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Title: Formation and disruption of pH gradients across biomembranes
Author: Daskalakis, Nikolaos Nektarios
ISNI:       0000 0004 2717 9733
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Membrane-bound redox enzymes utilise the free energy generated by electron transfer reactions to translocate protons across membranes, generating a transmembrane proton gradient. This is a means to store energy and living cells utilise it for ATP synthesis or other energy requiring processes. The molecular mechanism of proton translocation by proton pumps has been intensively studied but is still controversial. A new method is being developed that allows studying cytochrome b03 a ubiquinol oxidase from Escherichia coli (E. coli) in a native- like environment. Cytochrome b03 catalyses the oxidation of quinol and the four electron reduction of O2 to water and functions as a proton pump. Cytochrome b03 is incorporated in lipid vesicles in the form of a total Inner Membrane extract, avoiding purification steps. Ubiquinone-IO (UQ-IO) an analogue of the physiological electron donor of the enzyme UQ-8 is also incorporated in the vesicles with a pH-sensitive fluorescent probe. The vesicles are adsorbed on specifically functionalised Au electrodes so that they remain intact and retain fluorescene. Cytochrome b03 is electrochemically activated by applying a sufficiently negative potential to reduce the quinone pool. A pH gradient is then formed and is monitored in real time by monitoring fluorescence changes of the probe. The activity of the enzyme can be controlled by electrochemically tuning the redox state of the quinone pool. The overall aim is to study the pumping activity of cytochrome b03 as a function of the pH gradient formed. For that a spectro-electrochemical cell is designed that allows simultaneous monitoring of the catalytic (02 reduction) and proton pumping activity of the enzyme. The step-by-step development of this method will be described in the subsequent chapters. The results obtained were validated by using purified and vesicle-reconstituted cytochrome b03.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available