THE EFFECTS OF CALCIUM DEPLETION ON THE O-2-EVOLVING COMPLEX IN SPINACH PS-II - THE S-1(ASTERISK)-STATE S-2(ASTERISK)-STATE AND S-3(ASTERISK)-STATE AND THE ROLE OF THE 17-KDA AND 23-KDA EXTRINSIC POLYPEPTIDES

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples depleted of calcium by various NaCl treatments which inhibit oxygen evolution without displacement of manganese. Changes in the pre-edge feature due to 1s --> 3d transitions and shifts in the edge position of samples in the S-1*, S-2* and S-3* states indicate manganese oxidation for the S-1*-->S-2* and S-2*-->S-3* transitions. Analysis of the EXAFS shows changes on NaCl treatment compared to native PS II membranes which are further modified by the chelator, EGTA. The intensity of the Fourier transform peak at about 1.8 Angstrom, assigned to oxygen, increases with increasing S-state in agreement with oxidation state changes, although the average distance for this first shell remains constant. Each of the inhibitor-treated S-states have a short average Mn-O bond length, showing the retention of the mu-oxo bridges postulated to occur in native samples. The Mn-Mn shell, found at 2.7 Angstrom in native PS II membranes is split in NaCl-treated samples to give a 2.7 Angstrom Mn-Mn and 3.0 Angstrom Mn-X interaction (X = Mn,C/O/N). Splitting of the 2.7 Angstrom shell is most apparent in the higher S-states, S-3*>S-2*>S-1*. Although the scatterers at 3.0 Angstrom could not be uniquely identified, the intensity favours heavy scatterers, Mn/Ca, over light scatterers, C/O/N. The cluster appears to contain at least two inequivalent Mn-Mn pairs or shows multiple scattering from a ligand such as tyrosine/histidine. NaCl treatment results in a smaller 3.3/3.6 Angstrom intensity compared to untreated PS II samples which could be due to replacement of calcium scatterers at this distance and/or a structural rearrangement. EGTA addition results in an S-2* state with a modified EPR spectrum but has only a small effect on the XAS. The changes on removal of the 17 and 23 kDa extrinsic polypeptides are small compared to the effect of the calcium depletion/NaCl treatment, indicating a minor role for these polypeptides on the structure of the cluster. Changes in the electron spin lattice relaxation time, T-1 of the dark stable tyrosine radical Y-D, have also been studied using pulsed EPR. The T-1 relaxation times decreased with increasing modified S-state S-1*>S-2*>S-3*, indicating oxidation occurring at or near the manganese cluster.