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Title: Flavocytochrome b2 : role of the interdomain hinge region
Author: Sharp, R. Eryl
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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The two distinct domains of flavocytochrome b2 (L-lactate : cytochrome c 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 b2 and cytochrome c) 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 (kcat = 256 s-1, 276 s-1 and 400 s-1 for HΔ3, HΔ6, and HΔ9, respectively, compared to 400s-1 for the wild-type enzyme) and from stopped-flow experiments monitoring flavin reduction (kcat = 516 s-1, 520 s-1 and 715 s-1 for HΔ3, HΔ6 and HΔ9, respectively, compared to 600 s-1 for the wild-type enzyme). The global effect of these deletions is to lower the enzymes' effectiveness as cytochrome c reductases. This property of HΔ6 and HΔ9 flavocytochromes b2 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 c 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-1 to 91 s-1) and, secondly, the steady-state rate of cytochrome c reduction has fallen 5-fold (from 207 s-1 to 39 s-1). 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available