X-ray spectroscopy of the Mn4Ca cluster in the water-oxidation complex of Photosystem II

Abstract

The water-oxidation complex of Photosystem II (PS II) contains a heteronuclear cluster of 4 Mn atoms and a Ca atom. Ligands to the metal cluster involve bridging O atoms, and O and N atoms from amino acid side-chains of the D1 polypeptide of PS II, with likely additional contributions from water and CP43. Although moderate resolution X-ray diffraction-based structures of PS II have been reported recently, and the location of the Mn4Ca cluster has been identified, the structures are not resolved at the atomic level. X-ray absorption (XAS), emission (XES), resonant inelastic X-ray scattering (RIXS) and extended X-ray absorption fine structure (EXAFS) provide independent and potentially highly accurate sources of structural and oxidation-state information. When combined with polarized X-ray studies of oriented membranes or single-crystals of PS II, a more detailed picture of the cluster and its disposition in PS II is obtained.

Figure 1

Energy level diagrams showing the origin of Kα X-ray absorption near-edge structure (XANES) and Kβ X-ray emission spectra (XES) for elements in the first transition-metal series. Exchange interaction with ligand orbitals leads to a splitting of the Kβ transition, as seen in Figure 4.

Figure 2

Mn K-edge XANES spectra of individual S-states obtained by deconvolution of spectra obtained from PS II samples illuminated with single-turnover 9-ns flashes (Left upper frame). (Left lower frame) S-state XANES difference spectra, multiplied by factor 5 and vertically offset for clarity, are shown for adjacent pairs of states. The dashed vertical line is at the inflection point energy for the S₂-state spectrum. (Right frame) These spectra are the second derivatives of the S-state XANES edge spectra shown in the upper-left portion of the figure.

Figure 3

Mn-EXAFS of the S₁-state for a solution of PS II membranes. Composite peak I with components at 1.8 and 2.0 Å is assigned to O and N ligand atoms joined to Mn. Peak II at 2.7 Å results from the contribution of binuclear Mn di-μ-oxo bridged atoms. Peak III at 3.3–3.4 Å has contributions from both Mn–Mn and Mn–Ca interactions. Apparent distances shown on the horizontal axis must be increased owing to a phase factor associated with the Mn scattering atom.

Figure 4

Mn Kβ X-ray emission spectra (XES) of individual S-states produced by the method described in Figure 2. The Mn Kβ XES spectrum (upper left) shows the split emission band mentioned in the Figure 1 caption. Below it are expanded segments showing the Kβ_1,3 peak for individual S-states, normalized and overplotted for comparison purposes. Black and gray lettering relates to the corresponding curves for each of the three pairs. S₂ is shifted to lower energy relative to S₁ and S₀ is at higher energy relative to S₁; S₂ and S₃ are essentially the same. These comparisons are more easily seen in the XES difference spectra (right frame).

Figure 5

(a) A two-dimensional plot showing the resonant inelastic X-ray spectrum from a Mn(II)acetylacetonate complex. X-axis is the excitation energy across the 1s–3d energy range of the spectrum. The 1s–3d K-edge spectrum is plotted in the back of the 2-dimensional spectrum for reference. Y-axis is the difference between the excitation and emission energy. The deconvolution of the 1s–3d spectrum as shown in the 2D plot is significantly better than one can obtain from a simple K-edge spectrum. An integration of the 2D plot parallel to the y-axis yields L-edge like spectra, the more intense feature at 640 eV corresponds to transitions to J=3/2 like states (L₃ edges) and transitions to 655 eV correspond to J=1/2 final states (L₂ edges). Integrations parallel to the energy transfer axis sort the spectrum according to the final state. (b) Contour plots of the 1s2p_3/2 RIXS planes for three molecular complexes Mn^II(acac)₂(H₂O)₂, Mn^III(acac)₃, and Mn^IV(sal)₂(bipy) and PS II in the S₁- and S₂-state. One axis is the excitation energy and the other is the energy transfer axis. The L-edge like spectra are along the energy transfer axis and the 1s to 3d transition is along the excitation energy. The assignment of Mn(III₂, IV₂) for the S₁ state is apparent in these spectra (Glatzel et al. 2004).

Figure 6

Cluster arrangements of 4 Mn atoms bridged by O atoms and exhibiting three di-μ-oxo bridged Mn–Mn pairs.

Figure 7

Ca-EXAFS of Photosystem II membranes in the S₁-state. Sample containing approx. 2 Ca per PS II (solid curve). Sample treated with NH₂OH to remove Mn (broken curve). The disappearance of the peak labeled II in the EXAFS of the NH₂OH-treated sample indicates the presence of a Mn–Ca interaction at 3.3 Å.

Figure 8

Mn-EXAFS dichroism of PS II membranes in the S₁-state oriented by layering, as illustrated at the right. Samples oriented with the membrane normals at 15° (solid curve) and 75° (broken curve) to the electric vector of the incident X-ray beam. Peaks labeled I, II and III correspond to those described in the caption to Figure 3. The presence of dichroism is indicated by the different relative peak heights for measurements at the different angles.

Figure 9

Sr-EXAFS dichroism of PS II membranes from which Ca has been extracted and reconstituted with Sr. Oriented membranes and dichroism measured as described in the caption to Figure 8. Spectra for samples oriented at 10 and 80° are shown (left frame). The polar plot (right frame) summarizes the normalized intensity of peak II for samples measured at a variety of angles. (Measurements shown in the first quadrant are repeated in the other three quadrants to show the expected symmetry. The solid curve is fit to the data.) Results indicate that the Sr–Mn vector(s) at 3.5 Å are oriented close to the membrane normal.

Figure 10

Cluster arrangements of 4 Mn and 1 Ca bridged by O atoms and consistent with the Mn- and Sr-EXAFS dichroism.

Figure 11

Single-crystal Mn-EXAFS of [Mn₂(III,IV)O₂(phen)₄](PF₆)₃CH₃CN, where phen = 1,10-phenanthroline. A schematic of the molecular structure is shown together with projections onto the ac- and bc-planes of these orthorhombic crystals (upper frame). Mn EXAFS measured with the a- (dash-dot curve), b- (solid curve) and c-axes (dotted curve) aligned with the E-vector of the incident X-ray beam are shown (lower frame). The strong polarization of the 2.7 Å Mn–Mn peak along the c-axis is consistent with the crystal structure.