The role of fixX in electron bifurcation
Miller, Jacquelyn Marie
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Two 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.