One of the Substrate Waters for O Formation in Photosystem II Is Provided by the Water-Splitting MnCaO Cluster's Ca 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 MnCaO cluster's O5 oxo bridge. Considerable evidence identifies O5 as one of the two substrate waters that ultimately form O. The relocated oxygen, known as O6 or O, may be the second. It is currently debated whether O6 or O originates as the Mn-bound water denoted W2 or as the Ca-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/O. 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 Sr is substituted for Ca. Because Sr/Ca 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/O derives from the Ca-bound W3. This conclusion provides an important constraint for proposed mechanisms of O-O bond formation in Photosystem II.