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Title: The domains of flavocytochrome b2
Author: Brunt, Claire Elaine
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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Flavocytochrome b2 is a respiratory enzyme found in the intermembrane space of yeast mitochondria, where it catalyses the oxidation of L-lactate to pyruvate, transferring electrons to cytochrome c. The enzyme is a tetramer. Each subunit comprises two functionally-distinct domains, one containing flavin mononucleotide and the other containing protohaem IX. The lactate-binding site of the enzyme is located within the flavin-containing domain. This domain is essential for L-lactate dehydrogenase activity. The haem domain is essential for electron transfer to cytochrome c. The haem- and FMN-containing domains of flavocytochrome b2 have now been expressed independently in E.coli. This thesis describes the development of effective purification procedures for the two independently-expressed domains and their characterization by biophysical and biochemical techniques. The characterization of a point mutant of the isolated b2-flavin domain is also discussed. The independently-expressed haem domain was purified to a high level by column chromatography. The isolated protein was found to be monomeric with a molecular weight of 10,500. It had no detectable enzyme activity and failed to accept electrons from either the isolated b2-flavin domain or the holoenzyme. Isolated b2-haem domain was found to be stable over a wide temperature and pH range. Its redox potential was found to be -31+ /-2mV, in agreement with a previously-determined value for a similar fragment isolated from the holoenzyme by proteolytic methods. High-field 1H-NMR studies have been carried out on the isolated b2-haem domain. The pKA values of the haem propionates were found by NMR to be 4.8 and 4.6, consistent with these groups being exposed to solvent. NMR studies were also used to determine an electron self-exchange rate constant of 2.3x106M-1s-1 for the b2-haem domain. The interactions of cytochrome c with both the holoenzyme and the isolated haem domain were studied by NMR. It was found that, whereas cytochrome c bound to the holoenzyme, it failed to bind to the isolated haem domain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available