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 s amples depleted of calcium by various NaCl treatments which inhibit ox ygen evolution without displacement of manganese. Changes in the pre-e dge feature due to 1s --> 3d transitions and shifts in the edge positi on of samples in the S-1, S-2* and S-3* states indicate manganese oxi dation for the S-1-->S-2* and S-2*-->S-3* transitions. Analysis of th e EXAFS shows changes on NaCl treatment compared to native PS II membr anes which are further modified by the chelator, EGTA. The intensity o f 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 co nstant. 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). Spli tting of the 2.7 Angstrom shell is most apparent in the higher S-state s, S-3>S-2*>S-1*. Although the scatterers at 3.0 Angstrom could not b e 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 ligan d 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 c hanges on removal of the 17 and 23 kDa extrinsic polypeptides are smal l compared to the effect of the calcium depletion/NaCl treatment, indi cating a minor role for these polypeptides on the structure of the clu ster. 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-st ate S-1>S-2*>S-3*, indicating oxidation occurring at or near the mang anese cluster.