Is Mn-Bound Substrate Water Protonated in the S (2) State of Photosystem II?

In spite of great progress in resolving the geometric structure of the water-splitting Mn4O (x) Ca cluster in photosystem II, the binding sites and modes of the two substrate water molecules are still insufficiently characterized. While time-resolved membrane-inlet mass spectrometry measurements indicate that both substrate water molecules are bound to the oxygen-evolving complex (OEC) in the S (2) and S (3) states (Hendry and Wydrzynski in Biochemistry 41:13328-13334, 2002), it is not known (1) if they are both Mn-bound, (2) if they are terminal or bridging ligands, and (3) in what protonation state they are bound in the different oxidation states S (i) (i = 0, 1, 2, 3, 4) of the OEC. By employing O-17 hyperfine sublevel correlation (HYSCORE) spectroscopy we recently demonstrated that in the S (2) state there is only one (type of) Mn-bound oxygen that is water exchangeable. We therefore tentatively identified this oxygen as one substrate 'water' molecule, and on the basis of the finding that it has a hyperfine interaction of about 10 MHz with the electron spin of the Mn4O (x) Ca cluster, we suggest that it is bound as a Mn-O-Mn bridge within a bis-mu(2) oxo-bridged unit (Su et al. in J Am Chem Soc 130:786-787, 2008). Employing pulse electron paramagnetic resonance, H-1/H-2 Mims electron-nuclear double resonance and H-2-HYSCORE spectroscopies together with H-1/H-2-exchange here, we test this hypothesis by probing the protonation state of this exchangeable oxygen. We conclude that this oxygen is fully deprotonated. This result is discussed in the light of earlier reports in the literature.