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Title: Analysis of the catalytic cycle & manipulation of substrate specificity in flavocytochrome b2
Author: Daff, Simon N.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The physiological role of flavocytochrome b2 from Saccharomyes cerevisiae is to couple L-lactate dehydrogenation to respiration via the ubiquitous electron carrier cytochrome c. For each L-lactate molecule dehydrogenated two cytochrome c. molecules are reduced, and as such the enzyme acts as a 'bio-electrical transformer'. The mechanism through which this process occurs can be simplified into five separate electron transfer events which form the catalytic cycle. L-lactate dehydrogenation results in the two electron reduction of flavin at the enzyme's active site. These electrons are passed individually to the b2-haem (intramolecular electron transfer) and on to two cytochrome c molecules (intermolecular electron transfer). Using stopped-flow spectrophotometry, the intramolecular electron transfer steps have been investigated using several different experimental procedures. Electron transfer from fully reduced FMN to b2-haem has been studied by monitoring both haem reduction and haem re-reduction (following oxidation by cytochrome c). In each situation this step proved too fast to be observed, and appeared only as a slight lag (relative to flavin reduction) in the b2-haem reduction trace. Nevertheless a lower estimate for this rate constant was derived to be 1500±500 s-1. The second intramolecular electron transfer step takes place from flavin semiquinone to b2 haem and was observed as a component of the flavin oxidation process. This proved to be the slowest step in the catalytic cycle (at 120 s-1) and is therefore responsible for determining the overall turnover rate. The product, pyruvate, was found to be an inhibitor of the flavin oxidation reaction (Ki= 40 ± 15 mM) consistent with reports that it acts as a non-competitive inhibitor in the steady-state. Stopped-flow studies on the cytochrome c reductase activity of flavocytochrome b2 yielded a second-order rate constant of 35 μM-1s-1, which represents the rate constant for cytochrome c association.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available