Chitnis 9405325 Photosystem I, the light-dependent plastocyanin-ferredoxin oxidoreductase, is a multisubunit pigment-protein complex in the photosynthetic membranes of chloroplasts and cyanobacteria. Photosystem I contains at least twelve proteins, 100 chlorophylls, -carotenes and a series of electron transfer centers. To identify roles of individual polypeptides in the function and assembly of the complex, we initiated a program to clone and mutate genes that encode protein subunits of photosystem I of the naturally transformable cyanobacterium Synechocystis sp. PCC 6803. Our NSF-supported research has provided insights concerning the functions of several subunits of photosystem I. This proposal describes our plans to continue the investigations by focusing on (1) role of the accessory subunits, PsaL, PsaJ and PsaI in particular, in mediating and stabilizing assembly of photosystem I, and (2) mutational dissection and biochemical analysis of the reducing side of photosystem I. Recently we showed that PsaL is required for the formation of trimeric quaternary structure of photosystem I. PsaL binds calcium ions which stimulate trimerization. Site-specific mutations and biochemical characterization will be used to dissect the structural and regulatory basis for the role of PsaL. We postulate that the small (<4 kDa) hydrophobic subunits of photosystem I (PsaJ, PsaM, PsaI) function as 'nuts and bolts' in the structure of the complex by anchoring and stabilizing other proteins. Characterization of our PsaJ-less mutant shows that PsaJ is necessary for the stable assembly of PsaF, a lumen-exposed subunit, and possibly PsaE, a peripheral subunit on the n-side. We will examine the interactions of PsaJ by biochemical and mutational analysis. We will also generate mutants lacking PsaI and further test our hypothesis. The terminal reduction reaction of photosystem I involves electron transfer from the reducing site on the complex to ferredoxin. The reducing site on photosyst em I consists of surface-exposed domains of PsaD, PsaE, and PsaC subunits. PsaD and PsaE are required for reduction of ferredoxin and therefore form essential topological components of the docking site where ferredoxin interacts and accepts electrons from PsaC. We have identified the solvent-exposed regions of PsaD on the basis of protease accessibility and labeling with NHS-biotin. Site-specific mutations in these domains will be used to identify residues that interact with ferredoxin. Physiological, biochemical and spectroscopic analyses will be conducted to investigate roles of mutations in specific subunits onthe assembly and function of photosystem I. %%%Photosynthesis is the major source of biological energy and oxygen on the earth. Betterunderstanding of this process is essential for facing the food and environmental challenges of the future. Photosynthesis requires coordinated and efficient functioning of membrane-embedded andsoluble enzymes in cyanobacteria and plants. Photosystem I is one of two pigment-proteincomplexes in the photosynthetic membranes, that can utilize light energy to produce high-energybiochemicals. Photosystem I contains at least eleven proteins, 100 chlorophylls, -carotenes and series of electron transfer centers. The long-term research objective of our research is to determine the roles of different proteins of photosystem I in its function and organization. We initiated a program to genetically manipulate photosystem I of a naturally transformable cyanobacterium that is easily amenable to genetic engineering. Our NSF-supported research has provided insights concerning the functions of several photosystem I proteins. We plan to continue these investigations by focusing on how these proteins mediate and stabilize the assembly of photosystem I. We will also study the role of photosystem I proteins in the transfer of electrons from photosystem I to soluble proteins. An interdisciplinary approach that uses molecular genetics, physiology , biochemistry and biophysics will lead to a better understanding of how light energy is utilized by plants to generate chemical energy. ***
