The role of the extrinsic 33 kDa protein in Ca2+ binding in photosystem II

The role of the 33 kDa protein in Ca2+ binding was studied by comparing the EPR properties of photosystem II in the presence and absence of the 33 kDa protein and Ca2+. When the removal of the 33 kDa protein was carried out in the dark, a normal manganese multiline EPR signal could be observed when the S2 state was generated. In addition, the split S3 signal could not be generated by illumination at 273 K. Exposure of the 33 kDa protein-less photosystem II to room light did not lead to any change in the EPR properties of the S2 state, but the split S3 state signal at around g = 2 could then be generated, indicating that Ca2+ was released from this preparation during the exposure to light. Treatment of photosystem II lacking the 33 kDa protein with EGTA in the light led to a modification of the S2 state characterized by a dark-stable multiline EPR signal. Much lower EGTA concentrations were required in order to obtain this modification in the absence of the 33 kDa protein than was required when the 33 kDa protein was present. This indicates that the manganese cluster was more accessible to chelator binding when the 33 kDa protein was absent. When 33 kDa protein-less photosystem II was treated with EGTA in the dark, no modification of the multiline EPR signal of the S2 state of the manganese cluster occurred, nor was Ca2+ released as monitored by the inability to generate the split S3 signal. These chelator- and Ca(2+)-binding properties occurring in PSII lacking the 33 kDa protein are very similar to those observed previously in NaCl-washed PSII in which the 33 kDa protein is present (reviewed in Boussac & Rutherford, 1994a). It is concluded that the 33 kDa protein has little or no direct role in binding the Ca2+ ion which is required for oxygen evolution.