The two distinct domains of flavocytochrome <I>b<SUB>2</SUB> </I>(L-lactate : cytochrome <I>c</I> oxidoreductase) are connected by a typical hinge peptide. Kinetic experiments have illustrated the importance of maintaining the structural integrity of the hinge for efficient interdomain electron transfer. To probe the role of the hinge in a more subtle manner, three mutant enzymes have been constructed; HΔ3, HΔ6 and HΔ9 which have three, six and nine amino acids deleted from the hinge region, respectively. Intra- (interdomain) and inter-protein (between flavocytochrome <I>b<SUB>2</SUB> </I>and cytochrome <I>c</I>) electron-transfer was investigated by steady-state and stopped-flow kinetic analysis. All three hinge-deleted enzymes remained good L-lactate dehydrogenases as was evident from steady-state experiments with ferricyanide as electron acceptor (k<SUB>cat</SUB> = 256 s<SUP>-1</SUP>, 276 s<SUP>-1</SUP> and 400 s<SUP>-1</SUP> for HΔ3, HΔ6, and HΔ9, respectively, compared to 400s<SUP>-1</SUP> for the wild-type enzyme) and from stopped-flow experiments monitoring flavin reduction (k<SUB>cat</SUB> = 516 s<SUP>-1</SUP>, 520 s<SUP>-1</SUP> and 715 s<SUP>-1</SUP> for HΔ3, HΔ6 and HΔ9, respectively, compared to 600 s<SUP>-1</SUP> for the wild-type enzyme). The global effect of these deletions is to lower the enzymes' effectiveness as cytochrome <I>c</I> reductases. This property of HΔ6 and HΔ9 flavocytochromes <I>b<SUB>2</SUB><SUP> </SUP> </I>is manifested at the interdomain electron-transfer step, where the rate of haem reduction is the same within experimental error as the steady-state rate of cytochrome <I>c</I> reduction: interdomain electron-transfer has become rate-limiting in the case of these two hinge-deleted enzymes, compared to the wild-type enzyme, where H-abstraction from C2 of L-lactate is rate-limiting. The situation for HΔ3 is more complicated; the rate of haem reduction has fallen 35-fold compared with the wild-type enzyme (from 1500 s<SUP>-1</SUP> to 91 s<SUP>-1</SUP>) and, secondly, the steady-state rate of cytochrome <I>c</I> reduction has fallen 5-fold (from 207 s<SUP>-1</SUP> to 39 s<SUP>-1</SUP>). This implies that, for HΔ3, interdomain electron-transfer from fully reduced flavin to haem cannot be rate-limiting, as is the case for HΔ6 and HΔ9, but some other step, such as flavin semiquinone to haem electron-transfer must be involved. These data, along with the measured kinetic isotope effects imply that complete structural integrity within the hinge region is essential for efficient interdomain communication.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661804 |
| Date | January 1994 |
| Creators | Sharp, R. Eryl |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14398 |
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