Structure of oxidized poplar plastocyanin at 1.6 A resolution.

The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.