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

Significance

Photosynthesis—the biological process via which solar energy is stored in the form of energy-rich molecules—fuels life on Earth and provides the molecular oxygen we breathe. The crucial starting point for this reaction is the splitting of water, which is carried out by a unique catalyst in Photosystem II. Unraveling the details of this reaction provides the blueprint for how to extract protons and electrons from water using abundant and cheap metal catalysts—a pre-requisite for the sustainable production of green fuels and chemicals. In this study, we identify a key feature of nature’s water-splitting unit, the binding site of one of the two water molecules involved in making O ₂.

Abstract

Identifying the two substrate water sites of nature’s water-splitting cofactor (Mn ₄CaO ₅ 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 ₁ 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 H ₂ ¹⁸O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn ₄CaO ₅ cofactor after mixing with H ₂ ¹⁷O. 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 Ca ²⁺→Sr ²⁺ 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.