Location of the calcium binding site in Photosystem II: A Mn2+ substitution study

The whereabouts of the Ca2+ site in Photosystem II (PSII) was investigated by experiments in which Mn2+ was substituted for Ca2+. When stoichiometric amounts of Mn2+ ions were added to Ca2+-depleted PSII, the Mn2+ was not detected by EPR. The titration of Ca2+ back into Ca2+-depleted/Mn2+-containing PSII resulted in the simultaneous release of the Mn2+ and the loss of the two EPR signals which are characteristic of the Ca2+-depleted enzyme (i.e., the stable, modified S-2 multiline signal arising from the intrinsic Mn cluster and the split S-3 signal from an organic radical interacting with the Mn cluster). These results indicate that the Mn2+ occupies the functional Ca2+ site. The S-2 and S-3 EPR signal characteristic of this kind of Ca2+-depleted preparation were unaffected by the binding of the Mn2+. Since, from earlier results, it seems likely that the modification and stability of S-2 multiline signal in these PSII preparations is due to binding of chelator to or close to the Mn cluster, the present results indicate that the Ca2+ site (at least when occupied by Mn2+) does not overlap with the chelator binding site. Since Mn2+ binding does not effect the S-2 EPR signal from the Mn cluster, it can be concluded that the Mn2+ is not involved in detectable magnetic interactions with the cluster. This result indicates that the Mn2+-occupied Ca2+ binding site is outside the first co-ordination sphere of the Mn cluster. The relaxation properties of TyrD(.) were enhanced by the presence of the Mn2+ when the Mn cluster was in the S-1 state.