Photosystem II (PSII) is an integral membrane protein complex in which photochemistry, electron and proton transport, and catalysis of water oxidation/quinone reduction are facilitated and regulated by multiple polypeptide subunits, ionic cofactors, prosthetic groups, and the protein/lipid environment. The long-term objectives of this project are to develop an understanding at the molecular level of the structure and assembly of the tetranuclear Mn complex that forms the active site of water oxidation, the catalytic mechanism of water oxidation, the electron-transfer properties of PSII, and the damaging side reactions that occur in the process of water oxidation.
The specific aims of the proposed studies are to use electron paramagnetic resonance and flash-detection optical spectroscopies to study the electron-transfer reactions of PSII, the intermediate oxidation states of the tetranuclear Mn complex, and the redox-active tyrosine YZ. These studies will utilize new redox-active herbicides to produce two-electron oxidized intermediates of the O2-evolving complex in high yield in the concentrated samples needed for spectroscopic measurements. One objective will be to characterize the role of YZ as a electron-proton-transfer center in the water-oxidation chemistry of PSII by measuring its reactions with nitric oxide and H-atom donors, and the H/D isotope effects on the rates of reaction. Another objective will be to purify O2-evolving PSII complexes by using affinity chromatography with a herbicide derivative bound to agarose. Improvements in purification of PSII will facilitate the spectroscopic studies and may enable growth of crystals suitable for x-ray analysis.
A final aim i s to develop and apply new electrochemical methods to study the electron-donor side of PSII. These proposed studies will lead to a clearer picture of the water-oxidation chemistry and the electron-transfer reactions of PSII. They also have direct application to the structure and function of metal ion clusters and amino-acid radicals that function to transfer electrons and/or catalyze reactions in a variety of other proteins. Further, the insight gained on the mechanism of water oxidation and the damaging side reactions in PSII will be important to understand the mechanisms of all enzymes that utilize dioxygen or hydrogen peroxide as a substrate, such as cytochrome c oxidase, cytochrome P-450, peroxidases, and oxygenases.
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