One of the key reactions of photosynthesis is the conversion of water into the oxygen in the atmosphere, which is catalyzed by an enzyme system called photosystem II. Photosystem II binds the chlorophyll molecules that absorb the light energy required for oxygen production, as well as the atoms of manganese, calcium and chloride that form the site of oxygen production in the enzyme. The enzyme consists of a number of proteins that anchor it in a membrane, but it also contains three proteins that are not associated with the membrane. Removal of one of these proteins, called PsbO, or ''manganese-stabilizing protein'' from the enzyme causes a loss of calcium and chloride cofactors and a loss of oxygen production as well. This research project addresses the role of PsbO in oxygen production. A series of genetically modified PsbO proteins have been developed with prior support from NSF; some of these proteins exhibit an interesting modification in their ability to rebind to photosystem II. Normally, two copies of the protein are present. Mutant proteins are available that exhibit binding of two, only one or of no copies of PsbO. The consequences of these modified protein stoichiometries on cofactor binding and stability of manganese retention will be determined. In addition, mutations in PsbO that modify amino acids that may be required for calcium binding will be characterized, and experiments will be carried out to determine whether PsbO might form part of a channel that conducts water to the site of oxygen formation in the enzyme. Results of this research project will provide more information on the unique chemistry of oxygen evolution, a reaction that may some day be used in synthetic systems for energy production.
Broader Impacts A principle aim of this project is to provide hands on research experience for undergraduate students who are majoring in the life sciences. A number of the experiments in the project involve site-directed mutagenesis, protein overexpression and purification, and enzyme assays. The goal is to have two undergraduates working in the laboratory at any given time. The Co-P.I. will obtain experience in mentoring undergraduates as part of this effort, and the students will be encouraged to present their results at regional meetings. A junior faculty member who teaches chemistry at Spring Arbor University will collaborate in this project so as to provide him with research facilities that can be combined with his teaching activities. Finally, the P.I. teaches two undergraduate courses (biochemistry, plant molecular biology) in which lectures on photosynthesis, and discussions of the research literature form an important part of the offerings.
