Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn₄CaO₅ cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S₁ state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved ¹⁷O-Electron-electron Double resonance detected NMR (TR-¹⁷O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H₂¹⁸O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn₄CaO₅ cofactor after mixing with H₂¹⁷O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W_S) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W_S are both enhanced by Ca²⁺→Sr²⁺ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W_S. This strongly restricts options for the mechanism of water oxidation.