POTENTIAL METAL LIGANDS IN PHOTOSYNTHETIC WATER OXIDATION IDENTIFIED BY SITE-DIRECTED MUTAGENESIS OF SYNECHOCYSTIS SP PCC-6803

In order to identify amino acids that could serve as ligands to the ma nganese cluster, which comprises the active site of the oxygen-evolvin g complex (OEC), and the calcium ion that affects the stability of the OEC, we have generated a series of site-directed mutants in the D1 po lypeptide of the PS II reaction center. Our approach has been to empha size characterization of mutants with reduced PS II activity rather th an those that lack PS II activity altogether in order to address quest ions about the effects of the mutations on the functional properties o f the Mn ensemble. A number of mutants have been isolated with impaire d PS II activity and a characterization of in vivo photosynthesis from mutant and WT strains is presented. All of the mutants described in t his report produce nearly WT levels of PS II in the thylakoid membrane . Non-conservative mutation at Asp-103 (DN103D1), Glu-104 (EQ104D1) or Glu-333 (EQ333D1) results in moderate (similar to 50%) inhibition of oxygen evolution, whereas mutation at Glu-65 (EQ65D1) or His-337 (HF33 7D1) results in cells that retain only 10 to 20% of WT activity. In vi vo fluorescence induction kinetics are consistent with a lesion on the donor side of PS II in each of the mutants tested. We conclude that G lu-65 and His-337 are directly involved in binding manganese or calciu m, and that Asp-103 and Glu-104 may be involved in calcium binding but are not manganese ligands.