In the utilization of energy on earth, photosynthesis by higher plants has a profound effect. It is the source of hydrocarbon energy for nearly all life forms through the capture of the sun's light energy and it is almost the sole source of dioxygen for respiration. A thorough understanding of the photosynthetic process is central to our ability to provide for our own energy needs in the future. Photosystem II is one of the two major light absorbing reaction centers of higher plants and cyanobacteria. It also contains the site of oxygen evolution, an unusual Mn4Ca catalytic center. The mechanism of the multiple step conversion of water into dioxygen is still not well understood. Both calcium and chloride are important participants in this process and they appear to interact with each other during oxygen production. As a part of the Mn4Ca cluster, calcium probably has a direct role in oxygen formation. Although chloride is not directly coordinated to the Mn4Ca cluster, it is required for transitions to higher oxidation states and appears to stabilize the calcium ion. This project is intended to help clarify the role of calcium in photosystem II of higher plants and its relationship to activation by chloride. The approach used will be to characterize the effects of calcium depletion or disruption at the Mn4Ca catalytic center. Two methods for biochemically disturbing the function of calcium will be used: treatment with mildly reduced pH and moderately high sodium chloride concentration; and treatment with fluoride, a competitive inhibitor of chloride activation. The results of these treatments will be examined using electron paramagnetic resonance (EPR) spectroscopy, a technique that is used to study molecules with unpaired electrons. A major objective of this project is to clarify the effects of calcium and chloride on the EPR signals from the oxygen evolving complex. To complement these studies, a second major objective is to use enzyme kinetics to characterize the interdependence of calcium and chloride in the activation of oxygen evolution.
Broader Impact This project will be carried out in a department that has a major focus on involving undergraduate and Master's level graduate students in research. The university is a mid-sized public institution with a student population that is ethnically diverse and a traditional emphasis on undergraduate education. The university has recently experienced a growth in graduate programs, including a new PhD program in the department housing the project. The Principle Investigator has previously trained numerous beginning researchers, who have gone on to further graduate study or careers in industry, and the current project is designed to continue this type of training. The project involves a range of biochemical and biophysical techniques appropriate for various levels of experience. Beginning researchers can work fairly independently on experiments involving enzyme kinetics analyses and participate in the preparation and work-up of samples intended for EPR spectroscopy. Those more interested in the physical aspects of the research can participate directly in spectroscopic measurements and analysis. Overall, students gain exposure to both commonly used biochemical methods and more specialized spectroscopic techniques. Because of the great importance that photosynthetic processes have for supporting life in general, the presence on campus of a project that is focused on light energy conversion and oxygen production will help to raise awareness about energy utilization issues.
In the utilization of energy on earth, photosynthesis by higher plants and cyanobacteria has an essential role as the source of hydrocarbon energy for nearly all life forms and the source of dioxygen for respiration by higher life forms. A thorough understanding of the way photosynthetic systems capture the sun’s light energy is central to our ability to provide for our own energy needs in the future. Photosystem II, one of the two major light absorbing reaction centers of higher plants and cyanobacteria, produces dioxygen at an unusual catalytic center comprised of four manganese and one calcium (Mn4Ca). Under experimental conditions, access to both calcium and chloride ions is required for the multiple step conversion of water into dioxygen. As a part of the Mn4Ca cluster, calcium evidently has a direct role in oxygen formation. Chloride is located a bond distance away from the Mn4Ca cluster, but is required for transitions of the Mn4Ca cluster to higher oxidation states during catalysis. In this study, the roles of chloride and calcium in the production of dioxygen from water in photosystem II were explored. Enzyme kinetics methods were used to investigate the characteristics of the chloride site and the interdependence of calcium and chloride in promoting oxygen evolution. Biochemical methods that cause calcium to dissociate or disrupt the function of calcium were explored and the effects characterized. These included altering the pH and salt concentrations and treatment with the inhibitor fluoride. In addition, electron paramagnetic resonance spectroscopy was used to investigate the effects of the treatments on the catalytic Mn4Ca cluster and a nearby tyrosine residue that is also involved in catalysis. The results of these studies have revealed details about the binding site of calcium and how it is influenced by chloride, as well as further characteristics of the chloride and other anion binding sites. A major goal of the project was to train undergraduate and graduate students in research design and methods. This location of the project was in a department that has a long-standing commitment to training beginning researchers, as a part of the goal of producing future scientists and citizens with a view point that is well-balanced in today’s technology needs. The university is a mid-sized public institution with a student population that is ethnically diverse and a traditional emphasis on undergraduate education. During the course of the project, eleven undergraduate students and two graduate students received training. Each student had a project that he or she could develop independently, planning and carrying out experiments, data analysis and writing summary reports. Many of these students worked on their projects for two or more years, thereby making a substantial contribution to the progress of the overall goals of the grant. Of the nine research students who have graduated, four moved on to graduate programs at other universities and two went to pharmacy schools, with the remainder joining the work force in biochemical technology.
