Plastocyanin and photosystem I.
Ru-modification

Personnel

Former graduate students: Collaborators:

Description of the project

Plastocyanin (Pc) is a small (10.5 kDa) Cu-containing protein which acts as an electron carrier between the cytochrome b6f and photosystem 1 (PS1) complexes in the photosynthetic electron-transfer chain. Pc belongs to the group of so called type 1 copper proteins which are characterized by a strong blue color.

The major question that we are addressing is: How are electrons transferred in and out of the copper site? To answer this we make site-specific mutations in regions that are thought to be important for the function of plastocyanin, in the "northern" hydrophobic patch and in the "eastern" acidic patches.

We have constructed a series of mutants of spinach Pc by site-directed mutagenesis and expression in Escherichia coli. Particular attention has been paid to the Cu ligand His-87 and to amino-acid residues in the surrounding hydrophobic patch as well as to Tyr-83 and to the acidic residues 42-45 and 59-61 that surround Tyr-83.

For most of the mutants, optical absorption, EPR and 1H-NMR spectra are similar to the wild-type spectra suggesting that there are no gross alterations in the structure. When the copper ligand His-87 is mutated to a Gly, Asp, Asn, or Gln, the copper site is severely distorted to a non-blue (type 2) site. None of the His-87 mutants can transfer electrons to PS1, underlining the importance of this residue.

The electron transfer to photosystem I is monitored with flash-photolysis techniques. In most experiments we use PS1 particles solubilized with digitonin from spinach chloroplasts. Mutations of Leu-12 or Pro-36 in the hydrophobic patch result in a reduced reactivity towards PS1 that is too drastic to be related to changes in reduction potential or spectral properties. A curve-fitting analysis shows that these mutants lack the fast, 70 000 per sec, component present in the decay kinetics of photooxidized P700 for WT Pc. However, a fast phase is present for mutants in position 7, 8, 11, 35, 85 or 88, which are also in, or close to, the hydrophobic patch. It can be concluded that Leu-12 and Pro-36 are part of a small, critical, region around the Cu ligand His-87, whose conserved structure is very important for the binding to PS1.

The Tyr-83 residue, which is a neighbor to the Cu ligand Cys-84, has been suggested to provide an entrance point for electrons from cytochrome f. This residue has been mutated to a His, Phe or a Leu and all of these mutants display a fast electron transfer to PS1, thus excluding the involvement of this pathway in the reaction with PS1.

When residues in the acidic patches 42-45 and 59-61 are mutated to neutral or positively charged ones, the reactivity towards PS1 decreases. The effect is stronger for mutations in the 42-45 region compared with the 59-61 region, suggesting a specific electrostatic interaction between the former region and positive charges on PS1.

The decay kinetics of photooxidized P700 are biphasic for the WT and for all mutants except the Leu-12 and His-87 mutants, with a concentration independent fast phase and a slower phase with a rate constant that saturates at high Pc concentrations. The relative amplitude of the fast phase saturates at a value well below one. A model in which a rate-limiting conformational change occurs in the Pc-PS1 complex before the electron transfer can account for this behavior.

In addition to the above studies, certain surface-exposed residues (15, 79, 81, 83) have been replaced with histidines to which a photoactive ruthenium (Ru) complex has subsequently been attached. The properties of the different electron-transfer pathways between the Cu and Ru ions have been determined with a flash-photolysis technique. The results suggest a strong electronic coupling along the Cu-Cys84-His83 pathway.

This project is supported by the Swedish Natural Science Research Council.

Figure

Plastocyanin

A view of plastocyanin prepared with the MolScript program (Kraulis, P.J. (1991) J. Appl. Crystallogr. 24, 946-950). The arrows indicate strands of beta-sheet. The copper ion near the top is coordinated in an approx. tetrahedral symmetry to His-37, Cys-84, His-87 and Met-92. The sidechain of Tyr-83 is surrounded by the negatively charged residues 42-44 and 59-61. Leu-12 is part of a hydrophobic region on the top of the molecule. Other amino-acid sidechains have been left out.

References

ÖH 2000-09-05