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Mutagenesis of the oxygen evolving complex of photosynthesis

Reference	BB/C507037/1
Principal Investigator / Supervisor	Professor Peter Nixon
Co-Investigators / Co-Supervisors	Professor James Barber
Institution	Imperial College London
Department	Biological Sciences
Funding type	Research
Value (£)	240,112
Status	Completed
Type	Research Grant
Start date	01/05/2005
End date	30/11/2008
Duration	43 months

Abstract

The groups of Jim Barber and So Iwata have recently solved the crystal structure of a cyanobacterial photosystem two complex to a resolution of 3.5 Angstrom. This has allowed the location of the oxygen-evolving complex (OEC) to be determined and the ligands to the Ca2+ Mn4 cluster identified. The overall aim of this proposal is to combine site-directed mutagenesis with an array of biophysical techniques to probe the importance of specific animo-acid residues for the assembly, stability and function of the OEC. Objectives are to: (1) Test the role of residue CP43-Glu354 (Ts. elongatus numbering) in the function and robustness of photosynthetic oxygen evolution. This residue is a ligand to the Mn cluster. Mutants will be generated in the cyanobacterium Synechocystis 6803 and perturbations to PSII electron transfer will be assessed in vivo using non-invasive chlorophyll fluorescence assays. Effects on S-state transitions will be monitored in flash oxygen experiments. Detailed measurements of electron transfer in isolated His-tagged complexes will be performed by time-resolved optical spectroscopy. (2) Probe the role of residue CP43-Arg357 in dioxygen formation. This residue is located close to the proposed site of water-splitting and may have a role in stabilising intermediates during dioxygen formation. Mutants will be constructed in Synechocystis and will be examined using techniques indicated in objective 1. (3) Test the function of amino-acid residues implicated in a proton export pathway. Residues in D2 (Lys317 and Glu312) and D1 (Glu65) will be mutated and effects on electron transfer and advancement of the S-states assessed. (4) Test the involvement of residue D1-Glu333 in the high-affinity Mn-binding site. Residue D1-Glu333 in the C-terminal region of D1 is a ligand to the same Mn ion as D1-Asp170, which has already been shown to be important for formation of a high-affinity Mn-binding site in apo-PSII. The contribution of D1-Glu333 to the high-affinity site will be assessed in mutant PSII complexes, lacking bound Mn, by monitoring the rate of reduction of Yz. as a function of Mn concentration. (5) Investigate the relationship between D1 processing and assembly of the oxygen-evolving complex. The structure confirms that the C-terminus of mature D1 ligates the Ca2+ ion of the CaMn cluster so that C-terminal processing of D1 regulates ligation of Ca2+. To assess the impact of processing on the capping of the OEC by the extrinsic proteins, His-tagged PSII complexes will be isolated from a mutant lacking the processing protease and the binding of the extrinsic proteins assessed by immunoblotting.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology, 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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