Resolution and Reconstitution of Photosystem I in Cyanobacteria and Higher Plants; Molecular Biological and Physiochemical Studies

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This research program aims at studying the structure, function and organization of the Photosystem I reaction center complex in cyanobacteria and green plants. Our ability to resolve P700- FA/FB complex into the P700-FX core, the P700-A1 core, and P700-A0 core, and our ability to reconstitute each preparation with altered cofactors and genetically-modified polypeptides, has provided new research opportunities in Photosystem I. The proposed program is based on the ability to resolve and reconstitute the Photosystem I reaction center in terms of polypeptides and cofactors, and has five major objectives: 1) To determine the involvement of FA and/or FB in the photoreduction of NADP+. 2) To confirm the ligands to the FX iron-sulfur cluster on PsaA and PsaB. 3) To determine the roles of PsaD, PsaE and PsaF in Photosystem I function. 4) To solve the 3-dimensional structure of the PsaC, PsaD and PsaE proteins. 5) To measure the spectroscopic properties of A1 with reconstituted quinones. %%% Green plants are able to utilize sunlight as an energy source with the use of photosynthetic reaction centers to power the organism's growth, metabolism, and reproduction. Two different kinds of reaction centers are located in the chloroplasts of green plants, and each contains chlorophyll, proteins, and metals ions necessary for transforming solar energy into chemical energy. In the last several years, we have learned how to remove the proteins and metals from one of these reaction centers, and we have learned how to rebuild the reaction centers from the constituent parts. In this proposal, we will rebuild the reaction centers using proteins that have been altered using techniques of modern genetic engineering. We will obtain these proteins through a close collaboration with several molecular geneticists. We will then use sophisticated measuring techniques to determine the function of the individual proteins in the process of photochemical energy conversion. By altering one protein while retaining the function of the others, we will be able to determine which protein provides a certain function in the process of the photosynthesis. The proposal, therefore, relies on the marriage of two very powerful, but disparate techniques, genetics and biophysics, to solve the complicated problem of green plant photosynthesis.