DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

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The overall goal is to understand the operation of metal-ion clusters and electron transfer processes in biological systems. The specific goal is to understand the mechanism of photosynthetic water oxidation. Oxygenic photosynthesis provides the molecular oxygen and fixed carbon required to sustain animal life. The proposed work will identify the ligands to the manganese and calcium ions located at the catalytic site of photosynthetic water oxidation. Identification of these ligands will impose new constraints on models for the operation of the manganese cluster, and will enable such models to be placed in the context of protein structure. The information obtained will be applicable to other metal-ion clusters, and to the study of membrane proteins and biological electron transfer processes in general. Electron transfer can be studied more easily and precisely in photosynthetic than in mitochondrial systems, because electron transfer can be conveniently initiated with light and studied under single turn-over conditions. In oxygenic photosynthesis, Photosystem II (PSII) uses light to extract electrons from water and donate them into an electron transport chain that generates the chemical free energy and reducing equivalents required for carbon fixation. PSII photochemistry takes place in a heterodimer of two polypeptides known as D1 and D2. A cluster of four manganese ions accumulates four oxidizing equivalents in response to this photochemistry, and then uses them to oxidize two molecules of water in concerted mechanism that requires calcium and releases one molecule of oxygen as a by-product. The ligands for both manganese and calcium are believed to be predominantly carboxyl residues on the D1 and D2 polypeptides. The proposed work will identify the specific carboxyl ligands to manganese and calcium, and characterize their influence on oxygen evolution. This will be accomplished by site-directed mutagenesis of the psbA and psbD genes from the unicellular cyanobacterium Synechocystis sp. PCC 6803, which encode the D1 and D2 polypeptides, respectively. A mutagenic screening procedure will rapidly identify those carboxyl residues most likely to serve as ligands. Mutations of these residues will be characterized by biochemical and spectroscopic methods, while the remaining mutants will be archived for future analysis. I have previously employed site-directed mutagenesis of the psbA and psbD genes of Synechocystis sp. PCC 6803 to identify the two redox-active tyrosine residues in the PSII core that interact with the manganese cluster.