Chloride-depletion effects in the calcium deficient oxygen-evolving complex of photosystem II

The effects of Cl- depletion in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy after removal of the 17- and 23-kDa polypeptides and depletion of Ca2+ by NaCl treatment. When the salt treatment was done in the presence of a high concentration (5 mM) of the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), a modified dark-stable multiline signal was observed from the S2 state and a 13 mT wide S3 signal could be generated by illumination at 0 degrees C as reported previously for experiments conducted under these conditions [Boussac, A., Zimmermann, J.L., & Rutherford, A. W. (1990) FEBS Lett. 277, 69-74}. The modified S2 multiline signal was lost after a further Cl- depletion in the presence of a low EGTA concentration (50 micromolar). Upon Cl- reconstitution, a normal S2 multiline signal could be generated by continuous illumination at 200 K. In contrast, a lowering of the EGTA concentration (50 micromolar) alone, in the presence of Cl- (30 mM), had no effect on the modified S2 multiline signal. These results indicate that the modification of S2 is due to binding of the chelator to PS-II and that Cl- stabilizes the chelator binding. When Cl- depletion in Ca2+/- depleted PS-II was done in the presence of a high concentration of EGTA (5 mM), the modified S2 multiline signal disappeared but was regenerated by Cl- reconstitution in darkness. These results indicate that when Cl- depletion is done to the EGTA-modified PS-II, the S2 multiline signal disappears but the S2 state remains stable in the dark. Thus, EGTA binding and Cl- depletion appear to be additive phenomena. Cl- depletion also modified the S3 EPR signal, showing a narrrow signal (< 10 mT) around g = 2. This modification of the S3 signal was reversed by Cl- reconstitution, resulting in the reappearance of the 13 mT wide S3 signal. The modifications of S2 and S3 due to Cl- depletion observed in Ca2+-depleted membranes are similar to those observed following Cl- depletion in regular PS-II membranes, in which functional Ca2+ is thought to be present. These results, therefore, indicate that the modifications of the S2 and S3 EPR signals due to Cl- depletion are independent of Ca2+. Investigations of PS-II membranes which were salt-treated without EGTA revealed that the chemical 2-(N-morpholino)ethanesulfonic acid (MES), generally used as a pH buffer, was able to modify the S2 state, in a similar fashion to EGTA. In consideration of the components that are known to modify S2 [EGTA, (ethylenedinitrilo)tetraacetic acid (EDTA), citrate, pyrophosphate, and MES), the results indicate that the modification of S2 is due to binding of these components, by their anionic groups containing oxygen, near to or on the Mn cluster itself. The observed effects of Ca2+ and Cl- depletion in PS-II may be relevant to the proposed role(s) of Ca2+ and Cl- in controlling substrate binding in the functional charge accumulation cycle.