of Fd reactivity by testing the insulation of electron transfer between nitrogen fixation and AACD. These two pathways use homologous Fds to power energy-intensive reactions, but existing evidence shows that they do not share reducing power. I demonstrate that R. palustris can be used as a chassis to select for mutations in electron transfer components, generating strains which overcome the insulation of electron transfer. In characterizing one such laboratory-evolved strain, I discovered two mutations which worked together to break the insulation between AACD and nitrogen fixation. One substitution was in a Fd while the other was in a possible Fd reducing enzyme. By characterizing the Fd variants involved with these pathways, I found that the substitution did not change any properties known to control electron transfer through Fds. The Fd mutant had a unique electronic structure, implicating these properties of Fds in electron transfer insulation. Through protein modelling, site directed mutagenesis, and growth studies, I discovered that the Fd reducing enzyme is the electron donor for AACD and is part of a family of electron-bifurcating enzymes which had not previously been implicated in nitrogen fixation. Altogether this work advances our understanding of the properties of Fds and Fd-reducing enzymes that control selective electron transfer and helps identify a novel electron-bifurcating enzyme involved in AACD and nitrogen fixation.
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    Redox potential dependent respiration by Geobacter sulfurreducens
    (2015-10) Levar, Caleb
    Geobacter sulfurreducens is a Gram negative δ-proteobacteria with the ability to couple the internal oxidation of a carbon and electron donor with the external reduction of extracellular electron acceptors. Extracellular electron acceptors utilized by G. sulfurreducens include insoluble Fe(III)- and Mn(IV)-oxides, electrodes poised at accepting potentials, and a variety of soluble acceptors including humic acids and chelated metals. These substrates exist over a redox potential window greater than 0.5 V, suggesting that respiratory flexibility to efficiently take advantage of electron acceptors with different redox potentials would be a useful trait. The data presented within this thesis demonstrate that G. sulfurreducens uses multiple electron transfer pathways for the reduction of extracellular electron acceptors of different redox potentials.