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Title: An engineered inter-domain disulfide bridge in flavocytochrome b2 : insights into the role of domain mobility
Author: Drewette, Katy J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Flavocytochrome b2 from the yeast Saccharomyces cerevisiae is a L-lactate:cytochrome c oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 Å. Each subunit consists of two distinct domains; a small (100 residue) N-terminal cytochrome domain containing a b-type heme, and a larger (411 residue) C-terminal domain containing flavin mononucleotide (FMN). The two domains are connected by a hinge sequence, running from residues 89 to 103. It has been proposed that the most likely role of this hinge region is to confer inter-domain mobility, allowing movement of the cytochrome domain with respect to the flavin domain. In this work a disulphide-bridge was engineered in a position, such that the two domains would be fixed in close proximity. Site-directed mutagenesis was used to make the double mutation, N42C:K324C. The crystal structure of the N42C:K324C mutant enzyme was solved to 2.8 Å resolution. An inspection of this structure has confirmed the existence of the imposed disulfide-bridge. In addition, the four b2-heme domains of the tetramer are ordered, indicating their limiting mobility. Steady-state kinetic analyses with L-lactate, using ferricyanide [Fe(CN)6]3- and cytochrome c as electron acceptors were carried out. The formation of the disulfide-bridge causes a 15-fold decrease in kcat with both electron acceptors. Since [Fe(CN)6]3- can accept electrons from both the FMN and b2-heme while cytochrome c can only accept electrons from the b2-heme this indicates that it is the rate of FMN reduction by L-lactate that is primarily affected by disulphide-bridge formation. Pre-steady-state kinetic analyses with L-lactate are consistent with the steady-state data. The formation of the disulphide-bridge makes it impossible to measure the rate constant for FMN reduction directly while b2-heme reduction shows a rate constant some 450-fold less than in the open. If flavin to b2-heme electron transfer is much faster then b2-heme reduction will be limited by the rate of formation of reduced flavin. Thus, disulfide- bridge formation substantially lowers the rate of FMN reduction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available