Structure of the Photosynthetic Mn₄Ca Cluster Using X-ray Spectroscopy

Abstract

Single crystals of Photosystem II (PSII) isolated from thermophilic cyanobacteria have been studied by X-ray diffraction (XRD) with resolutions between 3 and 3.8 Å (Ferreira et al. 2004; Loll et al. 2005). These studies have localized electron density associated with the wateroxidizing Mn₄Ca cluster within the large complex of PSII peptides, but the limited resolution is short of what is needed to place individual metal atoms precisely in the cluster. Examination of the orientation dependence of the EXAFS of single crystals of PSII can provide structural information about the Mn sites at a resolution higher than that is presently available from single-crystal X-ray diffraction. We have successfully collected single crystal XANES and EXAFS data from the native S₁ state with the X-ray e-vector parallel to the a, b, and c axes of the crystal, under non-damaging conditions by monitoring the Mn K-edge for any X-ray induced Mn reduction. The EXAFS spectra show that the Fourier peaks are clearly dichroic, demonstrating an asymmetric Mn cluster. We have used the EXAFS dichroism to evaluate the Mn cluster geometry. Three Mn₄Ca models which satisfy the trend of EXAFS dichroism were further fit into the ligand environment obtained from XRD, in order to discriminate between the several symmetry-related orientations which arise from the crystal symmetry. Furthermore, single crystals in the S1 state were illuminated either by continuous illumination or by laser flashes to create intermediate S-states (S₂ and S₃). Polarized XANES and EXAFS spectra from these crystals show unique orientational dependence. Additionally, a review of how the resolution of traditional EXAFS techniques can be improved, using methods such as rangeextended EXAFS, is presented.