Roles of D1-Glu189 and D1-Glu329 in O Formation by the Water-Splitting MnCa Cluster in Photosystem II.

During the catalytic step that precedes O-O bond formation in Photosystem II, a water molecule deprotonates and moves next to the water-splitting MnCa cluster's O5 oxo bridge. The relocated oxygen, known as O6 or O, may serve as a substrate, combining with O5 to form O during the final step in the catalytic cycle, or may be positioned to become a substrate during the next catalytic cycle. Recent serial femtosecond X-ray crystallographic studies show that the flexibility of D1-E189 plays a critical role in facilitating the relocation of O/O. In this study, the D1-E189G and D1-E189S mutations were characterized with FTIR difference spectroscopy. The data show that both mutations support MnCa cluster assembly, substantially inhibit advancement beyond the S state, and alter the network of H bonds that surrounds the MnCa cluster. Previously, the D1-E189Q, D1-E189K, and D1-E189R mutations were shown to have little impact on the activity, electron transfer rates, or spectral properties of Photosystem II. A rationale for this behavior is presented. The residue D1-E329 interacts with water molecules in the O1 water network that has been suggested recently to supply substrate during the catalytic cycle. Characterization of the D1-E329A mutant with FTIR difference spectroscopy shows that this mutation does not substantially perturb the structure of PSII or the water molecules whose O-H stretching modes change during the catalytic cycle. This result provides additional evidence that the water molecules whose vibrational properties change during the S to S transition are confined approximately to the region bounded by D1-N87, D1-N298, and D2-K317.