Electron paramagnetic resonance (EPR) measurements were performed on photosystem II (PSII) membranes that were treated with 2 M NaCl to release the 17- and 23-kilodalton (kDa) polypeptides. By using 75 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to limit the photosystem II samples to one stable charge separation in the temperature range of 77–273 K, we have quantitated the EPR signals of the several electron donors and acceptors of photosystem II. It was found that removal of the 17- and 23-kDa polypeptides caused low potential cytochrome b559 to become fully oxidized during the course of dark adaptation. Following illumination at 77–130 K, one chlorophyll molecule per reaction center was oxidized. Between 130 and 200 K, both a chlorophyll molecule and the S1 state were photooxidized and, together, accounted for one oxidation per reaction center. Above 200 K, the chlorophyll radical was unstable. Oxidation of the Si state gave rise to the S2-state multiline EPR signal, which arises from the Mn site of the O2-evolving center. The yield of the S2-state multiline EPR signal in NaCl-washed PSII membranes was as high as 93% of the control, untreated PSII membranes, provided that both Ca2+ and Cl- were bound. Furthermore, the 55Mn nuclear hyperfine structure of the S2-state multiline EPR signal was unaltered upon depletion of the 17- and 23-kDa polypeptides. In NaCl-washed PSII samples where Ca2+ and/or Cl- were removed, however, the intensity of the S2-state multiline EPR signal decreased in parallel with the fraction of PSII lacking bound Ca2+ and Cl-. Reconstitution of Ca2+ was accomplished in a medium containing 100 mM NaCl, and the yield of the S2-state multiline EPR signal after Ca2+ reconstitution was comparable to that observed in NaCl-washed PSII membranes before removal of Ca2+. We conclude that Ca2+ and Cl-, and not the 17- and 23-kDa polypeptides, are the main factors governing the ability to observe the S2-state multiline EPR signal. Furthermore, removal of the 17- and 23-kDa polypeptides from photosystem II does not significantly perturb the environment of the Mn site of the O2-evolving center.
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