18O Isotope Exchange Measurements Reveal that Calcium Is Involved in the Binding of One Substrate-Water Molecule to the Oxygen-Evolving Complex in Photosystem II

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Abstract
Direct evidence is presented to show that calcium is inherently involved in the binding of one of the two substrate-water molecules to the oxygen-evolving complex in photosystem II. Previous rapid (millisecond range) 18O isotope exchange measurements between added H218O and the photogenerated O2 have shown that the two substrate−water molecules bind to separate sites throughout the S-state cycle, as revealed by their kinetically distinct rates of 18O exchange [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399−4405]. Upon extraction of the functionally bound calcium using a either a low-pH/citrate treatment or a NaCl/A23187/EGTA treatment and subsequent reconstitution of activity with strontium, we show for the first time a specific increase in the slow rate of 18O exchange by a factor of 3−4. This increase in the slow rate of exchange is consistently observed across the S1, S2, and S3 states. In contrast, the fast phase of 18O exchange in the S3 state appears to be affected little upon strontium reconstitution, while the fast phases of exchange in the S1 and S2 states remain largely unresolvable, at the detectable limits of the current techniques. The results are discussed in terms of a possible substrate bridging structure between the functional calcium and a catalytic manganese ion that gives rise to the slowly exchanging component.