| Photosystem II (PSH) is a trans-membrane protein complex that contains a number of redox-active species and carries out the initial steps in oxygenic photosynthesis. Its oxygen-evolving complex (OEC) contains a tetramanganese-oxo (Mn4) cluster, as well as the essential inorganic cofactors calcium and chloride, and catalyzes the oxidation of water to molecular oxygen. Following photon absorption, electrons are transferred from the Mn4 cluster at the donor side of PSII, via a nearby tyrosine residue (YZ) and the photoactive chlorophyll (P680), to a diffusable quinone at the acceptor side. Oxidizing equivalents accumulate at the Mn4 cluster in a cycle of four consecutive oxidative steps, progressing from the S0 to the S4 state. Molecular oxygen is released in the S4-to-S is not well understood, although specific steps in the oxidation cycle may require proton-coupled electron transfer (PCET) between Mn4 and oxidized YZ•. The present study utilizes electron paramagnetic resonance (EPR) techniques and enzyme-activity measurements to characterize the cofactors participating in water oxidation and to determine if certain steps in the oxidation process require PCET.; To address these questions, manganese-depleted and acetate-inhibited PSII samples are used. Manganese-depleted samples allow study of YZ • in the absence of Mn4, whereas in acetate-treated samples OEC turnover stalls in the S2YZ• state. EPR power-saturation measurements show that YZ • and Mn4 are in close enough proximity to affect each other's spin-relaxation behavior. The interaction between YZ • and the Mn4 cluster is characterized by analysis of EPR-signal lineshapes and spin-relaxation behavior in acetate-inhibited samples. It is demonstrated that the different EPR signals observed in acetate-treated PSH at temperatures ranging from 7 to 293 K all arise from the interacting S2YZ• state. To characterize the site of acetate inhibition, binding-competition studies are performed. Acetate is shown to bind to the OEC in competition with chloride, whose presence is required for O2-evolution activity. As acetate is known to bind near YZ•, this result places chloride in the immediate proximity of YZ and suggests a direct function of chloride in the electron-transfer mechanism between Mn4 and YZ. To further elucidate the mechanism of electron transfer at the donor side, pH effects on O2-evolution and electron-transfer activities are examined over a range of temperatures. In addition, temperature, pH, and deuterium-isotope dependence of competitive electron donation to P680+ from Y Z versus alternative electron donors are surveyed by EPR measurements of YZ photooxidation and re-reduction. The findings suggest that a PCET step is necessary for advancement of the Mn4 cluster in at least some S-state transitions. Electron transport may be enhanced by deprotonation of a nearby amino-acid side chain serving as proton acceptor to YZ. We conclude that the inhibitory effect brought about by replacement of chloride by acetate may stem from disruption of the hydrogen-bonding network between YZ, its nearby proton acceptor, chloride, and Mn4.; The results presented in this dissertation aid elucidation of the mechanism of electron transport in the vicinity of the OEC and allow new insights into the mechanism of water oxidation. We speculate about the role of YZ in water oxidation and the possible involvement of chloride in the electron-transfer pathway from Mn4 to YZ |
