Author(s): Yachandra, VK; Glatzel, P; Yano, J; Visser, H; Robblee, JH; Sauer, K; Cramer, SP; Bergmann, U

Acetoxymethyl-1,4-naphthoquinone (2- AcOMeNQ) binds with rapid kinetics and high affinity to the primary quinone QA site of reaction centers from Rhodopseu- domonas capsulata. Binding of 2-AcOMeNQ fully restores electron-transfer activity with kinetics that is similar, but not identical, to that seen with ubiquinone-50. When bound at the QA site, 2-AcOMeNQ preferentially labels the L subunit. This preference suggests that 2-AcOMeNQ labels primarily the region of a quinone-binding site that is close to the first isoprenoid unit of the side chain, which is expected from the location and structure of the reaction region of the molecule. In photosystem II particles from Synechococcus sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of only the 38-kDa polypeptide is sufficiently sensitive to 3-(3,4-dichlorophenyl)- 1,l-dimethylurea (DCMU) to conclude that it is involved in binding quinones on the acceptor side of photosystem II. Although we have not yet identified the 38-kDa protein, its properties suggest that it is the D2 protein. From the DCMU- sensitive labeling and from homologies to functionally impor- tant regions of the bacterial reaction-center subunits, we propose that the 38-kDa protein is intimately involved in binding the cofactors that mediate primary photochemistry.

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, S0through 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-S3and S3-to-S0transitions. 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 S2state 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 absorption spectroscopy has provided important insights into the structure and function of the Mn4Ca 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 Kα 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 0.09Å. The high resolution data obtained from oriented spinach PS II membranes in the S1 state show that there are three di-μ-oxo-bridged manganese-manganese distances of ∼2.7 and ∼2.8Å in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of ∼60° relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the ∼3.2Å manganese-manganese and the ∼3.4Å 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 ∼3.4Å is aligned along the membrane normal, while the ∼3.2Å manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn4Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models.

Detailed information about the orientation of particular Mn-Mn and Mn-Ca vectors in the oxygen evolving complex (OEC) of the Photosystem II in the S-1 state provide a critical starting point for the analysis of the structural changes in the OEC along the catalytic Si-state cycle. The method of olarized range-extended EXAFS is an important technical development, that allows: i) resolution of the 2.7 angstrom and 2.8 angstrom Mn-Mn interactions; ii) resolution of 3.2 angstrom MnMn and 3.4 angstrom Mn-Ca; iii) determination of 2.7 angstrom, 2.8 angstrom, 3.2 angstrom Mn-Mn and 3.4 angstrom Mn-Ca vectors orientation relative to the membrane normal.

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.

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.

Fig. 1. Mn XANES and EXAFS of single crystals of photosystem II as a function of x-ray dose. As the x-ray dose increases, Mn in PS II normally present as Mn4(III2,IV2) is reduced to Mn(II) as seen by the changes in XANES spectra (left bottom). The changes in the corresponding EXAFS spectra (right) show that the three Fourier peaks characteristic of Mn-bridging-oxo, Mn-terminal, and Mn-Mn/Ca interactions (dashed vertical line) are replaced by one Fourier peak characteristic of a Mn(II) environment. A PS II crystal subsequent to x-ray exposure is also shown (left top). The bright green color is from the chlorophyll molecules in PS II and the dark spots show the areas of the crystal used for x-ray diffraction data collection. Damage by X-rays: A Case Study for Metallo-Protein Crystallography

Group and had elected to work on photosynthesis.He became particularly interested in structure function relationships and ended up working with me.Actually, he worked largely on his own!John

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.

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.

The anisotropic g and hyperfine tensors of the Mn di-mu-oxo complex, [Mn-2(III,IV)O-2(phen)(4)](PF6)(3)(CH3CN)-C-., 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)](PF6)(3)(CH3CN)-C-. EPR spectra showed distinct resolved Mn-55 hyperfine lines in all crystal orientations, unlike single-crystal EPR spectra of other Mn-2(III,IV) di-mu-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-crystai 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(x)(III) = \171\ G, A(y)(III) = \176\ G, A(z)(III) = \129\ G, A(x)(IV) = \77\ G, A(y)(IV) = \74\ G, A(z)(IV) = \80\ G.

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.

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures Mn IV 3 O 4 X (X=Cl, F, or OH) that contain a di-μ-oxo and two mono-μ-oxo bridges or Mn IV 2 O 2 X (X=Cl, F, OH, OAc) that contain a di-μ-oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X=O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3 Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.

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 Mn4 cluster and in its internal organization during the accumulation of oxidizing equivalents leading to O2 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.

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.