Dugald J. MacLachlan

Modulation analysis of the electron spin echo signals of in vivo oxidised primary donor 14N chlorophyll centres in bacterial, P870 and P960, and plant Photosystem I, P700, reaction centres

Abstract The primary donors of two structurally characterised photosynthetic bacterial reaction centres, P870 + from Rhodobacter sphaeroides and P960 + from Rhodopseudomonas viridis , and the spinach Photosystem I primary donor P700 + were studied by electron spin echo modulation. The values obtained for the in vivo isotropic hyperfine interaction, a iso , were considerably smaller than literature reports from in vitro studies. The results for P960 + and P870 + are in accord with data from other spectroscopic techniques and reveal that the electron is more localised in one half of the chlorophyll dimer with a ratio of 2:1. Damping of the modulations was most severe for P960 + , which interacts with the non-haem iron of the reaction centre as well as a donor cytochrome, cyt c . The reduced hyperfine interactions obtained for P700 + indicate that P700 + is either a chlorophyll dimer with the electron extensively localised on one of the chlorophyll pair with a ratio of 3:1 or 4:1, depending on which of the published values of a iso for chlorophyll a + is used, or a monomer.

Investigation of the ammonium chloride and ammonium acetate inhibition of oxygen evolution by Photosystem II

Using EPR and EXAFS spectroscopies we show that high concentrations of ammonium cations at alkaline pH are required for (1) inhibition of oxygen evolution: (2) an alteration of the EPR properties of the oxygen evolving complex: (3) the ability to detect YZ; and (4) the slow reduction of the Mn complex leading to the appearance of EPR detectable Mn2+. The inhibition of S state cycling, slowing of YZ reduction, appearance of Mn2+ and the yield of a Hpp < 10 mT S3 type EPR signal are decreased by calcium addition. This indicates that these effects were probably associated with calcium depletion arising from the high concentration of ammonium cation. The ammonia-induced changes to the S2 multiline EPR signal are not affected by calcium addition. The appearance of Mn2+ is shown to be reversible on illumination, suggesting that the Mn reduced from the native state is located at or near the native site. Simulations of the interaction which give rise to the S3 EPR signal are also presented and discussed. These indicate that lineshape differences occur through small changes in the exchange component of the interaction between the manganese complex and organic radical, probably through minor structural changes between the variously treated samples.

The effects of calcium depletion on the O2-evolving complex in spinach PS II: The S∗1, S∗2 and S∗3 states 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 managanese. 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 managanese 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 A, 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 μ-oxo bridges postulated to occur in native samples. The Mn-Mn shell, found at 2.7 A in native PS II membranes is split in NaCl-treated samples to give a 2.7 A Mn-Mn and 3.0 A Mn-X interaction (X = Mn,C/O/N). Splitting of the 2.7 A shell is most apparent in the higher S-states, S∗3 >S∗2 >S∗1. Although the scatterers at 3.0 A 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 3.33.6 A 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, T1 of the dark stable tyrosine radical YD have also been studied using pulsed EPR. The T1 relaxation times decreased with increasing modified S-state S∗1 >S∗2 >S∗3, indicating oxidation occurring at or near the manganese cluster.

A XANES study of the manganese complex of inhibited PS II membranes indicates manganese redox changes between the modified S1, S2 and S3 states

Abstract Manganese K-edge X-ray spectra have been obtained for Photosystem II samples treated to inhibit oxygen evolution without displacement of manganese. Inhibition treatments included the use of ammonia, acetate and high concentrations of sodium chloride. These treatments affect the binding of calcium and chloride cofactors and result in either a block or slowing of OEC S-state cycling at the S3 → S0 transition. Following each inhibition treatment the S-states show characteristic K-edge energies and EPR spectra. The differences between each type of inhibitory treatment in the K-edge energy for each S-state may result from conformational and/or ligand changes to the manganese complex. However for each of the inhibitory treatments, the K-edge energies showed edge shifts between S-states consistent with manganese oxidation on both the S1 → S2 and S2 → S3 transitions.

Spectroscopic studies of the manganese complex of Photosystem II

Our recent EPR and EXAFS experiments investigating the structure of the oxygen-evolving complex of PS II are discussed. PS II treatments which affect the cofactors calcium and chloride have been used to poise samples in modified forms of the S-states, S1, S2 and S3. X-ray absorption studies indicate a similar overall structure for the manganese complex between treated and native samples although the influence of the treatments and cofactors is observed. Manganese oxidation (or oxidation of a ligand to the manganese cluster) is indicated to occur on each of the transitions S1 →S2 and S2 →S3 in these modified samples. The cluster appears to contain at least two inequivalent Mn-Mn pairs. In the native samples the Mn-Mn distance is 2.7 Å, but in samples where the calcium site is affected, one of the pairs has a 3.0 Å Mn-Mn distance. The intensity of the 3.3/3.6 Å interaction is reduced on sodium chloride treatment (calcium depletion) perhaps indicating calcium binding close to the manganese cluster. From EPR data we also propose that treatments which affect calcium and chloride binding cause a modification of the native S2 state, slow the reduction of Yz• and allow an S3 EPR signal to be observed following illumination. The origin of the S3 EPR signal, a modified S3 or S2 X• where X• is an organic radical of unknown charge, is discussed in relation to the results from the EXAFS studies.

An ENDOR study of structural changes in the environment of the dark stable tyrosine radical, YD, of Photosystem 2 induced by inhibition of the oxygen evolving complex

Abstract The oxygen evolving complex (OEC) of Photosystem II (PS II) may be inhibited without inducing the loss of the manganese cofactor. Methods include depletion of the calcium cofactor by high salt concentration and the addition of ammonium ions at alkaline pH or acetate ions at acidic pH. ENDOR spectra of the dark stable tyrosine radical Y D in PS II have been obtained for samples treated in these ways. These show that the orientation of the tyrosine aromatic ring relative to the attached β methylene protons is different in each case. The effects are specific to the inhibitory treatment employed and have been quantitated. Y D is constrained by its protein environment and as such these orientation effects show that the protein environment around this tyrosine is affected by such inhibitory treatments. Since the distance between the OEC and Y D has previously been estimated at 3–4 nm (Evelo, R.G., Styring, S., Rutherford, A.W. and Hoff, A.J. (1989) Biochim. Biophys. Acta 973, 428–442), the ENDOR data suggest that long-range protein-mediated communication occurs between them. The orientation of Y D is also shown to be sensitive to the removal of the light harvesting complex, LHC II.