(18)O Isotope Exchange Measurements Reveal That Calcium is Involved in the Binding of One Substrate-water Molecule to the Oxygen-evolving Complex in Photosystem II

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) (18)O isotope exchange measurements between added H(2)(18)O and the photogenerated O(2) 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 (18)O 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 (18)O exchange by a factor of 3-4. This increase in the slow rate of exchange is consistently observed across the S(1), S(2), and S(3) states. In contrast, the fast phase of (18)O exchange in the S(3) state appears to be affected little upon strontium reconstitution, while the fast phases of exchange in the S(1) and S(2) 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.