Award details

Formation of Tyrosine Radicals in haem proteins: their role in electron transfer

Reference	BB/F007663/1
Principal Investigator / Supervisor	Professor Mike Wilson
Co-Investigators / Co-Supervisors	Professor Christopher Cooper, Dr Brandon Reeder, Dr Dimitri Svistunenko
Institution	University of Essex
Department	Biological Sciences
Funding type	Research
Value (£)	379,918
Status	Completed
Type	Research Grant
Start date	01/01/2008
End date	31/12/2010
Duration	36 months

Abstract

Many enzymes use free radicals in important biochemical processes involving both catalysis and signalling. These free radicals, if not properly controlled, participate in unwanted reactions that can lead to a variety of pathological complications. The processes by which enzymes and proteins control, or indeed loose control of, the movement of radicals through intra and intermolecular mechanisms is a topic of central biological and biomedical importance. We propose to study electron transfer, and hence radical migration, in proteins using native, chemically modified and engineered haem proteins as models. Radicals will be produced by reaction with peroxides and studied by a combination of techniques including optical and EPR spectroscopies. The main aim of the study is to understand the relationship between protein structure and the pathways through which protein-bound radicals migrate. By reference to the known structures we will construct site-specific mutant proteins in which putative pathways are removed or introduced. We will concentrate particularly (but not exclusively) on tyrosyl radicals, which we are able to locate to a specific residue by analysis of their EPR signal using an algorithm to simulate the spectrum and which incorporates information about the residue configuration taken from the crystal structure. Our recent results strongly suggest that tyrosyl radicals act as true chemical intermediates in electron transfer to and from the exterior of the protein. We intend, therefore, to develop methods to determine/confirm the positions of surface exposed radicals using DMPO labelling and to discriminate between intra- and inter-molecular radical migration. By using organic reductants we will map electron migration pathways from surface exposed amino acids to the haem iron and use kinetic modelling to predict the rates and extent of radical migration. This has particular relevance to the understanding of the toxicity of haemoglobin-based blood substitutes.

Summary

Many enzymes use highly reactive free radicals to perform their catalytic functions. Free radicals are compounds containing unpaired electrons and play an important role in a number of biological processes, many of which are necessary to sustain life. However, because of their high reactivity free radicals can also participate in unwanted side reactions, causing cell damage that may lead to many diseases including arthritis, Alzheimer's disease and cancer. Some enzymes require that free radicals are moved over large distances, whereas some require that free radicals do not move from their point of creation. The way in which enzymes control free radical movement is of important scientific interest. We intend to examine the ways in which radicals move through enzymes and proteins by using model proteins systems, namely myoglobin and haemoglobin, in which we can control the formation of free radicals at will. These proteins are known to form and translocate free radicals when they react with peroxides. We intend to follow the migration of these free radicals, using various direct and indirect free radical detection methods, to discover whether radical movement is mainly within the protein or is transmitted to other proteins. Also, we intend to manipulate the pathways through which these radicals migrate by re-designing parts of the protein. As the structures of these proteins are known we can use this information together with genetic methods to remove existing pathways and to create potential new ones. The information gained will be valuable in understanding the mechanisms by which proteins control radicals and will also be of importance in understanding why these processes go wrong in some proteins such as artificially designed haemoglobin-based blood substitutes.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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