Flavocytochrome <I>b<SUB>2</SUB></I> from <I>Saccharomyces cerevisiae</I> is located in the mitochondrial intermembrane space where it catalyses the oxidation of L-lactate to pyruvate with concomitant electron transfer to cytochrome <I>c.</I> The enzyme is a homotetramer, each protomer having two distrinct domains; one of which binds flavin mononucleotide (FMN) and the other protohaem IX. The domains are connected by a region of polypeptide which constitutes the interdomain hinge. The pathway of electron transfer is from L-lactate to FMN to haem to cytochrome <I>c.</I> The work described in this thesis focuses on the latter two steps on the catalytic cycle, namely intramolecular electron transfer from FMN to haem and intermolecular electron transfer from flavocytochrome <I>b<SUB>2</SUB></I> to cytochrome <I>c. </I>Intramolecular electron transfer has been investigated by the generation of interspecies hybrid enzymes. These include hinge-swap<I>b<SUB>2</SUB></I>, in which the hinge region of the <I>S.cerevisiae </I>enzyme has been replaced with that from <I>Hansenula anomala</I>; and domain-swap-<I>b<SUB>2</SUB></I>, which comprises the flavodehydrogenase domain of <I>H.anomala</I> with the hinge region and haem domain from <I>S.cerevisiae.</I> For both hinge-swap enzymes, the most significant effect was a dramatic decrease in the rate of haem reduction, the rate constant for this decreases from 445<SUB>s</SUB>-<SUP>1</SUP> to 1.61<SUB>s</SUB>-<SUP>1</SUP> for the hinge-swap enzyme and to 0.99 <SUB>s</SUB>-<SUP>1</SUP> for domain swap-<I>b<SUB>2</SUB></I>. This indicates that flavin to haem electron transfer is severely affected in both hybrids, to the extent that this step is now rate-limiting, rather than <I>C-2</I> hydrogen abstraction as in the <I>S.cerevisiae</I> wild-type enzyme. Other kinetic parameters including deuterium kinetic isotope effects also support this. We can therefore conclude that the interdomain hinge, and more generally, the structural integrity of this region are crucial in ensuring recognition and efficient electron transfer between the domains.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663703 |
| Date | January 1993 |
| Creators | White, Patricia |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11555 |
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