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Title: A study of flavocytochrome b2 and flavocytochrome c3
Author: Moysey, Ruth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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Flavocytochrome c3 is a fumarate reductase expressed by the marine bacterium Shewanella frigidimarina during anaerobic growth in the presence of fumarate. This soluble periplasmic enzyme is composed of three domains; a flavin domain containing non-covalently bound FAD, a cytochrome domain and a clamp domain. The active site is located in the flavin domain at the interface with the clamp domain. The enzyme catalyses the reduction of fumarate to succinate with a kcat of 509 ± 15 s-1 and Km of 25 ± 2mM at pH 7.2, 25°C. Reduction of fumarate requires hydride transfer from the FAD and protonation by an active site acid. Residues implicated in catalysis have been studied using site directed mutagenesis. Substitution of Arg402 by alanine leads to complete loss of activity whereas neither of the two active site histidines (His504 and His365) is essential for catalysis. The H365A:H504A double mutant enzyme was found to have a kcat of 0.84 ± 0.05 s-1 at pH 7.2. Substitution of Arg402 by lysine, histidine or tyrosine led to a fall in kcat to 0.55 s-1, 0.091 s-1 and 0.05 s-1 respectively. Substrate specificity and inhibition studies have been carried out to probe the active site structure. Fcc3 was unable to catalyse the reduction of alternative enoates to fumarate. However, oxaloacetate, methylsuccinate and 3-nitropropinoate were found to be inhibitors of succinate oxidation with Ki values of 5.3 mM, 1.7 mM, and 0.5 mM respectively.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available