This research is designed to determine how the structure of photosystem II (PSII) and associated cofactors mediate electron transfer from water to the primary electron donor, P680. The focus of this project is on the role of the D1 reaction center polypetide of PSII in oxygen evolution. The powerful tools of molecular genetics are being used to mutagenize the chloroplast gene coding for D1, transform the mutant gene back into the choroplast of the green alga, Chlamydomonas reinhardtii, and characterize the transformants. In a directed approach, specific amino acid residues are being targeted for change. In a broader approach, specific portions of the gene are being altered, both by deletion and by random mutagenesis. These changes in D1 are being characterized to determine their effect on PSII stability, D1 stability, oxygen evolution and fluorescence induction kinetics. Transformants with defects in oxygen evolution are being genetically crossed with specific mutant strains of C. reinhardtii which are lacking photosynthetic membrane complexes, including Photosystem I (PSI), to produce strains which facilitate our isolation of relatively pure oxygen-evolving membrane preparations, free from PSI contamination and suitable for spectroscopic studies of PSII electron transfer. These strains are being further characterized in conjuction with Dr. Bruce Diner (DuPont de Nemours) who is performing detailed spectroscopic analyses and electron transfer and rates of oxygen evolution in transformants. EPR analyses are being performed on these strains in conjunction with Dr. Gary Brudvig (Yale University). Finally, suppressor mutants of selected transformant strains in which oxygen evolution is restored as a result of a second mutation are being identified and characterized. This technique provides further insight into inter- and intra-molecular structural relationships critical to PSII function. Terrestrial, aquatic and marine plants are the primary source of renewable biomass on earth. Through the process of photosynthesis plants convert radiant energy received from the sun into chemical energy. This energy drives the conversion of atomospheric carbon dioxide into sugars and other compounds, such as fats and proteins, which are essential components of all living organisms. Photosynthesis also results in the production of oxygen, and this oxygen is crucial for sustaining all forms of life that rely on respiration in order to provide the energy to keep them alive. The purpose of this research project is to gain an increased understanding of the biochemical and physical reactions that take place during photosynthesis by plants, resulting in the production of oxygen and consumption of carbon dioxide. Most of this research will be done on a simple unicellular plant which is well suited to analysis by modern molecular biology and therefore provides an excellent model system for study.
