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Page count: 172

Photosynthesis is the process that higher plants, algae, and cyanobacteria use to convert light energy into chemical energy in the form of carbohydrates. In this process, light energy is used to split water and molecular oxygen is released as a byproduct. This water splitting reaction occurs in the oxygen evolving complex of Photosystem II (PSII). The oxygen evolving complex is made up of a tetramanganese-calcium cluster, which also requires chloride, bound to the intrinsic PSII proteins D1 and CP43. The extrinsic PSII proteins (manganese stabilizing protein (MSP), cytoehrome C550, and PsbU) form a cap over the manganese cluster that protects the cluster and concentrates calcium and chloride at the active site of the oxygen-evolving complex. In this thesis, residues that affect the functioning of the oxygen evolving complex, and specifically, the chloride requirement, have been identified. Previously, the R305S mutant was extensively characterized by our laboratory and showed impaired oxygen evolving activity when grown under conditions of chloride depletion (Young et al, 2002). Isolated PSII particles fi-om the R305S mutant failed to bind the cytochrome C550 protein (Bricker et ah, 2002). Preliminary data showed that the D306N mutant also exhibited a chloride effect. Our hypothesis is that the R305 and D306 residues may help form part of a binding site for cytochrome C550. To determine whether these two residues function in concert or independently, the double mutant, RD305306SN, of Photosystem II in Synechocystis PCC 6803 was characterized in this thesis. The double mutant appeared to be more severely affected than either of the single mutants under conditions of chloride depletion. The double mutant failed to grow photoautotrophically and evolved oxygen to only 4% of the control rates under photoheterotrophic growth conditions. The double mutant assembled fewer PSII centers and the centers that were assembled were extremely sensitive to photoinhibition. Isolated PSII particles from double mutant lacked the extrinsic cytochrome C550 protein and also appeared to have reduced amounts of MSP. These results suggest that the two sites do not function independently and support the hypothesis that both residues R305 and D306 may be involved in the binding of cytochrome C550 to PSII.