(A) 0 mM up to 20 mM and from (B) 20 mM up to 100 mM performed on C12E8 dPSIIcc solutions, from (C) 0 mM to 100 mM performed on C12E8 dPSIIcc microcrystals. EPR spectra (D) of added Mn2+ with apo-WOC-PSIIcc using 10 mM MnCl2 washed with buffers (20 mM MES pH 6.0, 0.5 M betaine and 0.02% C12E8) with (black trace) and without CaCl2 (blue trace). Samples were measured at 20 K (microwave frequency, 9.22 GHz; field modulation amplitude, 32 G at 100 kHz; microwave power, 1 mW). Panel A: PSII solutions were treated with no NH2OH (red trace), 1 mM NH2OH (blue trace), 5 mM NH2OH (blue-green trace), 10 mM NH2OH (black trace) and 20 mM NH2OH (pink trace) and incubated for 10–15 min at RT in dark. Panel B: Samples were treated with 20 mM NH2OH (black trace), 30 mM NH2OH (blue-green trace), 50 mM NH2OH (red trace), 100 mM NH2OH (blue trace) and incubated for 10–15 min at RT in dark. Panel C: Microcrystals were treated with no NH2OH (yellow trace), 20 mM NH2OH (blue-green trace), 30 mM NH2OH (pink trace), 40 mM NH2OH (blue trace), 50 mM NH2OH (red trace), 100 mM NH2OH (black trace) and incubated for 10–15 min at RT in dark. Panel D: PSII solutions containing 10 mM MnCl2 were treated with buffers with (black trace) and without (blue trace) CaCl2. We observe a six-line signal at g ≈ 2 from Mn2+ bound to protein that is still present after repeated washing. The spectra have similar features as intact PSII treated with NH2OH. The sample washed with buffer with CaCl2 shows a weaker signal than the one without CaCl2. After Mn depletion, apo-PSII core complexes bind Mn2+ ions with low affinity at nonspecific sites when the concentration of the MnCl2 used is above the stoichiometric levels and in absence of other divalent ions like Ca2+ or Mg2+. In presence of an excess of another divalent cation, in our case Ca2+ (from CaCl2), the contribution from the Mn2+ ions with low binding affinity can be eliminated (black trace). It has been shown before that Ca2+ plays a role in preventing or reversing the binding of this low-affinity nonfunctional Mn2+ (Chen et al., 1995).