Structure and orientation of the Mn4Ca cluster in plant photosystem II membranes studied by polarized range-extended x-ray absorption spectroscopy

Abstract

X-ray absorption spectroscopy has provided important insights into the structure and function of the Mn(4)Ca cluster in the oxygen-evolving complex of Photosystem II (PS II). The range of manganese extended x-ray absorption fine structure data collected from PS II until now has been, however, limited by the presence of iron in PS II. Using a crystal spectrometer with high energy resolution to detect solely the manganese Kalpha fluorescence, we are able to extend the extended x-ray absorption fine structure range beyond the onset of the iron absorption edge. This results in improvement in resolution of the manganese-backscatterer distances in PS II from 0.14 to 0.09A(.) The high resolution data obtained from oriented spinach PS II membranes in the S(1) state show that there are three di-mu-oxo-bridged manganese-manganese distances of approximately 2.7 and approximately 2.8A in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of approximately 60 degrees relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the approximately 3.2A manganese-manganese and the approximately 3.4A manganese-calcium interactions in oriented PS II samples and determine their orientation relative to the membrane normal. The average of the manganese-calcium vectors at approximately 3.4A is aligned along the membrane normal, while the approximately 3.2A manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn(4)Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models.

FIGURE 1. Range-extended x-ray absorption spectroscopy

Left, x-ray fluorescence of manganese and iron; Above, manganese Kα₁ and Kα₂ fluorescence peaks, with natural line width of ~5 eV, split by 11 eV. The multicrystal monochromator with 1-eV resolution is tuned to the manganese Kα₁ peak. Below, fluorescence peaks of manganese and iron as detected using germanium detector. The fluorescence peaks are convoluted with the electronic window resolution of 150–200 eV of the germanium detector. This method of detection cannot resolve manganese Kα₁ and Kα₂ fluorescence peaks. Note different energy scales for the schemes shown above and below. Iron is an obligatory element in functional PS II complexes. Right, comparison of the PS II manganese K-edge EXAFS spectrum from an S₁ state PS II sample obtained with a traditional 30-element energy-discriminating germanium detector with a spectrum collected using the high resolution crystal monochromator. Use of the high resolution detector eliminates the interference of iron and removes the limit of the energy range for manganese EXAFS data collection.

FIGURE 2. Manganese K-edge spectra of oriented PS II membranes

Top, S₁ state manganese K-edge x-ray spectra of oriented PS II membranes. The membrane normal of the PS II samples was oriented at 15° (solid line) or 75° (dashed line) with respect to the x-ray e-vector during data acquisition. Bottom, the corresponding second derivatives of the manganese K-edge spectra shown above.

FIGURE 3. Manganese K-edge EXAFS spectra of oriented PS II membranes

Manganese K-edge EXAFS spectra (k³-weighted) from oriented PS II membrane samples in the S₁ state obtained with a high resolution spectrometer (range-extended EXAFS) at orientations of 15° (solid line) and 75° (dashed line) of the sample normal with respect to the x-ray e-vector. The dashed line at k = 11.5 Å⁻¹ denotes the spectral limit of a conventional EXAFS experiment due to the iron edge. The range-extended EXAFS method allows data collection above the iron edge.

FIGURE 4. Fourier transforms of the EXAFS spectra from oriented PS II membranes

A, FT of manganese K-edge EXAFS spectra (Fig. 3) from oriented PS II membrane samples in the S₁ state obtained with a high resolution spectrometer (range-extended EXAFS) at orientations of 15° (solid line) and 75° (dashed line) of the membrane normal with respect to the x-ray e-vector. The k range was 3.5–15.2 Å⁻¹. Fourier peaks in A and B appear at an apparent distance R′ that is shorter than the actual distance R by ~0.5 Å due to a phase shift. B, Fourier transforms of the same data as in A, but the k range was truncated at 11.5 Å⁻¹ for comparison with earlier published polarized EXAFS data obtained with conventional EXAFS. C, same as in A, but a phase correction was done using the EXAFSPAK suite of programs by Drs. Graham George and Ingrid Pickering (Stanford Synchrotron Radiation Laboratory), which results in the conversion of the apparent distance R′ into an approximate real distance R.

FIGURE 5. Fourier isolates of peak II and III from oriented PS II membranes

Fourier isolates of peak II (top) and peak III (bottom) of one-dimensionally oriented PS II samples in the S₁ state with the membrane normal at 15° and 75° with respect to the x-ray e-vector. The data are k³-weighted from 3.5 to 15.2 Å⁻¹ (see Fig. 4A). For the back transform the individual Fourier peaks II and III were isolated by applying a Hamming window to the first and last 15% of the chosen range, leaving the middle 70% untouched. For typical isolates the range was ~0.7 Å for peak II and ~0.8–0.9 Å for peak III.

FIGURE 6. Linear plot of polarized manganese EXAFS data from Fourier peak II

Linear plots of the x-ray absorption dichroism of Peak II for oriented PS II samples in the S₁ state. The N_app values (solid squares) are derived from two-shell curve fits of FT peak II (Fig. 4A and Table 1) and are plotted against 3cos²θ−1 (see supplemental Equation S6). Best fits are shown for the different manganese-manganese vectors as solid lines. Additional third points (open squares) were obtained from extended EXAFS measurements of isotropic PS II S₁ in solution (see supplemental Table S1). Fits with three data points including the isotropic values (dashed lines) are very close to those obtained using only oriented sample data.

FIGURE 7. Linear plot of polarized manganese EXAFS data from Fourier peak III

Linear plots of the x-ray absorption dichroism of the manganese-manganese (3.20 ± 0.02 Å) (black) and manganese-calcium (3.40 ± 0.02 Å) (gray) vectors for oriented PS II samples in the S₁ state. The N_app values were derived from one-shell fits (solid squares) of FT peak III (Fig. 4A and Table 2) and are plotted with respect to 3cos²θ−1 (estimation of N_app for manganese-manganese vector at θ = 15° and for manganese-calcium vector at θ = 75° is described in the text). The best fits of N_app versus θ to supplemental Equation S6 are shown for the manganese-manganese and manganese-calcium vectors (solid line) taking into account the experimentally determined mosaic spread of Ω = 20°. Additional third points (open squares) were obtained from range-extended EXAFS measurements of PS II S₁ solution (see supplemental Table S2). Fits with three data points including the isotropic values (dashed lines) are close to that obtained using only oriented sample data. The results from Figs. 6 and 7 and the polar plots in the supporting information are summarized in Table 3.

FIGURE 8. Cluster models compatible with polarized range-extended EXAFS

A, models for the Mn₄Ca cluster compatible with the range-extended manganese EXAFS data with three short 2.7–2.8 Å manganese-manganese distances and one longer manganese-manganese distance at 3.2 Å. B, Mn₄ models developed from Fig. 8A topological core structures and their proposed orientation relative to the membrane normal consistent with polarized range-extended EXAFS (Table 3). Note that in the membrane plane there is a rotational ambiguity which is always present for one-dimensionally oriented samples such as layered membranes. We emphasize that there may be other models that can be tested in this manner (this would include structural and optical isomers of listed models). C, the orientation of the average manganese-calcium vector in relation to the 3.2 Å manganese-manganese vector. The cones represent a range for the average manganese-calcium vector(s) along the membrane normal (~18°), and the 3.2 Å manganese-manganese vector toward the membrane plane (~20°), respectively.