Structural changes in the Mn4Ca cluster and the mechanism of photosynthetic water splitting.

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn-Mn and Ca(Sr)-Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn-Mn and Ca(Sr)-Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn-Mn and Mn-Ca(Sr) atoms in the O-O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.

X-Ray spectroscopy of the photosynthetic oxygen-evolving complex.

Water oxidation to dioxygen in photosynthesis is catalyzed by a Mn(4)Ca cluster with O bridging in Photosystem II (PS II) of plants, algae and cyanobacteria. A variety of spectroscopic methods have been applied to analyzing the participation of the complex. X-ray spectroscopy is particularly useful because it is element-specific, and because it can reveal important structural features of the complex with high accuracy and identify the participation of Mn in the redox chemistry. Following a brief history of the application of X-ray spectroscopy to PS II, an overview of newer results will be presented and a description of the present state of our knowledge based on this approach.

The water-oxidation complex in photosynthesis.

Studies of the photosynthetic water-oxidation complex of photosystem II (PS II) using spectroscopic techniques have characterized not only important structural features, but also changes that occur in oxidation state of the Mn(4) cluster and in its internal organization during the accumulation of oxidizing equivalents leading to O(2) formation. Combining this spectroscopic information with that from the recently published relatively low-resolution X-ray diffraction studies, we have succeeded in limiting the range of likely cluster arrangements. This evidence strongly supports several options proposed earlier by DeRose et al. [J. Am. Chem. Soc. 116 (1994) 5239] and these can be further narrowed using compatibility with electron paramagnetic resonance (EPR) data.

Structural Change of the Mn Cluster during the S2→S3 State Transition of the Oxygen-Evolving Complex of Photosystem II. Does It Reflect the Onset of Water/Substrate Oxidation? Determination by Mn X-ray Absorption Spectroscopy.

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 (S0-S4) 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 S1 and S2 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 S1 and S2 states. This report describes the application of X-ray absorption spectroscopy to S3 samples created under physiological conditions with saturating flash illumination. Significant changes are observed in the Mn-Mn distances in the S3 state compared to the S1 and the S2 states. The two 2.7 Å Mn-Mn distances that characterize the di-µ-oxo centers in the S1 and S2 states are lengthened to ~2.8 and 3.0 Å in the S3 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 S3 state. Mn-centered oxidation is evident during the S0→S1 and S1→S2 transitions. We propose that the changes in Mn-Mn distances during the S2→S3 transition are the result of ligand or water oxidation, leading to the formation of an oxyl radical intermediate formed at a bridging or terminal position. The reaction of the oxyl radical with OH-, H2O, or an oxo group during the subsequent S state conversion is proposed to lead to the formation of the O-O bond. Models that can account for changes in the Mn-Mn distances in the S3 state and the implications for the mechanism of water oxidation are discussed.

Comparison of the Manganese Cluster in Oxygen-Evolving Photosystem II with Distorted Cubane Manganese Compounds through X-ray Absorption Spectroscopy.

X-ray absorption spectroscopy has been employed to assess the degree of similarity between the oxygen-evolving complex (OEC) in photosystem II (PS II) and a family of synthetic manganese complexes containing the distorted cubane [Mn(4)O(3)X] core (X = benzoate, acetate, methoxide, hydroxide, azide, fluoride, chloride, or bromide). These [Mn(4)(&mgr;(3)-O)(3)(&mgr;(3)-X)] cubanes possess C(3)(v)() symmetry except for the X = benzoate species, which is slightly more distorted with only C(s)() symmetry. In addition, Mn(4)O(3)Cl complexes containing three or six terminal Cl ligands at three of the Mn were included in this study. The Mn K-edge X-ray absorption near edge structure (XANES) from the oxygen-ligated complexes begin to resemble general features of the PS II (S(1) state) spectrum, although the second derivatives are distinct from those in PS II. The extended X-ray absorption fine structure (EXAFS) of these Mn compounds also displays superficial resemblance to that of PS II, but major differences emerge on closer examination of the phases and amplitudes. The most obvious distinction is the smaller magnitude of the Fourier transform (FT) of the PS II EXAFS compared to the FTs from the distorted cubanes. Curve fitting of the Mn EXAFS spectra verifies the known core structures of the Mn cubanes, and shows that the number of the crucial 2.7 and 3.3 Å Mn-Mn distances differs from that observed in the OEC. The EXAFS method detects small changes in the core structures as X is varied in this series, and serves to exclude the distorted cubane of C(3)(v)() symmetry as a topological model for the Mn catalytic cluster of the OEC. Instead, the method shows that even more distortion of the cubane framework, altering the ratio of the Mn-Mn distances, is required to resemble the Mn cluster in PS II.

High-resolution structure of the photosynthetic Mn4Ca catalyst from X-ray spectroscopy.

The application of high-resolution X-ray spectroscopy methods to study the photosynthetic water oxidizing complex, which contains a unique hetero-nuclear catalytic Mn4Ca cluster, is described. Issues of X-ray damage, especially at the metal sites in the Mn4Ca cluster, are discussed. The structure of the Mn4Ca catalyst at high resolution, which has so far eluded attempts of determination by X-ray diffraction, X-ray absorption fine structure (EXAFS) and other spectroscopic techniques, has been addressed using polarized EXAFS techniques applied to oriented photosystem II (PSII) membrane preparations and PSII single crystals. A review of how the resolution of traditional EXAFS techniques can be improved, using methods such as range-extended EXAFS, is presented, and the changes that occur in the structure of the cluster as it advances through the catalytic cycle are described. X-ray absorption and emission techniques (XANES and Kbeta emission) have been used earlier to determine the oxidation states of the Mn4Ca cluster, and in this report we review the use of X-ray resonant Raman spectroscopy to understand the electronic structure of the Mn4Ca cluster as it cycles through the intermediate S-states.

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

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.

Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster.

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.

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

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.

High-resolution Mn EXAFS of the oxygen-evolving complex in photosystem II: structural implications for the Mn4Ca cluster.

The biological generation of oxygen by the oxygen-evolving complex in photosystem II (PS II) is one of nature's most important reactions. The recent X-ray crystal structures, while limited by resolutions of 3.2-3.5 A, have located the electron density associated with the Mn4Ca cluster within the multiprotein PS II complex. Detailed structures critically depend on input from spectroscopic techniques, such as EXAFS and EPR/ENDOR, as the XRD resolution does not allow for accurate determination of the position of Mn/Ca or the bridging and terminal ligand atoms. The number and distances of Mn-Mn/Ca/ligand interactions determined from EXAFS provide important constraints for the structure of the Mn4Ca cluster. Here, we present data from a high-resolution EXAFS method using a novel multicrystal monochromator that show three short Mn-Mn distances between 2.7 and 2.8 A and, hence, the presence of three di-mu-oxo-bridged units in the Mn4Ca cluster. This result imposes clear limitations on the proposed structures based on spectroscopic and diffraction data and provides input for refining such structures.

X-ray damage to the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography.

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca 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 Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca 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.

A possible evolutionary origin for the Mn4 cluster of the photosynthetic water oxidation complex from natural MnO2 precipitates in the early ocean.

The photosynthetic water oxidation complex consists of a cluster of four Mn atoms bridged by O atoms, associated with Ca2+ and Cl-, and incorporated into protein. The structure is similar in higher plants and algae, as well as in cyanobacteria of more ancient lineage, dating back more than 2.5 billion years ago on Earth. It has been proposed that the proto-enzyme derived from a component of a natural early marine manganese precipitate that contained a CaMn4O9 cluster. A variety of MnO2 minerals are found in nature. Three major classes are spinels, sheet-like layered structures, and three-dimensional networks that contain parallel tunnels. These relatively open structures readily incorporate cations (Na+, Li+, Mg2+, Ca2+, Ba2+, H+, and even Mn2+) and water. The minerals have different ratios of Mn(III) and Mn(IV) octahedrally coordinated to oxygens. Using x-ray spectroscopy we compare the chemical structures of Mn in the minerals with what is known about the arrangement in the water oxidation complex to define the parameters of a structural model for the photosynthetic catalytic site. This comparison provides for the structural model a set of candidate Mn(4) clusters-some previously proposed and considered and others entirely novel.

Single crystal X- and Q-band EPR spectroscopy of a binuclear Mn(2)(III,IV) complex relevant to the oxygen-evolving complex of photosystem II.

The anisotropic g and hyperfine tensors of the Mn di-micro-oxo complex, [Mn(2)(III,IV)O(2)(phen)(4)](PF(6))(3).CH(3)CN, were derived by single-crystal EPR measurements at X- and Q-band frequencies. This is the first simulation of EPR parameters from single-crystal EPR spectra for multinuclear Mn complexes, which are of importance in several metalloenzymes; one of them is the oxygen-evolving complex in photosystem II (PS II). Single-crystal [Mn(2)(III,IV)O(2)(phen)(4)](PF(6))(3).CH(3)CN EPR spectra showed distinct resolved (55)Mn hyperfine lines in all crystal orientations, unlike single-crystal EPR spectra of other Mn(2)(III,IV) di-micro-oxo bridged complexes. We measured the EPR spectra in the crystal ab- and bc-planes, and from these spectra we obtained the EPR spectra of the complex along the unique a-, b-, and c-axes of the crystal. The crystal orientation was determined by X-ray diffraction and single-crystal EXAFS (Extended X-ray Absorption Fine Structure) measurements. In this complex, the three crystallographic axes, a, b, and c, are parallel or nearly parallel to the principal molecular axes of Mn(2)(III,IV)O(2)(phen)(4) as shown in the crystallographic data by Stebler et al. (Inorg. Chem. 1986, 25, 4743). This direct relation together with the resolved hyperfine lines significantly simplified the simulation of single-crystal spectra in the three principal directions due to the reduction of free parameters and, thus, allowed us to define the magnetic g and A tensors of the molecule with a high degree of reliability. These parameters were subsequently used to generate the solution EPR spectra at both X- and Q-bands with excellent agreement. The anisotropic g and hyperfine tensors determined by the simulation of the X- and Q-band single-crystal and solution EPR spectra are as follows: g(x) = 1.9887, g(y) = 1.9957, g(z) = 1.9775, and hyperfine coupling constants are A(III)(x) = |171| G, A(III)(y) = |176| G, A(III)(z) = |129| G, A(IV)(x) = |77| G, A(IV)(y) = |74| G, A(IV)(z) = |80| G.

Structural Effects of Calcium Depletion on the Manganese Cluster of Photosystem II: Determination by X-ray Absorption Spectroscopy.

The structural consequences of calcium depletion of Photosystem II (PS II) by treatment at pH 3.0 in the presence of citrate has been determined by Mn K-edge X-ray absorption spectroscopy. X-ray absorption edge spectroscopy of Ca-depleted samples in the S1', S2', and S3' oxidation states reveals that there is Mn oxidation on the S1'-S2' transition, although no evidence of Mn oxidation was found for the S2'-S3' transition. This result is in keeping with the results from EPR studies where it has been found that the species oxidized to give the S3' broad radical signal found in Ca-depleted PS II is tyrosine Yz. The S2' state can be prepared by two methods: illumination followed by dark adaptation and illumination in the presence of DCMU to limit to one turnover. Illumination followed by dark adaptation was found to yield a lower Mn K-edge inflection-point energy than illumination with DCMU, indicating vulnerability to reduction of the Mn complex, even over the relatively short times used for dark adaptation (~15 min). EXAFS measurements of Ca-depleted samples in the three modified S states (referred to here as S' states) reveals that the Fourier peak due to scatterers at ~3.3 Å from Mn is strongly diminished, consistent with our previous assignment of a Ca-scattering contribution at this distance. Even after Ca depletion, there is still significant amplitude in the third peak, further supporting our conclusions from earlier studies that the third peak in native samples is comprised of both Mn and Ca scattering. The Mn-Mn contributions making up the second Fourier peak at ~2.7 Å are largely undisturbed by Ca-depletion, but there is some evidence that S1'-state samples contain significant amounts of reduced Mn(II), which is then photooxidized in the preparation of higher S' states.

FTIR spectra and normal-mode analysis of a tetranuclear manganese adamantane-like complex in two electrochemically prepared oxidation states: relevance to the oxygen-evolving complex of photosystem II.

The IR spectra and normal-mode analysis of the adamantane-like compound [Mn(4)O(6)(bpea)(4)](n+) (bpea = N,N-bis(2-pyridylmethyl)ethylamine) in two oxidation states, Mn(IV)(4) and Mn(III)Mn(IV)(3), that are relevant to the oxygen-evolving complex of photosystem II are presented. Mn-O vibrational modes are identified with isotopic exchange, (16)O-->(18)O, of the mono-micro-oxo bridging atoms in the complex. IR spectra of the Mn(III)Mn(IV)(3) species are obtained by electrochemical reduction of the Mn(IV)(4) species using a spectroelectrochemical cell, based on attenuated total reflection [Visser, H.; et al. Anal. Chem. 2001, 73, 4374-4378]. A novel method of subtraction is used to reduce background contributions from solvent and ligand modes, and the difference and double-difference spectra are used in identifying Mn-O bridging modes that are sensitive to oxidation state change. Two strong IR bands are observed for the Mn(IV)(4) species at 745 and 707 cm(-1), and a weaker band is observed at 510 cm(-1). Upon reduction, the Mn(III)Mn(IV)(3) species exhibits two strong IR bands at 745 and 680 cm(-1), and several weaker bands are observed in the 510-425 cm(-1) range. A normal-mode analysis is performed to assign all the relevant bridging modes in the oxidized Mn(IV)(4) and reduced Mn(III)Mn(IV)(3) species. The calculated force constants for the Mn(IV)(4) species are f(r)(IV)= 3.15 mdyn/A, f(rOr) = 0.55 mdyn/A, and f(rMnr) = 0.20 mdyn/A. The force constants for the Mn(III)Mn(IV)(3) species are f(r)(IV)= 3.10 mdyn/A, f(r)(III)= 2.45 mdyn/A, f(rOr) = 0.40 mdyn/A, and f(rMnr) = 0.15 mdyn/A. This study provides insights for the identification of Mn-O modes in the IR spectra of the photosynthetic oxygen-evolving complex during its catalytic cycle.

Strontium EXAFS Reveals the Proximity of Calcium to the Manganese Cluster of Oxygen-Evolving Photosystem II.

The oxygen-evolving complex of Photosystem II (PS II) in green plants and algae contains a cluster of four manganese atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. A key unresolved question is whether Ca is close to the Mn cluster, within about 3.5 Å. To further test and verify this finding, we substituted strontium for Ca and probed from the Sr point-of-view for any nearby Mn. Sr has been shown to replace Ca and still maintain enzyme activity (about 40% of normal rate). The extended X-ray absorption fine structure (EXAFS) of Sr-PS II probes the local environment around the Sr cofactor to detect any nearby Mn. We focused on the functional Sr by removing nonessential, loosely bound Sr in the protein environment. For comparison, an inactive sample was prepared by treating the intact PS II with hydroxylamine to disrupt the Mn cluster and to produce nonfunctional enzyme. Sr EXAFS results indicate major differences in the phase and amplitude between the functional (intact) and nonfunctional (NH2OH-treated) samples. In intact samples, the Fourier transform of the Sr EXAFS shows a peak that is missing in inactive samples. This Fourier peak II is best simulated by two Mn neighbors at a distance of 3.5 Å. Thus, with X-ray absorption studies on Sr-reconstituted PS II, we confirm the proximity of Ca (Sr) cofactor to the Mn cluster and show that the active site is a Mn-Ca heteronuclear cluster.

Orientation of calcium in the Mn4Ca cluster of the oxygen-evolving complex determined using polarized strontium EXAFS of photosystem II membranes.

The oxygen-evolving complex of photosystem II (PS II) in green plants and algae contains a cluster of four Mn atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. The current study using polarized Sr EXAFS on oriented Sr-reactivated samples shows that Fourier peak II, which fits best to Mn at 3.5 A rather than lighter atoms (C, N, O, or Cl), is dichroic, with a larger magnitude at 10 degrees (angle between the PS II membrane normal and the X-ray electric field vector) and a smaller magnitude at 80 degrees . Analysis of the dichroism of the Sr EXAFS yields a lower and upper limit of 0 degrees and 23 degrees for the average angle between the Sr-Mn vectors and the membrane normal and an isotropic coordination number (number of Mn neighbors to Sr) of 1 or 2 for these layered PS II samples. The results confirm the contention that Ca (Sr) is proximal to the Mn cluster and lead to refined working models of the heteronuclear Mn(4)Ca cluster of the oxygen-evolving complex in PS II.

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

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.