The project's overall goal is to describe the molecular mechanism of photosynthetic water oxidation. The catalytic site is located in Photosystem II (PSII) and contains a (Mn)4-Ca cluster that interacts with a redox-active tyrosine residue known as Yz. The Yz radical extracts electrons and protons from the (Mn)4-Ca cluster, leading to the oxidation of water and the release of 02 as a by-product. The project's specific goals are to characterize the substrate, inhibitor, and cofactor binding properties of the (Mn)4 cluster in its dark-stable S1 oxidation state and to further characterize the environmental factors that control the reactivity of tyrosine Yz. These objectives build on our recent discoveries that (1) the (Mn)4 cluster in its S1 oxidation state exhibits a parallel polarization multiline EPR signal, and (2) the rate of electron transfer from YD to P680+ is five orders of magnitude faster than previously believed (YD, a second redox-active tyrosine in PSII, does not participate in the primary electron transfer reactions of water oxidation). The proposed studies will exploit the S1 state multiline EPR signal as a spectroscopic probe of the (Mn)4 cluster in its S1 oxidation stale in the same manner that the S2 state multiline EPR signal has been exploited over the last two decades as a probe of the (Mn)4 cluster in its S2 oxidation state. Our observation that electron transfer from YD to P680+ takes place rapidly has important mechanistic implications and suggests a variety of hypotheses regarding the factors that govern the reactivity of tyrosine Yz and, possibly, the factors that govern location of the P680+ cation among the chlorophyll molecules that constitute P680. The proposed studies will test these hypotheses. Understanding the mechanism of photosynthetic water oxidation should provide insight into the mechanisms of two important classes of enzymes that are currently the subjects of intensive bio-medical research: metallo-radical enzymes and enzymes whose mechanisms involve proton-coupled electron transfer reactions. Photosystem II possesses unique advantages for studying the initiation of catalysis in these two classes of enzymes because catalysis in PSII can be initiated with a flash of light, thereby facilitating kinetic studies of reaction cycle intermediates with high time resolution.
