Calcium EXAFS establishes the Mn-Ca cluster in the oxygen-evolving complex of photosystem II

Abstract

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 A, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is approximately 3.5 A distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster.

Figure 1

Ca EXAFS spectrum in k-space (k³-weighted) from intact Chelex-treated PS II in the S₁ state (—) and the corresponding NH₂OH-treated samples (---). After conversion into k-space and background removal (with a five-domain cubic spline), the spectra from two types of samples are presented. These represent the average of 130 scans from intact samples and 160 scans from inactive samples. Subtle but clear and reproducible differences in phase and amplitudes are visible in the range of k = 6 – 7 Å⁻¹ and k = 9 – 10 Å⁻¹. These differences become more pronounced when contribution from the extraneous Ca not involved in oxygen evolution is subtracted (data not shown). The differences become more obvious in the Fourier transform shown in Fig. 2.

Figure 2

Fourier Transform of Ca EXAFS from Chelex-treated, layered samples with 2 Ca/PS II (k³-weighted, k = 2.5 – 10.5 Å⁻¹). Intact (—) and inactive (---) 2 Ca/PSII samples are shown. These are the FTs of the EXAFS spectra in the previous figure. Apparent distance ‘R’ is less than the actual distance by about 0.5 Å. The peak at ‘R’ < 1 Å is an artifact of incomplete background removal. Fourier peak I consists of nearest-neighbor oxygen atoms while Fourier peak II fits best to Mn scattering and is absent in inactive samples where the Mn cluster is disrupted by NH₂OH treatment. The pattern of peaks can be compared to that in the inset. The Fourier transform magnitudes are in arbitrary units. Inset: Fourier Transform of Sr EXAFS from isotropic Sr-substituted PS II samples (k³-weighted, k = 2.7 – 11.7 Å⁻¹). Apparent distance ‘R’ is less than the actual distance by about 0.5 Å. Fourier peak I consists of nearest-neighbor oxygen atoms while Fourier peak II, which is present in the intact Sr-PS II (—) and absent in inactive samples (---), fits best to Mn scattering atoms. Adapted from an earlier Sr EXAFS study (32).

Figure 3

Model of Ca-binding site of the oxygen-evolving complex in PS II. From the results of the Ca EXAFS studies on PS II, the Ca cofactor is linked by single-O bridging to two Mn. The oxygens can be provided by water, hydroxyl or protein residues (carboxylate, phenolate). Other bridging that may be present (single-O or bidentate 3-atom) or other ligands to Ca are not depicted for clarity. The arrangement shown here is not unique as other placements of the two Mn around the Ca are conceivable.