Abstract
The light-driven oxidation of water is catalyzed by the Mn4CaO5 oxygen-evolving complex (OEC) of Photosystem II (PS II). The dioxygen formed as a result of water splitting is released and maintains the earth’s oxygenic environment. During biogenesis of PS II, PsbM and PsbT, both membrane-spanning single helix proteins, remain associated with the chlorophyll-binding CP47 pre-assembly complex and are eventually located at the monomer-monomer interface of the mature dimeric photosystem. The first objective of this study was to investigate the role of removing PsbT or PsbM in the absence of lumenal extrinsic proteins that contribute to dimer stability, the stability of the OEC and to several channels that allow substrate access and product egress from the OEC. The redox active cofactors of PS II are chiefly bound by two reaction centre proteins, D1 and D2, and these are flanked by CP43 and CP47 that bind chlorophyll to form a core antenna. D1 and CP43 are also involved in preparing the active site for water splitting and maintain the coordination environment for the catalytic activity of the OEC. The hydrogen bond networks present between water molecules and nearby residues help substrate delivery to the OEC, and the removal of by-products, through channels in the core proteins and the extrinsic proteins leading to and from the lumen. A second objective was to investigate the role of ligands in the secondary ligand sphere of the OEC in photoactivation and optimizing its catalytic activity. For this purpose, the CP43 residues Gly340, Thr342, Met343 and Phe345 in a conserved GETMRF motif were selected for a site-directed mutagenesis study. In addition, a hydrophilic loop of CP43 (loop E) serves as an anchoring point for extrinsic proteins potentially contributing to substrate delivery and exit pathways ultimately merging with the extrinsic proteins. To investigate this putative role of CP43, two sets of residues present in loop E were selected. One set consisting of Leu388, Gly389 and Ser390 is present ~10 Å away from the OEC and the other set consisting of Val397, Ile398 and Thr399 was present at the interface of loop E and the PsbV extrinsic protein.