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

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.