The exchange of the fast substrate water in the S(2) state of photosystem II is limited by diffusion of bulk water through channels – implications for the water oxidation mechanism

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn(4)CaO(5) cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the ‘slow exchanging substrate water (W(s))’, while, in the S(2) state, the terminal water ligands W2 and W3 are both discussed as the ‘fast exchanging substrate water (W(f))’. A critical point for the assignment of W(f) is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn(4)CaO(5) cluster. In this study, we measured the rates of H(2)(16)O/H(2)(18)O substrate water exchange in the S(2) and S(3) states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of W(f) and W(s) were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn(4)CaO(5) cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that W(f) exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S(2) state, namely the indifference of the rate of W(f) exchange towards Ca/Sr substitution.