INVESTIGATING STRUCTURE-FUNCTION RELATIONSHIPS IN THE PHOTOSYNTHETIC REACTION CENTRE

K. E. McAuley-Hecht1, P.K. Fyfe3, J.P. Ridge3, M. R. Jones3, R.J. Cogdell2, N.W. Isaacs1

1Department of Chemistry, Joseph Black Building and
2Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, United Kingdom.
3Kreb's Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2UH, United Kingdom .

Email:kat@chem.gla.ac.uk

Keywords: membrane protein, site-directed mutants, X-ray crystallography

The purple bacterial photosynthetic reaction centre is an integral membrane protein that performs electron transfer across the membrane following absorption of light (1). It consists of three protein subunits, designated L, M and H and ten co-factor pigment molecules. Although the electron transfer occurs via the pigment molecules, the protein is involved in fine-tuning the energy levels of the pigments and so plays an important role in the charge separation process. The structure of this protein complex has previously been determined for the reaction centres from Rhodopseudomonas viridis (2, 3) and Rhodobacter sphaeroides (3-15).

We have used a combined approach of site-directed mutagenesis and X-ray crystallography to investigate the role of protein-pigment interactions in the reaction centre from Rb. sphaeroides. A mutation at residue M197 (Phe to Arg) was designed to add a hydrogen bond to the bacteriochlorophyll dimer, and the effects of this mutation on the structure of the complex and the electron transfer process have been studied in some detail (12,14). Another mutation was constructed to exclude the primary quinone from its binding site (15). This was achieved by replacing an alanine residue (M260) with a tryptophan so the mutated residue partially fills the space normally occupied by the quinone head-group. Some structural changes were observed in the region around the site of mutation, affecting residues M256 to M260. The cavity formed by the exclusion of the quinone molecule allows a greater flexibility of the protein in this region and alternative conformations of the amino acids have been identified. The effect of this mutation on the secondary quinone will also be discussed.

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