Fumarate reduction in <i>Shewanella </i>is catalysed by a fumarate reductase known as flavocytochrome <i>c<sub>3</sub></i> (Fcc<sub>3</sub>). This enzyme consists of three domains: a cytochrome domain containing four <i>c</i>-type heme groups; a flavin domain containing a non-covalently bound FAD; and a mobile clamp domain. Fumarate is saccinate by hydride transfer from the flavin N5 and protonation by the active site acid, Arg402. Access of substrate to the active site in Fcc<sub>3</sub> was believed to be controlled by movement of the clamp domain. To test this assumption, site-directed mutagenesis has been used to create a disulfide bond between the clamp and flavin domains via the double mutation A251C:S430C. The disulfide bond in the mutant enzyme has been confirmed by both crystal structure and Ellman analysis. When the disulfide bond is formed both the steady-state and pre-steady-state rate constants for fumarate reduction fall to 25 and 30% of the wild-type values respectively whilst <i>K<sub>M</sub></i> values for fumarate are unaffected. Deuterium solvent kinetic isotope effects in the mutant enzyme are unchanged from wild-type (8.2 ±0.4 at pL 7.2), indicating that proton and/or hydride transfer is still rate-limiting. These results suggest that clamp domain mobility has little role in controlling fumarate reduction. The reduction of fumarate also requires the delivery of reducing equivalents to the active site. This is facilitated by the four bis-histidine-ligated heme groups within the cytochrome domain. Hemes I, II and III are solvent exposed and therefore able to collect electrons from the electron donor CymA, and deliver them to the FAD via heme IV. Mutations to His61, a ligand to the iron of heme IV, results in a lowering of both the steady-state and pre-steady-state rate constants for fumarate reduction (WT>H61Y>H61M>H61A). Crystal structures of H61A and H61M show that there is an exogenous ligand bound to the heme iron in both cases; acetate and water respectively.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661417 |
| Date | January 2004 |
| Creators | Rothery, Emma L. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14338 |
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