The physiological role of flavocytochrome <I>b<SUB>2</SUB></I> from <I>Saccharomyes cerevisiae </I>is to couple L-lactate dehydrogenation to respiration via the ubiquitous electron carrier cytochrome <I>c</I>. For each L-lactate molecule dehydrogenated two cytochrome <I>c.</I> 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 <I>b<SUB>2</SUB></I>-haem (intramolecular electron transfer) and on to two cytochrome <I>c</I> 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 <I>b<SUB>2</SUB></I>-haem has been studied by monitoring both haem reduction and haem re-reduction (following oxidation by cytochrome <I>c</I>). In each situation this step proved too fast to be observed, and appeared only as a slight lag (relative to flavin reduction) in the <I>b<SUB>2</SUB></I>-haem reduction trace. Nevertheless a lower estimate for this rate constant was derived to be 1500±500 s<SUP>-1</SUP>. The second intramolecular electron transfer step takes place from flavin semiquinone to <I>b<SUB>2</SUB></I> 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<SUP>-1</SUP>) and is therefore responsible for determining the overall turnover rate. The product, pyruvate, was found to be an inhibitor of the flavin oxidation reaction (K<SUB>i</SUB>= 40 ± 15 mM) consistent with reports that it acts as a non-competitive inhibitor in the steady-state. Stopped-flow studies on the cytochrome <I>c</I> reductase activity of flavocytochrome <I>b<SUB>2</SUB></I> yielded a second-order rate constant of 35 μM<SUP>-1</SUP>s<SUP>-1</SUP>, which represents the rate constant for cytochrome <I>c</I> association.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:649069 |
| Date | January 1996 |
| Creators | Daff, Simon N. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12172 |
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