Discrete extraction of the Ca atom functional for O2 evolution in higher plant photosystem II by a simple low pH treatment

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Abstract
Treatment of spinach PS II membranes with a citrate solution at pH 3.0 totally inactivated O2 evolution concomitant with a 50% decrease in Ca abundance. Notably, neither the abundance of Mn and extrinsic proteins nor the activity of DPC photooxidation was at all affected by the treatment. The treated membranes evolved O2 at a high rate in the presence of exogenous Ca2+, but the activity was sensitive to EDTA. However, when the treated membranes were incubated with Ca2+ for a few tens of minutes, the O2‐evolving activity became EDTA‐resistant, suggesting a firm re‐ligation of Ca2+ to the Ca‐binding site. It was indicated that spinach PS II contains two Ca atoms per reaction center, and that the low pH citrate treatment selectively removes one of the two Ca atoms that is specifically functional for O2 evolution, even in the presence of all three extrinsic proteins.