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The disulfide reduction pathway of system II cytochrome c maturation

ReferenceBB/C503597/1
Principal Investigator / Supervisor Professor Nicolas Le Brun
Co-Investigators /
Co-Supervisors
Institution University of East Anglia
DepartmentBiological Sciences
Funding typeResearch
Value (£) 221,720
StatusCompleted
TypeResearch Grant
Start date 01/01/2005
End date 31/12/2007
Duration36 months

Abstract

C-type cytochromes play essential roles in processes as diverse as respiratory electron transport and programmed cell death. Despite a wealth of information about the proteins themselves, only now are we beginning to understand how they are synthesised in the cell. Remarkably, three different systems have been discovered in different organisms. We are interested in the cytochrome c maturation (CCM) system II pathway found in Gram-positive and some Gram-negative bacteria, as well as in chloroplasts. Using Bacillus subtilis as a model, we have recently shown that this bacterium has three specific CCM proteins, ResA, ResB and ResC. This proposal focuses on ResA, which is a low potential thiol-disulfide oxidoreductase anchored to the outside of the cytoplasmic membrane. The recently solved X-ray structures of ResA in both redox states provide significant insight into the function of the protein. Our working model predicts that ResA accepts electrons from the integral membrane protein CcdA (required for CCM, but not specific for it) and subsequently reduces a disulfide-bonded form of the apo-cytochrome polypeptide, in preparation for heme attachment. We will test and develop the model by investigating in detail, using biochemical and structural methods: (1) factors that control the reactivity of ResA, including the pKa of the N-terminal Cys of the CXXC motif, the importance of a hydrophobic cavity located near the active site that is only present in the reduced protein, and the importance of the cysteines for in vivo function; (2) the requirement for two cysteines in the apo-cytochrome, using a thermophilic c-type cytochrome system developed by Stuart Ferguson and colleagues in Oxford; (3) the in vivo and in vitro interaction between reduced ResA and oxidised apo-cytochrome c. Part of this work will connect with the work of Ferguson and colleagues, who are investigating aspects of apo-cytochrome c; (4) the interaction between ResA and CcdA, including the functional importance of the insertions present in the ResA structure compared to the classic thioredoxin fold, the importance of the ResA anchor, and attempts to purify CcdA to enable in vitro studies with ResA. This work will extend the fruitful collaborations between the UEA group and the Hederstedt group in Lund, Sweden.

Summary

unavailable
Committee Closed Committee - Biochemistry & Cell Biology (BCB)
Research TopicsX – not assigned to a current Research Topic
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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