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In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680 + ) precedes the water-oxidation chemistry of the S-state cycle of the Mn 4 Ca cluster. Here we investigate these early events, observable within about 10 ns to 10 ms after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310-1890 cm -1 for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80 ns, 500 ns, and 10 µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680 + was used to trace largely specifically the P680 + reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680 + transients with a sum of four exponentials, revealing two nanosecond phases (30-46 ns and 690-1110 ns) and two microsecond phases (4.5-8.3 µs and 42 µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2023. The Author(s).)

Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today's oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S 4 state-which was postulated half a century ago 1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O 2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O 2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S 4 state as the oxygen-radical state; its formation is followed by fast O-O bonding and O 2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O 2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems., (© 2023. The Author(s).)

Diiron cofactors in enzymes perform diverse challenging transformations. The structures of high valent intermediates (Q in methane monooxygenase and X in ribonucleotide reductase) are debated since Fe-Fe distances of 2.5-3.4 Å were attributed to "open" or "closed" cores with bridging or terminal oxido groups. We report the crystallographic and spectroscopic characterization of a Fe III 2 (μ-O) 2 complex (2) with tetrahedral (4C) centres and short Fe-Fe distance (2.52 Å), persisting in organic solutions. 2 shows a large Fe K-pre-edge intensity, which is caused by the pronounced asymmetry at the T D Fe III centres due to the short Fe-μ-O bonds. A ≈2.5 Å Fe-Fe distance is unlikely for six-coordinate sites in Q or X, but for a Fe 2 (μ-O) 2 core containing four-coordinate (or by possible extension five-coordinate) iron centres there may be enough flexibility to accommodate a particularly short Fe-Fe separation with intense pre-edge transition. This finding may broaden the scope of models considered for the structure of high-valent diiron intermediates formed upon O 2 activation in biology., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

In soluble methane monooxygenase enzymes ( s MMO), dioxygen (O 2 ) is activated at a diiron(II) center to form an oxodiiron(IV) intermediate Q that performs the challenging oxidation of methane to methanol. An analogous mechanism of O 2 activation at mono- or dinuclear iron centers is rare in the synthetic chemistry. Herein, we report a mononuclear non-heme iron(II)-cyclam complex, 1 - trans , that activates O 2 to form the corresponding iron(IV)-oxo complex, 2 - trans , via a mechanism reminiscent of the O 2 activation process in s MMO. The conversion of 1 - trans to 2 - trans proceeds via the intermediate formation of an iron(III)-superoxide species 3 , which could be trapped and spectroscopically characterized at -50 °C. Surprisingly, 3 is a stronger oxygen atom transfer (OAT) agent than 2 - trans ; 3 performs OAT to 1 - trans or PPh 3 to yield 2 - trans quantitatively. Furthermore, 2 - trans oxidizes the aromatic C-H bonds of 2,6-di- tert -butylphenol, which, together with the strong OAT ability of 3 , represents new domains of oxoiron(IV) and superoxoiron(III) reactivities.

Information on binding and rearrangement of pivotal water molecules could support understanding of light-driven water oxidation at the catalytic Mn 4 CaO 5 cluster of photosystem II (PSII). To address this point, the binding of ammonia (NH 3 )-a possible substrate-water analogue-has been investigated and discussed in the context of putative reaction mechanisms. By time-resolved detection of O 2 formation after light-flash excitation, we discriminate three NH 3 /NH 4 + binding sites jointly characterized by a K m value around 25 mM (of NH 4 + ), but differing in their influence on the O 2 -formation step. At 100 mM NH 4 Cl (pH 7.5), we observe (1) a PSII fraction with complete inhibition of O 2 -formation, (2) fast O 2 -formation with a time constant of 1.7 ms at 20 °C (Fast-PSII), and (3) slow O 2 -formation with a time constant of 36 ms at 20 °C (Slow-PSII). For the Fast-PSII, we determine an activation enthalpy of 223 ± 11 meV. Activation enthalpy and entropy of the Fast-PSII are essentially identical to the corresponding figures in the absence NH 3 /NH 4 + binding. For the Slow-PSII, the activation enthalpy is 323 ± 11 meV and thus significantly increased, whereas the activation entropy remains essentially unchanged. We conclude: (1) The fully-inhibitory binding site could relate to bound NH 3 replacing one of the two substrate-water molecules. (2) The Fast-PSII may relate to NH 3 /NH 4 + binding in the S 2 -state of PSII followed by unbinding before onset of the OO bond formation step, but also more intricate mechanisms are not excluded. (3) In the Slow-PSII, NH 3 /NH 4 + binding increases the energetic barrier of the OO bond formation step significantly., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn 4 CaO 5 -cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn 4 CaO 5 -cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn 4 CaO 5 -cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn 4 CaO 5 -cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.

Photosynthetic splitting of water into oxygen by plants, algae, and cyanobacteria is catalyzed by the oxygen-evolving center (OEC). Synthetic mimics of the OEC, which is composed of an asymmetric manganese-calcium-oxygen cluster bound to protein groups, may promote insight into the structural and chemical determinants of biological water oxidation and lead to development of superior catalysts for artificial photosynthesis. We synthesized a Mn4Ca-cluster similar to the native OEC in both the metal-oxygen core and the binding protein groups. Like the native OEC, the synthetic cluster can undergo four redox transitions and shows two magnetic resonance signals assignable to redox and structural isomerism. Comparison with previously synthesized Mn3CaO4-cubane clusters suggests that the fourth Mn ion determines redox potentials and magnetic properties of the native OEC., (Copyright © 2015, American Association for the Advancement of Science.)

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H(2)) in the presence of dioxygen (O(2)) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O(2)-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O(2)-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron-sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 Å Fe-Fe distances and additional longer vectors of ≥3.4 Å, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni-μO bonds due to Ni-Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Ni(ia)-S) of the MBH. We propose that the O(2) tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H(2) and O(2).

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 μs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ∼140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 μs at pH 7.0 vs τ(lag) of 470 ± 80 μs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.

The atmospheric dioxygen (O(2)) is produced at a tetramanganese complex bound to the proteins of photosystem II (PSII). To investigate product inhibition at elevated oxygen partial pressure (pO(2) ranging from 0.2 to 16 bar), we monitored specifically the redox reactions of the Mn complex in its catalytic S-state cycle by rapid-scan and time-resolved X-ray absorption near-edge spectroscopy (XANES) at the Mn K-edge. By using a pressure cell for X-ray measurements after laser-flash excitation of PSII particles, we found a clear pO(2) influence on the redox reactions of the Mn complex, with a similar half-effect pressure as determined (2-3 bar). However, XANES spectra and the time courses of the X-ray fluorescence collected with microsecond resolution suggested that the O(2) evolution transition itself (S(3)-->S(0)+O(2)) was not affected. Additional (nonstandard) oxidation of the Mn complex at high pO(2) explains our experimental findings more readily. Our results suggest that photosynthesis at ambient conditions is not limited by product inhibition of the O(2) formation step.

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.

The energetics of the individual reaction steps in the catalytic cycle of photosynthetic water oxidation at the Mn(4)Ca complex of photosystem II (PSII) are of prime interest. We studied the electron transfer reactions in oxygen-evolving PSII membrane particles from spinach by a photothermal beam deflection technique, allowing for time-resolved calorimetry in the micro- to millisecond domain. For an ideal quantum yield of 100%, the enthalpy change, DeltaH, coupled to the formation of the radical pair Y(Z)(.+)Q(A)(-) (where Y(Z) is Tyr-161 of the D1 subunit of PSII) is estimated as -820 +/- 250 meV. For a lower quantum yield of 70%, the enthalpy change is estimated to be -400 +/- 250 meV. The observed nonthermal signal possibly is due to a contraction of the PSII protein volume (apparent DeltaV of about -13 A(3)). For the first time, the enthalpy change of the O(2)-evolving transition of the S-state cycle was monitored directly. Surprisingly, the reaction is only slightly exergonic. A value of DeltaH(S(3)-->S(0)) of -210 meV is estimated, but also an enthalpy change of zero is within the error range. A prominent nonthermal photothermal beam deflection signal (apparent DeltaV of about +42 A(3)) may reflect O(2) and proton release from the manganese complex, but also reorganization of the protein matrix.

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.

In oxygenic photosynthesis, a complete water oxidation cycle requires absorption of four photons by the chlorophylls of photosystem II (PSII). The photons can be provided successively by applying short flashes of light. Already in 1970, Kok and coworkers [Photochem Photobiol 11:457-475, 1970] developed a basic model to explain the flash-number dependence of O2 formation. The third flash applied to dark-adapted PSII induces the S3-->S4-->S0 transition, which is coupled to dioxygen formation at a protein-bound Mn4Ca complex. The sequence of events leading to dioxygen formation and the role of Kok's enigmatic S4-state are only incompletely understood. Recently we have shown by time-resolved X-ray spectroscopy that in the S3-->S0 transition an interesting intermediate is formed, prior to the onset of O-O bond formation [Haumann et al. Science 310:1019-1021, 2005]. The experimental results of the time-resolved X-ray experiments are discussed. The identity of the reaction intermediate is considered and the question is addressed how the novel intermediate is related to the S4-state proposed in 1970 by Bessel Kok. This leads us to an extension of the classical S-state cycle towards a basic model which describes sequence and interplay of electron and proton abstraction events at the donor side of PSII [Dau and Haumann, Science 312:1471-1472, 2006].

In oxygenic photosynthesis, water is split at a Mn(4)Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I(0), eight successive steps of alternate proton and electron removal lead to I(8) and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.

Plants and cyanobacteria produce atmospheric dioxygen from water, powered by sunlight and catalyzed by a manganese complex in photosystem II. A classic S-cycle model for oxygen evolution involves five states, but only four have been identified. The missing S4 state is particularly important because it is directly involved in dioxygen formation. Now progress comes from an x-ray technique that can monitor redox and structural changes in metal centers in real time with 10-microsecond resolution. We show that in the O2-formation step, an intermediate is formed--the enigmatic S4 state. Its creation is identified with a deprotonation process rather than the expected electron-transfer mechanism. Subsequent electron transfer would give an additional S4' state, thus extending the fundamental S-state cycle of dioxygen formation.

The atmospheric dioxygen is produced by photosynthetic organisms. This light-driven process culminates in what appears as one step: a four-electron abstraction from two water molecules bound to the Mn4Ca complex of photosystem II. Recently, an intermediate of the O2-producing reaction sequence was stabilized by elevated oxygen backpressure and detected by UV flash photometry [Clausen, J., and Junge, W. (2004) Nature 430, 480]. We scrutinized its properties by delayed chlorophyll fluorescence measurements. Half-suppression of oxygen evolution was observed at a similar O2 pressure of 2.3 bar, as previously, now with photosystem II membrane particles from spinach, without artificial electron acceptors, and at a high signal-to-noise ratio. The data are tentatively interpreted as the stabilization of a 2-fold oxidized state of the catalytic center (S2*) with bound peroxide and its slow conversion into the normal S2 state by the release of peroxide.

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 degrees C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O2-evolution, followed by the slower release of (2 + 2) MnII ions [Pospisil P et al. (2003) Biophys J 84: 1370-1386]. Here, the predicted biphasic MnII release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn4Ca complex in the dark is due to the loss of the 18 kDa protein. The S2-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.

Structural changes and redox transitions at the metal atoms of the active site are essential for the understanding of the catalytic mechanisms of biological metalloenzymes. First steps towards studying these processes by time-resolved X-ray absorption spectroscopy on protein samples (BioXAS) are reported. Photosystem II (PSII) catalyses the light-driven oxidation of bound water molecules at a tetranuclear manganese complex yielding the molecular oxygen of the atmosphere. In this work, first time-resolved XAS results under quasi-physiological conditions (at room temperature, non-crystalline samples) on PSII are presented. Perspectives of time-resolved BioXAS are discussed.

Photosystem II (PS II) membrane particles are particularly well suited for various types of spectroscopic investigations on the PS II manganese complex. Here we present: (1) a preparation protocol for PS II membrane particles of higher plants, which yields exceptionally high oxygen-evolution activity due to the use of glycinebetaine as a PS II-stabilizing agent; (2) preparation protocols for highly active PS II membrane particles for the green algae Scenedesmus obliquus and Chlamydomonas reinhardtii; (3) a determination of pH dependence of oxygen evolution for spinach and Scenedesmus; (4) a comparison of the EPR multiline signal observed in the S2-state of green algae and higher plants of PS II membrane particles. A clearly broader type of multiline EPR signal is observed in green algae.

X-ray absorption spectroscopy at the Mn K-edge has been performed on multilayers of photosystem II-enriched fragments of the native thylakoid membrane prepared from a higher plant (spinach) and a unicellular green alga (Scenedesmus obliquus). Spectra collected for various angles between the prevailing orientation of the thylakoid membrane normal and the X-ray electric field vector contain information on the atomic structure of the tetranuclear manganese complex of photosystem II (PS II) and its orientation with respect to the membrane normal. The previously used approach for evaluation of the dichroism of extended X-ray absorption fine structure (EXAFS) spectra [George, G. N., et al. (1989) Science 243, 789-791] is modified, and the following results are obtained for PS II in its dark-stable state (S1-state): (1) structure and orientation of the PS II manganese complexes of green algae and higher plants are highly similiar or fully identical; (2) two 2.7-A vectors, which, most likely, connect the Mn nuclei of a planar Mn2(mu-O2) structure, are at an average angle of 80 degrees +/- 10 degrees with respect to the thylakoid normal; (3) the plane of the Mn2(mu-O2) structures is rather in parallel with the thylakoid plane than perpendicular. Structural models for the oxygen-evolving manganese complex and its orientation in the thylakoid membrane are discussed within the context of the presented results.