**8. Conclusion**

Site-directed mutagenesis has been employed extensively to probe the function of the CP43 protein of PSII. In particular, substitutions at Arg320 have revealed a potential role of this amino acid in the interaction of CP43 with the extrinsic PSII protein, cytochrome c550. In the present study, we have further defined the requirement at position 320 by constructing and characterizing additional mutant strains R320K and R320D. The results indicate that both the R320K and R320D strains are defective in their ability to utilize chloride to carry out efficient oxygen evolution and the effect is magnified under low chloride conditions. Spectrophotometrically detectable cytochrome c550 is lacking in these mutants. Additionally, we have preliminary evidence that these mutants also lack PsbU (Bricker and Putnam-Evans, unpublished). The phenotype of the R320S mutant previously described is intermediate between that of the K and D mutants. Thus, the order of effectiveness of amino acids at this site in supporting normal PSII function is R>K>S>D. A positively charged residue appears to be required at this site for proper function. The 1.9 Å crystal structure reveals a number of amino acid residues within both cytochrome c550 and PsbU that are potential hydrogen bonding partners to Arg320. Our operating hypothesis is that CP43, cytochrome c550 and PsbU interact via a hydrogen-bonding network that is involved in promoting a stable environment for chloride incorporation into the active site. While the position and ligands of chloride around the manganese cluster are now known, what other proteins bind chloride, how chloride is funneled to the active site, and where potential chloride channels occur remain unclear. Continuing studies to address these questions will no doubt expand our knowledge of the protein-protein and protein-ligand interactions in PSII that contribute to the proper functioning of the oxygen-evolving complex.
