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The light-induced oxidation of water to O is catalyzed by a four-manganese atom cluster associated with Photosystem II (PS II). This chapter summarizes ongoing investigations of the oxidation state, the structure and the associated cofactors calcium and chloride of the catalytic Mn cluster using X-ray and electron paramagnetic resonance (EPR) spectroscopy. Manganese K-edge X-ray spectroscopy, Kβ X-ray emission spectroscopy (XES), and extended X-ray absorption fine structure (EXAFS) studies have not only determined the oxidation states and structural features, but also changes that occur in oxidation state of the Mn cluster and in its structural organization during the accumulation of oxidizing equivalents leading to O formation. Combining X-ray spectroscopy information with X-ray diffraction studies, and consistent with the available EPR data, we have succeeded in limiting the range of likely structures of the Mn cluster. EXAFS studies at the strontium and calcium K-edges have provided evidence that the catalytic center is a Mn/Ca heteronuclear complex. Based on the X-ray spectroscopy data, models for the structure and a mechanism for O evolution are presented.

Water oxidation in Photosystem II (PS II) takes place under severe constraints. Because little surplus free energy is available, the midpoint potential of the (Mn) cluster must be tightly controlled to prevent it from increasing substantially as oxidizing equivalents are accumulated. In addition, the release of toxic, highly reactive intermediates of water oxidation must be minimized. To operate under these constraints, the reactivity of the (Mn)-Ca-Y complex is carefully controlled by its protein environment. This chapter describes the site-directed mutagenesis studies that have been undertaken to identify the amino acid residues that are responsible for this control. These residues include those that ligate the Mn and Ca ions and those that influence the electron and proton transfer reactions of Y and the (Mn)-Ca metal cluster. Most characterizations relied initially on non-invasive methods that were conducted in vivo. With improved methods for purifying PS II, characterizations increasingly involve newer developments in magnetic resonance, vibrational, and optical absorption spectroscopy. On the basis of the mutagenesis studies that are described in this chapter, possible ligands of the (Mn) cluster were proposed to include D1-Asp170, D1-His332, D1-Glu333, D1-His337 and D1-Asp342, plus the carboxyl-terminus of the D1 polypeptide at D1-Ala344. Possible ligands of the Ca ion were proposed to include D1-Asp59, D1-Asp61 and D1-Asp342. In addition, D1-Glu189 was proposed to participate in a network of hydrogen bonds that facilitates electron transfer from the (Mn) cluster to Y during the higher S state transitions and D1-His190 was proposed to serve as the proton acceptor for Y. The recent 3.2 Å and 3.5 Å X-ray crystallographic structural models of PS II support some of these proposals, but conflict with others, most notably the ligation of Ca and the roles of D1-Glu189 and, in the 3.5 Å structural model, the role of the carboxyl-terminus of the D1 polypeptide. Two surprising aspects of the structural models are that one Mn ion is ligated by both D1-Asp170 and D1-Glu333 and another is ligated by both D1-Glu189 and D1-His332. The properties of the D1-Asp170 and D1-Glu333 mutants differ markedly, as do the properties of the D1-Glu189 and D1-His332 mutants. The main points of agreement and disagreement between the mutagenesis studies and the recent X-ray crystallographic structural models are discussed.

Recent spectroscopic studies on the water oxidizing complex (WOC) of Photosystem II are discussed in terms of the possible nature and structures for the Mn containing catalytic site. Emphasis is given to the various electron paramagnetic resonance techniques which have been increasingly employed, as well as examination of complementary data from optical and X-ray absorption spectroscopies. All of the quasi-stable intermediate states of the catalytic turnover cycle of the WOC (S to S) are now accessible spectroscopically. We show that the available data may be rationalized by a scheme in which the ‘active,’ catalytically cycling component of the water oxidizing site contains a pair of coupled, oxo bridged Mn ions, closely associated with a Ca ion. The structure of the active Mn site resembles the structure of the dinuclear Mn catalase. During functional turnover, oxidizing equivalents are stored both in Mn ions and ligand groups and one Mn ion has a unique, low symmetry ligand environment.

Single atoms of Ca and Cl are closely associated with the tetranuclear Mn cluster of Photosystem II (PS II). Extraction of either cofactor blocks advancement of the S-state cycle beyond S. In the case of Cl depletion, this modification has been proposed to result from replacement of the anion as a Mn ligand. This would cause a decrease of the potential, and thereby localization of the Mn(IV) state on the Mn to which Cl was bound. The implications of this hypothesis, and of the notion that the ligand replacing Cl in the modified S state is most likely hydroxyl (OH), are discussed and found to provide a plausible explanation for apparently conflicting reports in the literature. The location of Ca with respect to the Mn cluster is less certain. Although the metal is positioned so as to interfere with the attack of a small ligand, such as hydroxylamine (NHOH), on the Mn cluster, the distance between Ca and atoms of the Mn cluster is not resolved at the present time. Calcium can be shown to reinforce the stability of Mn ligation by PS II. However, its role in water oxidation must extend beyond structural effects to account for the block in electron transfer at S observed in Ca-depleted PS II, and the upward shift in the minimum temperature at which the transition from S to S occurs. The inactivation of PS II caused by replacement of Ca with lanthanides suggests that it functions as a site for binding of water molecules destined for oxidation at the Mn site in PS II.

Photosystem II (PS II) is the location for the antagonistic interactions between bicarbonate and monovalent anions such as formate. Incubation of PS II-containing samples with formate results in the inhibition of electron flow activity, which can be restored only by addition of bicarbonate. This bicarbonate effect exists on both the acceptor as well as on the donor side of PS II. The bicarbonate interaction on the acceptor side is located between the primary and secondary quinones and can be demonstrated in intact cells or leaves as well as in isolated thylakoid or core preparations. At physiological pH, bicarbonate is suggested to ligate to the non-heme iron between the D1 and D2 proteins and form hydrogen bonds to several amino acids of the D1 and D2 proteins. On the one hand, bicarbonate may stabilize, through conformational means, the reaction center proteins by protonation of certain amino acids near the secondary quinone electron acceptor; while on the other hand, it may play a significant role in the assembly and functioning of the water-oxidizing complex. A probable functional role in vivo is that it controls PS II electron flow in order to cope with stress conditions leading to, for instance, photoinhibition or thermoinactivation.

β-Carotene (Car), cytochrome (Cyt) and a monomeric chlorophyll (Chl) designated as chlorophyll Z, all undergo oxidation in Photosystem (PS) II under some illumination conditions. These components are not part of the direct electron transfer that leads to water oxidation and plastoquinone reduction and are thus designated ‘side-path electron donors.’ Under the usual conditions of PS II function, the quantum yield for the oxidation of these components is low; however, under certain experimental conditions, particularly low temperatures, the dominant reactions can be those involving the side-path donors. Car is a branch point in the side-path electron donation, being oxidized by P (the kinetically competent Chl cation radical), and reduced by Cyt , which is itself reduced by electrons from the pool of plastoquinol, possibly through the Q site. This all occurs on the D2-side of the reaction center. When the Cyt is pre-oxidized, Car is reduced by Chl Z. There are two candidates for Chl Z, the more obvious candidate on the D2 side and the less straightforward candidate on D1 side of the reaction center. The side-pathway is usually rationalized as a photoprotective cycle aimed at removing long-lived P and thus limiting oxidative damage. Based on the low quantum yields, we consider this unlikely. Instead we suggest that the side-path constitutes a photoprotective cycle in which the aim is to reduce the Car cation, rather than P, returning the carotene cation to its unoxidized state, preventing adventitious reactions and allowing it to play its a role as a singlet O quencher in the heart of PS II.

Vibrational spectroscopy, which includes infrared and Raman spectroscopies, provides structural information of molecules by detecting molecular vibrations based on chemical bonds and interactions. These methods have been applied to the study of various cofactors in Photosystem II. In particular, light-induced Fourier transform infrared (FTIR) difference spectroscopy has proven to be a powerful method to reveal detailed structures of the binding sites of cofactors including protein moieties and water molecules. Information available by FTIR difference spectroscopy includes hydrogen bonding and protonation state of chemical groups, which play an essential role in proton transfer and also in controlling redox reactions, but are often not available by X-ray crystallography. The FTIR investigations cover all the redox cofactors of Photosystem II in both the main and peripheral electron-transfer pathways, i.e., the manganese-cluster, the redox-active tyrosines Y and Y, the primary donor P680, the primary acceptor pheophytin, the quinone acceptors Q and Q, the non-heme iron, cytochrome b, chlorophyll Z, and β-carotene. This article reviews how the structures and reactions of these cofactors have been studied using mainly FTIR spectroscopy with the assistance of Raman spectroscopy.

Orientations and relative positions of paramagnetic intermediates in protein complexes of Photosystem (PS) II can be studied by electron paramagnetic resonance (EPR) spectroscopy. The EPR spectroscopic approaches and results on components of the electron transfer chain in PS II are presented. Where possible the data from EPR spectroscopy are compared to structural data from X-ray analysis. This comparison shows that the EPR-derived orientations and distances between the redox partners in PS II are in general corroborated by the recent X-ray crystallographic models. Furthermore, specific experiments that complement information available from crystallography are discussed.

Electron microscopy has contributed greatly to the structural biology of Photosystem II (PS II) in higher plants and green algae from the level of its in vivo organization, within the thylakoid membrane, through to the determination of the structure of light-harvesting complex II (LHCII) at 3.4 Å. Freeze-fracture and freeze-etch techniques provided the first visualization of PS II and its antenna systems in vivo. Subsequently a range of PS II and PS II-antenna, super- and mega-complexes were purified from thylakoid membranes using mild detergent solubilization and these were structurally characterized by single particle analysis. In particular these studies showed the structural linkage between the PS II core and a range of bound macromolecular light-harvesting antennae, as well as the overall shape of the extrinsic oxygen-evolving complex. Electron crystallography extended the resolution range, revealing the positioning of PS II subunits and the transmembrane helix organization of both PS II and antenna proteins. This technique also identified many of the chlorin cofactors in the reaction center proteins of D1 and D2, and also in the inner antenna, CP47, and outer antenna of LHCII. Future work will involve obtaining more highly resolved structures of supercomplexes and megacomplexes, using electron cryo-microscopy and including structural information emerging from X-ray and electron crystallography, with the view to gaining a near atomic resolution model of higher plant/green algal PS II as it exists in the native thylakoid membrane.

In the first part of this chapter structural elements of Photosystem II (PS II) and their functional behavior which have been elucidated primarily through special spectroscopic techniques are described. A transmembrane charge separation was identified as the primary act of light-energy conversion. The chlorophyll-containing P680 complex was discovered as the electron donor of PS II at the lumenal side of the membrane while the bound plastoquinone Q was discovered as the first stable electron acceptor and localized at the stromal side of the membrane. This membrane-spanning chlorophyll/quinone couple represents the PS II reaction center (RC) that drives water oxidation. This couple also provides a model for the RC of Photosystem I (PS I) and of other photosystems which drive different redox reactions. Two intermediate chlorin molecules located between the chlorophyll/quinone couple were found to function in the path of fast electron transfer from P680 across the membrane to Q while a pool of plastoquinones was found to function in a transmembrane path for electrons from reduced Q to PS I and for protons from the stroma to the membrane lumen. Primary electron donors located at the membrane base of PS II and PS I were found to be organized as chlorophyll pairs. Electron microscopy identified PS II as a dimer and PS I as a trimer. Based on the sequence homologies between PS II, PS I and the bacterial RC, predictions were made on the helical structure of the PS II complex. The preceding results served as the essential basis for the analysis and interpretation of the 3D crystal structures of PS I and PS II. In the second part of this chapter, the first PS II crystals capable of water oxidation are described. Based on the X-ray structure analysis of these crystals at 3.6 – 3.8 Å resolution, the framework of PS II, the architecture of the antenna system, the electron transfer chain, and the manganese cluster are discussed. The manganese environment is considered in terms of the more recent structure at 3.2 Å resolution. In the third part, functional events are described, especially changes in manganese valences, deprotonations, and the water states which were followed spectroscopically during the quaternary cycling of the water-oxidizing complex and which are summarized in a functional model. Finally, the implication of the high oxidation potential of the PS II RC is discussed as well as the functional cooperation between the dimeric electron donor P680, the monomeric electron donor chlorophyll D1 and the pheophytin D1 within the electron transfer chain.