9728754 Burnap The focus of this project is on the structure and assembly of the catalytic site of the photosynthetic water-oxidation reaction. The work combines molecular genetic, biochemical and biophysical approaches to clarify the process of photoactivation, which is the sequential light-dependent assembly of the catalytic tetramer of manganese atoms that forms the core of the water-oxidation complex. Photoactivation represents a fascinating problem of metalloprotein assembly. It is a multi-quantum process involving a sequence of kinetically resolvable molecular rearrangements that are stabilized by the photooxidation of manganese and binding of calcium. The molecular events occurring during this process remain obscure and the major object of this research is to define these events in terms of the influence of the protein groups providing the coordination environment for the incoming manganese and calcium ions during the assembly sequence. The research is facilitated by the availability of numerous partially impaired site-directed mutants of putative manganese and calcium ligands in the intrinsic portion of the complex and mutants of the extrinsic polypeptides, which have been shown to modulate the efficiency of the assembly process. Polarographic and fluorescence methods of monitoring the assembly process are utilized. Earlier studies of photoactivation using intact mutant cyanobacterial cells are now extended to in vitro studies where analysis of the cofactor requirements of normal and genetically modified reaction center core complexes can be studied in greater detail. Photosynthesis is a globally important process that has a number of crucial unresolved features. Among the most puzzling aspects of the photosynthetic mechanism is oxygen production by the PSII water-splitting enzyme. PSII is of critical importance from both the agricultural and ecological perspectives because it is responsible both for generating the vast majority of hydrogen used to fix carbon into the earth's biosphere and for releasing oxygen necessary for respiration. The understanding of this process takes on added importance given the recognition of anthropomorphic changes such as global warming and ozone depletion because PSII is potentially quite sensitive to these environmental stresses. The project is basic research into the enzyme mechanism of PSII and because PSII is also a membrane protein, the knowledge gained from this project should also positively impact the field of membrane biology, which has many important applications.
