The oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn 4 Ca cluster, the structure of which remains elusive. Polarized extended x-ray absorption fine structure (EXAFS) measurements on PSII single crystals constrain the Mn 4 Ca cluster geometry to a set of three similar high-resolution structures. Combining polarized EXAFS and x-ray diffraction data, the cluster was placed within PSII, taking into account the overall trend of the electron density of the metal site and the putative ligands. The structure of the cluster from the present study is unlike either the 3.0 or 3.5 angstrom-resolution x-ray structures or other previously proposed models.Oxygen, which makes up about 20% of Earth's atmosphere, comes mostly from photosynthesis that occurs in cyanobacteria, green algae, and higher plants (1). These organisms have within photosystem II (PSII) an oxygen-evolving complex (OEC), in which the energy of sunlight is used to oxidize water to molecular oxygen. The heart of the OEC is a cluster of four Mn atoms and one Ca atom (Mn 4 Ca) connected by mono-μ-oxo, di-μ-oxo, and/or hydroxo bridges. The specific protein environment and one chloride ion are also essential for the water-splitting activity (1). During the oxidation of water, the OEC cycles through five different oxidation states, which are known as S i states (where i ranges from 0 to 4), that couple the one-electron photochemistry of the PSII reaction center with the fourelectron chemistry of water oxidation (2).The structure of the Mn 4 Ca cluster and its role in the mechanism of water oxidation have been investigated with the use of spectroscopic methods (1), especially electron ‡ To whom correspondence should be addressed. messinger@mpi-muelheim. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript paramagnetic resonance and electron nuclear double-resonance spectroscopy (3-9), x-ray spectroscopy (10), and Fourier transform infrared (FTIR) spectroscopy (11). In addition, recent x-ray diffraction (XRD) studies of single crystals of PSII provide critical information about its structure at 3.8 to 3.0 Å resolution (12-16). However, even XRD data of the highest resolution presently available are insufficient to accurately determine the positions of Mn, Ca, and the bridging and terminal ligands. This is reflected by the differences in the placement of the metal ions and putative ligands in the 3.0 (16) and 3.5 Å (14) structures. Furthermore, at the x-ray dose and temperature used in the XRD studies, the geometry of the Mn 4 Ca cluster is disrupted, initiated by the rapid reduction of Mn(III) and Mn(IV) present in the dark-stable S 1 state to Mn(II), as shown by Mn x-ray absorption near-edge structure (XANES) studies and Mn x-ray absorption fine structure (EXAFS) studies of PSII single crystals (17).EXAFS experiments with PSII require a substantially lower x-ray dose than XRD measurements (17), and the onset of radiation damage can be precisely determined and controlled by monitoring the Mn K-edge positio...

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn4Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn4(III2,IV2) oxo-bridged Mn4Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.manganese ͉ oxygen evolution ͉ water oxidation ͉ x-ray spectroscopy

In the photosynthetic evolution of oxygen, water oxidation occurs at a catalytic site that includes four manganese atoms together with the essential cofactors, the calcium and chlorine ions. A structural model and a determination of the manganese oxidation states based on x-ray absorption spectroscopy are presented. The salient features, in both higher plants and cyanobacteria, are a pair of di-mu-oxo bridged manganese binuclear clusters linked by a mono-mu-oxo bridge, one proximal calcium atom, and one halide. In dark-adapted samples, manganese occurs in oxidation states (III) and (IV). Data from oriented membranes display distinct dichroism, precluding highly symmetrical structures for the manganese complex.

Intense femtosecond X-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) of microcrystals of Photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD/XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.

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The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flashinduced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR)

The oxygen-evolving complex of Photosystem II in plants and cyanobacteria catalyzes the oxidation of two water molecules to one molecule of dioxygen. A tetranuclear Mn complex is believed to cycle through five intermediate states (S 0 -S 4 ) to couple the four-electron oxidation of water with the one-electron photochemistry occurring at the Photosystem II reaction center. We have used X-ray absorption spectroscopy to study the local structure of the Mn complex and have proposed a model for it, based on studies of the Mn K-edges and the extended X-ray absorption fine structure of the S 1 and S 2 states. The proposed model consists of two di-μ-oxo-bridged binuclear Mn units with Mn-Mn distances of ~2.7 Å that are linked to each other by a mono-μ-oxo bridge with a Mn-Mn separation of ~3.3 Å. The Mn-Mn distances are invariant in the native S 1 and S 2 states. This report describes the application of X-ray absorption spectroscopy to S 3 samples created under physiological conditions with saturating flash illumination. Significant changes are observed in the Mn-Mn distances in the S 3 state compared to the S 1 and the S 2 states. The two 2.7 Å Mn-Mn distances that characterize the di-μ-oxo centers in the S 1 and S 2 states are lengthened to ~2.8 and 3.0 Å in the S 3 state, respectively. The 3.3 Å Mn-Mn and Mn-Ca distances also increase by 0.04-0.2 Å. These changes in Mn-Mn distances are interpreted as consequences of the onset of substrate/water oxidation in the S 3 state. Mn-centered oxidation is evident during the S 0 →S 1 and S 1 →S 2 transitions. We propose that the changes in Mn-Mn distances during the S 2 →S 3 transition are the result of ligand or water oxidation, leading to the Supporting Information Available: E-space S 3 state EXAFS spectrum; the data in k-space and the background that was removed to reduce the low-frequency contributions that show up as peaks at <1 Å in the Fourier transform; and the Fourier isolate of the k-space S 3 spectrum, shown overplotted on the S 3 EXAFS spectrum (PDF). This material is available free of charge via the Internet at http:// pubs.acs.org. NIH Public Access

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and free from radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract statistically significant high-resolution signals from fewer diffraction measurements.