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 (MnCaO 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 S state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved O-Electron-electron Double resonance detected NMR (TR-O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of HO to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the MnCaO cofactor after mixing with HO. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W are both enhanced by Ca→Sr substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W. This strongly restricts options for the mechanism of water oxidation.