An Alternative Method for Calcium Depletion of the Oxygen Evolving Complex of Photosystem II As Revealed by the Dark-Stable Multiline EPR Signal

The dark-stable multiline EPR signal of photosystem II (PSII) is associated with a slow-decaying S, state that is due to Ca2+ loss from the oxygen evolving complex. Formation of the signal was observed in intact PSI I in the presence of 100-250 mM NaCl at pH 5.5. Both moderately high NaCl concentration and decreased pH were required for its appearance in intact PSII. It was estimated that only a portion of oxygen evolving complexes was responsible for the signal (about 20% in 250 mM NaCl), based on the loss of the normal S-2-state multiline signal. The formation of the dark-stable multiline signal in intact PSII at pH 5.5 could be reversed by addition of 15 mM Ca2+ in the presence of moderately high NaCl, confirming that it was the absence of Ca2+ that led to its appearance. Formation of the dark-stable multiline signal in NaCl-washed PSII, which lacks the PsbP (23 kDa) and PsbQ (17 kDa) subunits, was observed in about 80% of the sample in the presence of 150 mM NaCl at pH 5.5, but some signal was also observed under normal buffer conditions. In both intact and NaCl-washed PSII, the S2YZ. signal, which is also characteristic of Ca2+ depletion, appeared upon subsequent illumination. Formation of the dark-stable multiline signal took place in the absence of Ca2+ chelator or polycarboxylic acids, indicating that the signal did not require their direct binding as has been proposed previously. The conditions used here were milder than those used to produce the signal in previous studies and included a preillumination protocol to maximize the dark-stable S-2 state. Based on these conditions, it is suggested that Ca2+ release occurred through protonation of key residues that coordinate Ca2+ at low pH, followed by displacement of Ca2+ with Na+ by mass action at the moderately high NaCl concentration.