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 .
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.