One of the Substrate Waters for O2 Formation in Photosystem II Is Provided by the Water-Splitting Mn4CaO5 Cluster's Ca2+ Ion

During the catalytic step immediately prior to O-O bond formation in Photosystem II, a water molecule deprotonates and moves next to the water-splitting Mn4CaO5 cluster's O5 oxo bridge. Considerable evidence identifies 05 as one of the two substrate waters that ultimately form O-2. The relocated oxygen, known as O6 or Ox, may be the second. It is currently debated whether O6 or Ox originates as the Mn-bound water denoted W2 or as the Ca2+-bound water denoted W3. To distinguish between these two possibilities, we analyzed the D-O-D bending mode of the water molecule that deprotonates/relocates to become O6/Ox. We show that this D-O-D bending mode is not altered by the D1-S169A mutation. Previously, we showed that this D-O-D bending mode is altered substantially when Sr2+ is substituted for Ca2+. Because Sr2+/Ca2+ substitution alters this D-O-D bending mode but the D1-S169A mutation does not, we conclude that the water-derived oxygen that relocates and becomes O6/Ox derives from the Ca2+-bound W3. This conclusion provides an important constraint for proposed mechanisms of O-O bond formation in Photosystem II.