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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 S 4 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 O 2 -formation step, an intermediate is formed—the enigmatic S 4 state. Its creation is identified with a deprotonation process rather than the expected electron-transfer mechanism. Subsequent electron transfer would give an additional S 4 ′ state, thus extending the fundamental S-state cycle of dioxygen formation.

Modification of the Co-oxo cores of cobalt-polyoxometalate water oxidation catalysts is detectable by X-ray absorption spectroscopy (XAS) as demonstrated by comparison of Na10[Co4(H2O)2(PW9O34)2] (1) and Na17[((Co(H2O))Co2PW9O34)2(PW6O26)] (2). XAS reveals the integrity of 1 uncompromised by oxidant-driven water oxidation, which proceeds without formation of catalytic cobalt oxide.

Mixed-valent amorphous MnOx catalysts suitable for photochemical and electrochemical water oxidation are prepared by treatment of nanostructured catalytically inactive MnO precursors with (NH4)2Ce(NO3)6 solution for 15 min under vigorous stirring.

Tyr D is a redox-active amino acid of the Photosystem II (PS II) D2-protein. Upon oxidation it forms an unusually stable neutral radical with characteristic EPR properties. We prepared highly active BBY particles of spinach and the green alga Scenedesmus obliquus. The X-band EPR spectra are similar with a peak to trough linewidth of 19.3 G for spinach and 19.0 G for Scenedesmus. Power saturation experiments were performed at 20 K. For unknown reasons, the signal of spinach satures slightly faster than that of the alga. Partially oriented PS II membrane particles exhibit comparable orientation-dependent four-line spectra. This indicates that the orientation of tyr D with respect to the membrane normal is similar in both species.

Thylakoid membranes were isolated from various cultures of the green alga Scenedesmus obliquus which differed in the extent of the observable high-energy-state quenching of chlorophyll fluorescence (qE). In uncoupled thylakoid membranes the pH-dependence of the fluorescence parameters F M and F 0 exhibits two distinct phases: (I) for pH-values ranging from 6.5 to 5.7, acidification results in a pronounced decrease of F M and F 0 ; (2) for pH-values below 5.7, acidification results in a slight decrease of F M and a significant increase of F 0 . The extent of the first phase of F M - and F 0 -quenching corresponds to the extent of qE in intact cells; the variability of qE of intact cells is not explainable by a pK-shift. Simulations on basis of the reversible radical pair model using pH-dependent molecular rate constants indicate the occurrence of a cooperative transition to a state with an enhanced rate of non-radiative decay of excited chlorophyll-singlet states (Hill coefficient n 1 ≥ 4, pK 1 6.1 for all cultures). For pH-values below 5.7, a pH effect on Photosystem II electron transfer reactions is involved (n 2 1, pK 2 4.5).

The Photosystem II (PS II) exciton dynamics at room temperature was investigated by steady-state and picosecond fluorescence measurements on PS II membrane particles of spinach. In the closed as well as in the open-reaction center state, emission and excitation spectra were found to be almost independent of the excitation (λex) and emission wavelength (λem), respectively. This holds even for anti-Stokes spectra (λem 690 nm indicating excitation energy transfer with a time constant of 15 ps. A model for the PS II exciton dynamics in the open reaction center state is proposed which describes the fluorescence relaxation in terms of three phases: (I) ultra-fast exciton equilibration (time constants < 15 ps) within a peripheral pigment pool consisting of LHC pigments and within an inner pigment pool presumably consisting of PS II core pigments, (II) rapid exciton equilibration (time constant of 15 ps) between these two pigment pools and (III) excited state decay by primary charge separation according to the reversible radical pair model (time constants of 131 ps and 322 ps), but maintenance of the exciton equilibrium distribution. Because the equilibration time of 15 ps is fast in comparison to the mean exciton lifetime of 220 ps, the steady-state fluorescence properties are determined mainly by the thermal equilibrium distribution of excited states on the PS II chlorophylls (Chl a and Chl b).

The redox state of the primary quinone acceptor (Q A ) of photosystem II (PS II) and specific non-photochemical effects determine the relation between PS II photochemistry and chlorophyll fluorescence. For the unicellular green alga Scenedesmus obliquus , the fluorescence yield ( F ), the fluorescence yield in the presence of reduced Q A ( F M ′) and the yield of respiration-corrected oxygen evolution (ф ox , evolution rate divided by light intensity) were determined during exposure to actinic light of various intensities (0.1 – 1600 μE m −2 s −1 ). Irrespective of the growth conditions, the parameter ф p = (F M ′ − F)/F M ′ was found to be proportional to ф ox for each algal culture. This finding is in full agreement with non-photochemical fluorescence quenching by increased thermal deactivation of excited antenna states, but is difficult to reconcile with non-photochemical quenching due to modified PS II electron transfer reactions. In particular, no indications of cyclic PS II electron flow were found.

The electron exit pathway of photosystem II (PSII) in plants and cyanobacteria comprises two quinone molecules (QA and QB) working in a series and in between a non-heme iron atom with a carboxyl ligand. Currently, the role of the Fe atom and its ligand in the QA-Fe-QB triad is insufficiently understood. We investigated the changes in the oxidation state and the coordination environment of the Fe in PSII upon quinone reduction using density-functional theory calculations (DFT). The electron transfer from QA − to QB in PSII was not accompanied by an oxidation state change of Fe(II), as previously also found for bacterial reaction center. Instead, DFT on large geometry-optimized cluster models of the Fe site and its surrounding environment based on the crystal structure suggested a transition from 6-coordinated Fe(II) to 5-coordinated Fe(II) due to a switch from bidentate to monodentate ligation (carboxylate shift) of bicarbonate at the Fe, which is induced by QA − formation. We propose that the non-heme Fe serves an essential function in the QA − →QB reaction: it steers charge densities and hydrogen-bonding within the iron-bicarbonate-quinone complex, facilitating rapid and high-yield QB reduction.

In photosynthesis, water is oxidized at a protein-bound Mn4Ca complex. Artificial water-oxidation catalysts that are similarly efficient and based on inexpensive and abundant materials are of great interest. A recently reported inorganic cobalt catalyst (CoCat) forms by electrodeposition as an amorphous layer on inert anodes, starting from an aqueous solution of cobalt ions and buffered salts such as potassium phosphate (KPi). X-ray absorption spectroscopy (XAS) indicates that the central unit of the CoCat is a cluster of edge-sharing CoIII(μ-O)6 octahedra. We find that the apparent cluster nuclearity is higher for film formation at anode voltages below the water-oxidation threshold. These films exhibit only minimally lower cobalt oxidation states than the films of the same thickness but deposited at voltages supporting water-oxidation. The similarities in structure, function, and oxidative self-assembly of the all-inorganic CoCat and the photosynthetic Mn4Ca complex are striking, despite stark differences in the chemical environment.

Humanity is on the threshold of a technological revolution that will allow all human structures across the earth to undertake photosynthesis more efficiently than plants; making zero carbon fuels by using solar energy to split water (as a cheap and abundant source of hydrogen) or other products from reduced atmospheric carbon dioxide. The development and global deployment of such articial photosynthesis (AP) technology addresses three of humanity’s most urgent public policy challenges: to reduce anthropogenic carbon dioxide (CO2) emissions, to increase fuel security and to provide a sustainable global economy and ecosystem. Yet, despite the considerable research being undertaken in this eld and the incipient thrust to commercialisation, AP remains largely unknown in energy and climate change public policy debates. Here we explore mechanisms for enhancing the policy and governance prole of this frontier technology for energy sustainability, even in the absence of a global project on articial photosynthesis. Globalizing AP – a first principles argument The argument for globalising articial photosynthesis (AP) appears simple from rst principles. Most of the our energy (particularly for transport) at present comes from burning ‘archived photosynthesis’ fuels (i.e., carbon-intensive oil, coal and natural gas) in a centralised and protable distribution network with decades long turnaround on high levels of private corporate investment and a well-honed capacity to prolong its existence through innovations such as coal-seam gas ‘fracking’ and shale oil extraction, despite its signicant contribution to critical problems such as atmospheric greenhouse gas emissions and climate change, ocean acidication and geopolitical instability. 1,2 Molecular hydrogen (H2) is an obvious alternative, its conversion into electricity or heat yielding only H2O, with no CO2 being produced. Currently 500 � 109 standard cubic

The mode of proton relocation from the water-oxidizing Mn complex of photosystem II (PSII) toward the aqueous phase is of key importance in photosynthetic water oxidation. An adequate description of the interrelation of proton and electron removal from the Mn complex is still lacking. We reinvestigate the influence of the pH on the rate constants of the redox transitions of the Mn complex (S-state transitions). For high-activity PSII membrane particles from spinach, near-UV absorption transients (at 360 nm) induced by trains of ns-Laser flashes were analyzed. To obtain the rate constants of the ‘pure’ S-state transitions, a stringent deconvolution of the raw transients was carried out. The transients of the S1→S2 and S2→S3 transitions exhibit mono-exponential behavior whereas the transients of the ‘S3→S0 + O2’ transition display a lag-phase behavior that is assignable to formation of a reaction intermediate by deprotonation. The rate constants of the electron transfer (ET) in the S1→S2 and S3→S0 transitions exhibit a negligible pH-dependence only. The rate constant of the S2→S3 transition decreases below pH 6 significantly, but clearly less than those of the lag-phase in the S3→S0 transition. Two models for the coupling of electron and proton transfer in the S2→S3 transition are discussed.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. Manganese based precious metal-free electrocatalysts for the oxygen evolution reaction (OER) are promising materials for energy storage systems based on dark or photo-coupled water electrolysis, because they are active, inexpensive and of low toxicity. In this work, atomic scale structure–activity relationships of two different nano-structured manganese oxides, MnOx, are established using a combination of X-ray absorption, diffraction and electrochemistry. Prepared by chemical symproportionation (s-MnOx) and impregnation (i-MnOx), the s-MnOx catalyst consisted of a layered structure similar to δ-MnO2 while the i-MnOx catalyst displayed a mixture of tunnelled, 3D cross-linked β- and defective γ-MnO2 structures. During electrocatalytic oxygen evolution the structural motifs of both MnOx remain largely unchanged, but the oxidation state of Mn increases from 3.5 to 3.9–4. Kinetic parameters of the electrocatalytic oxygen evolution reaction were extracted using Tafel slope analysis and pH titration experiment, and the role of the protons abstracted was analyzed. The study reveals fundamental differences of general importance in the catalytic activity between layered and cross-linked structures. The exclusive presence of di-μ-oxo-bridged Mn ions in the layered structure is coupled to a pronounced redox and charge capacity behaviour. This ensured efficient use of surface and bulk active sites, and resulted in a relatively large Tafel slope. Consequently, the intrinsic OER activity is especially high in s-MnOx. In contrast, 3D cross-linked structures with both mono- and di-μ-oxo-bridged Mn ions resulted in lower intrinsic activity but smaller Tafel slope, and thus favourable activity at technological water-splitting rates. The insights from this comparative study will provide guidance in the structural design and optimization of other non precious metal oxide OER catalysts.

Water oxidation by Photosystem II (PSII) is a process of fundamental importance for atmosphere (O2 production) and biosphere (primary biomass formation). In order to understand this basic biological process and to promote the rationale designs of artificial systems that mimic photosynthetic water oxidation, it is important to understand the energetic and kinetic parameters of intermediates formed in the course of the reaction cycle (S-state cycle). In the present study, we use time-resolved measurements of the delayed chlorophyll fluorescence to estimate rate constants, activation energies, free energy differences, and to discriminate between the enthalpic and the entropic contributions. Using a novel joint-fit simulation approach, kinetic parameters are determined for intermediates in the S2→S3 and in the S3→S0 + O2 transitions. The estimated parameters provide evidence for intermediate formation by deprotonation processes that take place already before the electron transfer from the tetra-manganese complex to the light-oxidized TyrZ, in both of the above S-state transitions.

The structure and orientation of the manganese complex in NH3-treated photosystem II (PS II) membrane particles of spinach are being studied by X-ray absorption spectroscopy. On the basis of earlier work by our group, a structure for the tetranuclear manganese complex of PS II, which consists of two di-mu-oxo-bridged binuclear Mn units linked by a mono-mu-oxo group, has been proposed [Yachandra, V. K., et al. (1993) Science 260, 675-679]. The extended X-ray absorption fine structure (EXAFS) of the complex modified by NH3 binding in the S2-state is suggestive of an increase in the Mn-Mn distance of one of these units from 2.72 +/- 0.02 to 2.87 +/- 0.02 A, whereas the Mn-Mn distance of the second unit seems to be unaffected by NH3 treatment. The elongation of one binuclear center could result from the replacement of one bridging mu-oxo by an amido group. The lengthening of one Mn-Mn distance means that, by NH3 treatment, the distance degeneracy of the 2.7 A Mn-Mn EXAFS interaction is removed. Consequently, the orientation of individual binuclear units with respect to the membrane normal becomes resolvable by EXAFS spectroscopy of partially oriented PS II membrane particles. The angle between the normal of the PS II-containing membrane and the Mn-Mn vector is determined to be 67 degrees +/- 3 degrees for the 2.87 A distance and 55 degrees +/- 4 degrees for the 2.72 A distance. Only small effects on position, shape, and orientation dependence of Mn K-edge spectra result from NH3 treatment, indicating that the Mn oxidation state, the symmetry of the Mn ligand environment, and the orientation of the complex remain essentially unaffected in the annealed NH3 S2-state. Therefore, it seems likely that the angles determined for the ammonia-modified manganese complex are similar to the respective angles of the untreated complex. The structure of the manganese complex and its orientation in the membrane are discussed.

Acetyl-CoA synthase (ACS) is involved in the bacterial carbon oxide conversion pathway. The binuclear nickel sites in ACS enzyme and two biomimetic synthetic compounds containing a Ni(II)Ni(II) unit (1 and 2) were compared using XAS/XES. EXAFS analysis of ACS proteins revealed similar Ni-N/O/S bond lengths and Ni-Ni/Fe distances as in the crystal structure in oxidized ACS, but elongated Ni-ligand bonds in reduced ACS, suggesting more reduced nickel species. The XANES spectra of ACS and the dinickel complexes showed overall similar shapes, but less resolved pre-edge and edge features in ACS, attributed to more distorted square-planar nickel sites in particular in reduced ACS. DFT calculation of pre-edge absorption and Kβ2,5 emission features reproduced the experimental spectra of the synthetic complexes, was sensitive even to the small geometry differences in 1 and 2, and indicated low-spin Ni(II) sites. Comparison of nickel sites in proteins and biomimetic compounds is valuable for deducing structural and electronic differences in response to ligation and redox changes.

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel–production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese–calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S 2 → S 3 transition of the classical S -state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant ( ). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein–water interface is characterized by a high activation energy ( E a = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S 0 → S 1 transition are similar (τ, approximately 100 µs; E a = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

Photosynthetic water oxidation and O2 formation are catalyzed by a Mn4Ca complex bound to the proteins of photosystem II (PSII). The catalytic site, including the inorganic Mn4CaOnHx core and its protein environment, is denoted as oxygen-evolving complex (OEC). Earlier and recent progress in the endeavor to elucidate the structure of the OEC is reviewed, with focus on recent results obtained by (i) X-ray spectroscopy (specifically by EXAFS analyses), and (ii) X-ray diffraction (XRD, protein crystallography). Very recently, an impressive resolution of 1.9 Å has been achieved by XRD. Most likely however, all XRD data on the Mn4CaOnHx core of the OEC are affected by X-ray induced modifications (radiation damage). Therefore and to address (important) details of the geometric and electronic structure of the OEC, a combined analysis of XRD and XAS data has been approached by several research groups. These efforts are reviewed and extended using an especially comprehensive approach. Taking into account XRD results on the protein environment of the inorganic core of the Mn complex, 12 alternative OEC models are considered and evaluated by quantitative comparison to (i) extended-range EXAFS data, (ii) polarized EXAFS of partially oriented PSII membrane particles, and (iii) polarized EXAFS of PSII crystals. We conclude that there is a class of OEC models that is in good agreement with both the recent crystallographic models and the XAS data. On these grounds, mechanistic implications for the Osingle bondO bond formation chemistry are discussed. This article is part of a Special Issue entitled: Photosystem II.

The future of energy supply depends on innovative breakthroughs regarding the design of cheap, sustainable and efficient systems for the conversion and storage of renewable energy sources. The production of hydrogen through water splitting seems a promising and appealing solution. We found that a robust nanoparticulate electrocatalytic material, H(2)-CoCat, can be electrochemically prepared from cobalt salts in a phosphate buffer. This material consists of metallic cobalt coated with a cobalt-oxo/hydroxo-phosphate layer in contact with the electrolyte and mediates H(2) evolution from neutral aqueous buffer at modest overpotentials. Remarkably, it can be converted on anodic equilibration into the previously described amorphous cobalt oxide film (O(2)-CoCat or CoPi) catalysing O(2) evolution. The switch between the two catalytic forms is fully reversible and corresponds to a local interconversion between two morphologies and compositions at the surface of the electrode. After deposition, the noble-metal-free coating thus functions as a robust, bifunctional and switchable catalyst.

The rapid response of the photosynthetic system to changes in light intensity (response within less than 30 min) is condidered. Variations in light intensity result in concentration changes in photosynthetic intermediates or protein states. These changes in turn affect Photosystem II (PS II) by a ‘backpressure effect’, resulting in the accumulation of PS II products (reduced plastoquinone, lumen protons). The product backpressure produces PS II states especially susceptible to photoinactivation and photodamage. By activation of special adaptation mechanisms, the efficiency of the photosynthetic system is optimized and photodamage is minimized. The following aspects are discussed: 1. long-term vs. short-term adaptation; 2. analysis of short-term adaptation by measurement of chlorophyll a fluorescence and photosynthetic oxygen evolution; 3. kinetics of the response of the photosynthetic system to changes in light intensity (induction curves, assignment of phases, time constants); 4. the ‘product backpressure’ on PS II (accumulation of reduced Q A , lumen pH effect on PS II donor side reactions, thylakoid voltage effect on PS II photochemistry); 5. the molecular mechanisms of short-term adaptation (pH-dependent energy quenching, reversible inactivation of the manganese complex, light-harvesting complex (LHC) phosphorylation); 6. induction of photoinactivation and photodamage; 7. relation between product backpressure, adaptation and photodamage.

The structure of the manganese cluster in the S2 state with the g approximately 4 EPR signal (S2-g4 state) generated by 130 K illumination of photosystem II (PSII) membranes prepared from spinach has been investigated by X-ray absorption spectroscopy. The Mn X-ray absorption K-edge spectra of the S2-g4 state not only show a shift of the inflection point to higher energy from the S1 state but also reveal a different edge shape from that of the S2 state with the multiline signal (S2-MLS state). Extended X-ray absorption fine structure (EXAFS) studies of the Mn K-edge show that the structure of the Mn cluster in the S2-g4 state is distinctly different from those in the S2-MLS or S1 states. In the S2-g4 state, the second shell of back-scatters from the Mn absorber is found to contain two Mn-Mn distances of 2.73 and 2.85 A. We interpret this to indicate the presence of two nonequivalent di-mu-oxo-bridged Mn binuclear structures in the Mn cluster of the S2-g4 state. The third shell of the S2-g4 state at about 3.3 A also contains increased heterogeneity. By contrast, very little distance disorder was found to exist in the second shell of the S1 or S2-MLS states. A mechanism is proposed to explain these results in the context of our model for the Mn cluster and the EPR properties of the Mn complex in the S2 state.

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.

The geometry and electronic structure of cis-[Ru(II)(bpy)(2)(H(2)O)(2)](2+) and its higher oxidation state species up formally to Ru(VI) have been studied by means of UV-vis, EPR, XAS, and DFT and CASSCF/CASPT2 calculations. DFT calculations of the molecular structures of these species show that, as the oxidation state increases, the Ru-O bond distance decreases, indicating increased degrees of Ru-O multiple bonding. In addition, the O-Ru-O valence bond angle increases as the oxidation state increases. EPR spectroscopy and quantum chemical calculations indicate that low-spin configurations are favored for all oxidation states. Thus, cis-[Ru(IV)(bpy)(2)(OH)(2)](2+) (d(4)) has a singlet ground state and is EPR-silent at low temperatures, while cis-[Ru(V)(bpy)(2)(O)(OH)](2+) (d(3)) has a doublet ground state. XAS spectroscopy of higher oxidation state species and DFT calculations further illuminate the electronic structures of these complexes, particularly with respect to the covalent character of the O-Ru-O fragment. In addition, the photochemical isomerization of cis-[Ru(II)(bpy)(2)(H(2)O)(2)](2+) to its trans-[Ru(II)(bpy)(2)(H(2)O)(2)](2+) isomer has been fully characterized through quantum chemical calculations. The excited-state process is predicted to involve decoordination of one aqua ligand, which leads to a coordinatively unsaturated complex that undergoes structural rearrangement followed by recoordination of water to yield the trans isomer.

The atomic structure of an electrodeposited Ni catalyst film is dominated by extensive di-μ-oxido bridging between Ni(III/IV) ions, as revealed by X-ray absorption spectroscopy. The structure is surprisingly similar to that of an analogous Co-based film and colloidal Mn-based catalysts. Structural requirements for water oxidation are discussed.

For the production of nonfossil fuels, water oxidation by inexpensive cobalt-based catalysts is of high interest. Films for the electrocatalysis of water oxidation were obtained by oxidative self-assembly (electrodeposition) from aqueous solutions containing, apart from Co, either K, Li or Ca with either a phosphate, acetate or chloride anion. X-ray absorption spectroscopy (XAS) at the Co K-edge revealed clusters of edge-sharing CoO(6) octahedra in all films, but the size or structural disorder of the Co-oxido clusters differed. Whereas potassium binding is largely unspecific, CaCo(3) O(4) cubanes, which resemble the CaMn(3) O(4) cubane of the biological catalyst in oxygenic photosynthesis, may form, as suggested by XAS at the Ca K-edge. Cyclic voltammograms in a potassium phosphate buffer at pH 7 revealed that no specific combination of anions and redox-inactive cations is required for catalytic water oxidation. However, the anion type modulates not only the size (or order) of the Co-oxido clusters, but also electrodeposition rates, redox potentials, the capacity for oxidative charging, and catalytic currents. On these grounds, structure-activity relations are discussed.

In oxygenic photosynthesis, solar energy drives the oxidation of water catalyzed by a Mn(4)Ca complex bound to the proteins of Photosystem II. Four protons are released during one turnover of the water oxidation cycle (S-state cycle), implying thermodynamic limitations at low pH. For proton concentrations ranging from 1 nm (pH 9) to 1 mm (pH 3), we have characterized the low-pH limitations using a new experimental approach: a specific pH-jump protocol combined with time-resolved measurement of the delayed chlorophyll fluorescence after nanosecond flash excitation. Effective pK values were determined for low-pH inhibition of the light-induced S-state transitions: pK(1)=3.3 ± 0.3, pK(2)=3.5 ± 0.2, and pK(3)≈pK(4)=4.6 ± 0.2. Alkaline inhibition was not observed. An extension of the classical Kok model facilitated assignment of these four pK values to specific deprotonation steps in the reaction cycle. Our results provide important support to the extended S-state cycle model and criteria needed for assessment of quantum chemical calculations of the mechanism of water oxidation. They also imply that, in intact organisms, the pH in the lumen compartment can hardly drop below 5, thereby limiting the ΔpH contribution to the driving force of ATP synthesis.

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Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H2) in the presence of dioxygen (O2) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O2-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 (Niia-S) of the MBH. We propose that the O2 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 H2 and O2. © 2011 American Chemical Society., We thank Drs. W. Meyer-Klaucke (EMBL at DESY, Hamburg) and F. Schäfers and M. Mertin (beamline KMC-1 at BESSY, Helmholtz-Zentrum Berlin) for excellent technical support. M.H. thanks Prof. C. Limberg (HU-Berlin) for discussion and Dr. P. Chernev (FU-Berlin) for providing the GloFit program.

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m2, and supplied with 0.03 and 3 % CO2 enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO2, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO2 concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L30R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO2 adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO2 adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L60CM and L75CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.

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.

Striving for new solar fuels, the water oxidation reaction currently is considered to be a bottleneck, hampering progress in the development of applicable technologies for the conversion of light into storable fuels. This review compares and unifies viewpoints on water oxidation from various fields of catalysis research. The first part deals with the thermodynamic efficiency and mechanisms of electrochemical water splitting by metal oxides on electrode surfaces, explaining the recent concept of the potential-determining step. Subsequently, novel cobalt oxide-based catalysts for heterogeneous (electro)catalysis are discussed. These may share structural and functional properties with surface oxides, multinuclear molecular catalysts and the catalytic manganese–calcium complex of photosynthetic water oxidation. Recent developments in homogeneous water-oxidation catalysis are outlined with a focus on the discovery of mononuclear ruthenium (and non-ruthenium) complexes that efficiently mediate O2 evolution from water. Water oxidation in photosynthesis is the subject of a concise presentation of structure and function of the natural paragon—the manganese–calcium complex in photosystem II—for which ideas concerning redox-potential leveling, proton removal, and OO bond formation mechanisms are discussed. The last part highlights common themes and unifying concepts.

The D1 protein (PsbA) of photosystem II (PSII) from Thermosynechococcus elongatus is encoded by a psbA gene family that is typical of cyanobacteria. Although the transcription of these three genes has been studied previously (Kós, P. B., Deák, Z., Cheregi, O., and Vass, I. (2008) Biochim. Biophys. Acta 1777, 74-83), the protein quantification had not been possible due to the high sequence identity between the three PsbA copies. The successful establishment of a method to quantify the PsbA proteins on the basis of reverse phase-LC-electrospray mass ionization-MS/MS (RP-LC-ESI-MS/MS) enables an accurate comparison of transcript and protein level for the first time ever. Upon high light incubation, about 70% PsbA3 could be detected, which closely corresponds to the transcript level. It was impossible to detect any PsbA2 under all tested conditions. The construction of knock-out mutants enabled for the first time a detailed characterization of both whole cells and also isolated PSII complexes. PSII complexes of the ΔpsbA1/psbA2 mutant contained only copy PsbA3, whereas only PsbA1 could be detected in PSII complexes from the ΔpsbA3 mutant. In whole cells as well as in isolated complexes, a shift of the free energy between the redox pairs in the PsbA3 complexes in comparison with PsbA1 could be detected by thermoluminescence and delayed fluorescence measurements. This change is assigned to a shift of the redox potential of pheophytin toward more positive values. Coincidentally, no differences in the Q(A)-Q(B) electron transfer could be observed in flash-induced fluorescence decay or prompt fluorescence measurements. In conclusion, PsbA3 complexes yield a better protection against photoinhibition due to a higher probability of the harmless dissipation of excess energy.

For molecular systems which are partially ordered with respect to one macroscopic axis, for example, the sample-surface normal, X-ray absorption spectroscopy (XAS) with linearly polarized synchrotron radiation can provide information on structure and orientation of the X-ray absorbing site (polarized or linear-dichroism XAS). Examples for such partially oriented systems are protein-carrying membrane particles deposited in the form of multilayers on surfaces, layered minerals, surface films or imperfect 2D crystals, planar electrodes or catalytic surfaces. For electric dipole transitions, equations are derived describing how partial unidirectional orientation determines the linear dichroism (LD). The approach presented facilitates description of 1) LD in multiple-scattering contributions of the extended X-ray absorption fine-structure (EXAFS) and 2) of LD in the X-ray absorption near-edge structure (LD-XANES). Structural and orientation information becomes accessible by combination with ab initio XANES calculations. The LD-XANES approach is applied to the water-oxidizing Mn complex of photosystem II. The results suggest that the (mu-O)-(mu-O) vectors of the Mn-(mu-O)(2)-Mn units are at an average angle to the membrane normal of 30-45 degrees. The best-fit structure in connection with crystallographic data suggests a specific oxidation-state assignment: Mn(1)(III)Mn(2)(III)Mn(3)(IV)Mn(4)(IV). However, currently this assignment remains uncertain. In future studies, the LD-XANES analysis should play an important role in construction of unequivocal atomic-resolution model of the photosynthetic Mn complex.

Photosynthesis in plants and cyanobacteria involves two protein-cofactor complexes which are denoted as photosystems (PS), PSII and PSI. These solar-energy converters have powered life on earth for approximately 3 billion years. They facilitate light-driven carbohydrate formation from H(2)O and CO(2), by oxidizing the former and reducing the latter. PSII splits water in a process driven by light. Because all attractive technologies for fuel production driven by solar energy involve water oxidation, recent interest in this process carried out by PSII has increased. In this Account, we describe and apply a rationale for estimating the solar-energy conversion efficiency (eta(SOLAR)) of PSII: the fraction of the incident solar energy absorbed by the antenna pigments and eventually stored in form of chemical products. For PSII at high concentrations, approximately 34% of the incident solar energy is used for creation of the photochemistry-driving excited state, P680*, with an excited-state energy of 1.83 eV. Subsequent electron transfer results in the reduction of a bound quinone (Q(A)) and oxidation of the Tyr(Z) within 1 micros. This radical-pair state is stable against recombination losses for approximately 1 ms. At this level, the maximal eta(SOLAR) is 23%. After the essentially irreversible steps of quinone reduction and water oxidation (the final steps catalyzed by the PSII complex), a maximum of 50% of the excited-state energy is stored in chemical form; eta(SOLAR) can be as high as 16%. Extending our considerations to a photosynthetic organism optimized to use PSII and PSI to drive H(2) production, the theoretical maximum of the solar-energy conversion efficiency would be as high as 10.5%, if all electrons and protons derived from water oxidation were used for H(2) formation. The above performance figures are impressive, but they represent theoretical maxima and do not account for processes in an intact organism that lower these yields, such as light saturation, photoinhibitory, protective, and repair processes. The overpotential for catalysis of water oxidation at the Mn(4)Ca complex of PSII may be as low as 0.3 V. To address the specific energetics of water oxidation at the Mn complex of PSII, we propose a new conceptual framework that will facilitate quantitative considerations on the basis of oxidation potentials and pK values. In conclusion, photosynthetic water oxidation works at high efficiency and thus can serve as both an inspiring model and a benchmark in the development of future technologies for production of solar fuels.

Across the thylakoid membrane of spinach chloroplasts a diffusion potential of defined magnitude can be created by rapid changes in the KCl concentration. The resulting electric field leads to an increase in the yield of photosystem II (PS II) fluorescence for positive membrane voltages — positive in the inner thylakoid space — and a decrease for negative membrane voltages (Dau and Sauer, 1991, BBA 1098, 49–60). We have how studied this phenomenon by measurement of picosecond fluorescence decay. Based on the kinetic PS II model of Schatz et al. (1988, Biophys. J. 54, 397–405), the fluorescence decays were interpreted in terms of the rate constant of primary charge separation (formation of reaction center cation radical and pheophytin anion radical) primary charge recombination (recombination of primary biradical to chlorophyll singlet state) and secondary charge separation (reduction of primary quinone acceptor). For increasingly positive thylakoid voltages, the results are indicative of a relatively small but significant decrease in the rate constant of primary charge separation (by about 8% per + 100 mV thylakoid voltage) and a much larger increase (by about 50% per + 100 mV) of the rate constant of primary charge recombination. Nevertheless, due to the high sensitivity of the PS II fluorescence yield to changes in the rate constant of primary charge separation, the field-induced increase of fluorescence yield results mainly from the decreased rate constant of primary charge separation, and to a smaller extent (about one third of the increase) from the increased rate constant of charge recombination. The free energy difference of the primary radical pair was found to change by 17 meV per 100 mV thylakoid voltage. The relation between the rate constant of primary charge separation and the free energy difference appears to be linear within the accessible range of free energy changes (−15 meV to +22 meV); the rate constant of primary charge separation increases with increasingly negative free energy differences by 6% per 10 meV. This free energy dependence is compared with model calculations for a sequential two-step and for a super-exchange model of the primary PS II charge separation.

In photosynthesis, water is oxidized at a protein-bound Mn(4)Ca complex. Artificial water-oxidation catalysts that are similarly efficient and based on inexpensive and abundant materials are of great interest. Recently, assembly of a catalyst as an amorphous layer on inert cathodes by electrodeposition starting from an aqueous solution of cobalt ions and potassium phosphate has been reported. X-ray absorption spectroscopy on the cobalt catalyst film (CoCF) suggests that its central structural unit is a cluster of interconnected complete or incomplete Co(III)-oxo cubanes. Potassium ligation to Co-bridging oxygens could result in Co(3)K(mu-O)(4) cubanes, in analogy to the Mn(3)Ca(mu-O)(4) cubane motif proposed for the photosynthetic Mn complex. The similarities in function and oxidative self-assembly of CoCF and the catalytic Mn complex in photosynthesis are striking. Our study establishes a close analogy also with respect to the metal-oxo core of the catalyst.

Across the thylakoid membrane of spinach chloroplasts a diffusion potential was created by rapid changes in the KCl concentration (salt-jumps) employing a flow and mixing technique. The analysis of salt-induced electrochromic absorbance changes indicates that under appropriate experimental conditions the salt-jump technique facititates the establishment of a well-defined thylakoid voltage. This approach appears to be well suited for quantitative studies of electric field effects on thylakoid reactions. In the open reaction center state, for positive salt-induced thylakoid voltages (positive at the lumenal side) the fluorescence yield of spinach thylakoids increases; for negative voltages it decreases. A linear relation between fluorescence yield and thylakoid voltage is observed in the range from −70 mV to +165 mV, characterized by a change of the fluorescence yield by 9% (± 2%) per 100 mV. Analysis of the fluorescence emission spectra reveals that the electric field effect (i) does not originate from a Stark shift of the emission spectrum and (ii) reflects an influence on the Photosystem II (PS II) fluorescence. With increasing degree of PS II trap-closure, the extent of the electric field effect decreases, and in the closed reaction center state no effect is detectable. The trap-closure sensitivity suggests that the electric field effect on the fluorescence yield originates from an electric field dependence of the PS II charge separation/recombination reactions. We propose that the observed fluorescence yield-thylakoid voltage relation reflects the free energy dependence of the rate constants of the PS II charge transfer reactions.

The millisecond kinetics of light-induced changes in thylakoid voltage as detected by absorbance changes at 520 nm were compared with the kinetics of the fluorescence yield on Aegopodium podagraria leaves. Frequency response measurements with red actinic light and the analysis of the response to far-red light flashes reveal that the light induced increase in thylakoid voltage is coupled to an increase in fluorescence yield. An increase in fluorescence yield of 5.4% per 10 mV increase in thylakoid voltage is found after calibration of the 520 nm absorbance changes by single-turnover flashes which are assumed to induce an increase in thylakoid voltage by 25 mV. The measured voltage sensitivity of the fluorescence yield compares well to calculations based on the assumption that the rate constant of PS II charge separation is sensitive to electric fields as proposed by Schatz et al. (Biophys. J. 54 (1988) 379–405). Based on the results of the frequency response measurements, it is estimated that about 7% of the O-I phase of the fluorescence kinetics and 10% of the fluorescence response to single-turnover flashes are due to the voltage effect. It is discussed whether transthylakoid voltage might be of relevance for the control of PS II electron flux.

Its superior quantum efficiency renders PSII a model for biomimetic systems. However, also in biological water oxidation by PSII, the efficiency is restricted by recombination losses. By laser-flash illumination, the secondary radical pair, P680(+)Q(-) (A) (where P680 is the primary Chl donor in PSII and Q(A), primary quinone acceptor of PSII), was formed in close to 100% of the PSII. Investigation of the quantum efficiency (or yield) of the subsequent steps by time-resolved delayed (10 micros to 60 ms) and prompt (70 micros to 700 ms) Chl fluorescence measurements on PSII membrane particles suggests that (1) the effective rate for P680(+) Q(-) (A) recombination is approximately 5 ms(-1) with an activation energy of approximately 0.34 eV, circumstantially confirming dominating losses by reformation of the primary radical pair followed by ground-state recombination. (2) Because of compensatory influences on recombination and forward reactions, the efficiency is only weakly temperature dependent. (3) Recombination losses are several-fold enhanced at lower pH. (4) Calculation based on delayed-fluorescence data suggests that the losses depend on the state of the water-oxidizing manganese complex, being low in the S(0)-->S(1) and S(1)-->S(2) transition, clearly higher in S(2)-->S(3) and S(3)-->S(4)-->S(0). (5) For the used artificial electron acceptor, the efficiency is limited by acceptor-side processes/S-state decay at high/low photon-absorption rates resulting in optimal efficiency at surprisingly low rates of approximately 0.15-15 photons s(-1) (per PSII). The pH and S-state dependence can be rationalized by the basic model of alternate electron-proton removal proposed elsewhere. A physiological function of the recombination losses could be limitation of the lifetime of the reactive donor-side tyrosine radical (Y(.) (Z)) in the case of low-pH blockage of water oxidation.

The water-oxidizing manganese complex bound to the proteins of photosystem II (PSII) was studied by X-ray absorption spectroscopy on PSII membrane particles. An extended range for collection of EXAFS data was used (up to 16.6 A−1). The EXAFS suggests the presence of two Mn-Mn distances close to 2.7 A (per Mn4Ca complex); the existence of a third Mn-Mn distance below 2.9 A is not indicated by the EXAFS data. A distance of 3.7 A is clearly resolved in the extended-range data and tentatively assigned to a Mn-Mn distance. Taking into account the above EXAFS results (inter alia), we present a model for the structure of the PSII manganese complex, which differs from previous atomic-resolution models. Emphasizing the hypothetical character, we propose for all semi-stable S-states (i) a structure of the Mn4Ca(μ-O)n core, (ii) a model of the amino-acid environment, and (iii) assignments of distinct Mn oxidation states to the individual Mn ions. This specific working model may permit discussion, verification, and invalidation of its various features by comparison with experimental and theoretical findings.

Investigations On Photosystem II (Psii) Electron Transfer Processes by Observing Transient Absorption Changes Are Challenging. They Require High Psii Concentrations, Extended Averaging and are Negatively Affected By The Strong Light Scattering of Various Sample Types. Avoiding These Problems, The Analysis of the Time-Resolved Delayed Fluorescence (Df) measurements has become an important tool to study quantitatively light-induced electron transfer as well as associated Processes (E.G. Proton Movements) at the Donor Side Of Psii. Inter Alia This method can provide insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The delayed emission of chlorophyll fluorescence was measured on both wild type and mutant of green algae psii membrane particles in the time domain from 10 μs to 60 ms after each flash of a train of nanosecond laser pulses. The influence of the psii-associated carbonic anhydrase (ca) on the donor side reactions was studied for a Chlamydomonas Reinhardtii Wild type and Ca-Free Mutant.

In Oxygenic Photosynthesis, Water Is Oxidized At A Mn4Ca Complex Bound To The Proteins Of Photosystem Ii (Psii). The Water-Oxidation Cycle Of The Mn Complex May Involve Nine Distinct States Of The Mn Complex (Dau And Haumann 2006), But Only Four Of These Are Stable For Several Seconds (S2, S3), Several Hours (0), Or Fully Dark-Stable (S1). The Four Transitions Between These Semi-Stable S-States Have Been Studied By A Variety Of Spectroscopic Techniques. Due To The Pioneering Work Of Several Groups, Today Also Ftir Spectroscopy Plays A Prominent Role Frequently In Fruitful Conjunction With Mutagenesis Studies (See, E.G., Studies Of Debus And Coworkers). After Application Of Ns Or μS Flashes Of Saturating Light And Subsequent Ftir Measurements, Difference Spectra Can Be Calculated Which Are Mostly Interpreted As S-State Difference Spectra. However, Potential Complications Come From (I) Acceptor Side Contributions To The Ftir Spectra, (Ii) Psii With A Partially Defect Donorside, And (Iii) Significant Mixing Of S-State Populations Caused By So-Called Miss Events. We Have Measured Ftir Spectra On Psii Membrane Particles In The Intervals Between Laser Flashes Of An Extended Flash Sequence. Taking Into Account Also Complementary Results Obtained By Time-Resolved Prompt And Delayed Fluorescence Measurements, We Attempt A Correction (Or Deconvolution) For (I) To (Iii). These Investigations Aim At A Rationale To Get The Pure S-State Difference Spectra In Ftir Investigations On The Reaction Cycle Of Psii Water Oxidation. First Results Are Presented; Problems And Perspectives Are Discussed.

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 water-oxidizing manganese complex bound to the proteins of photosystem II (PSII) was studied by X-ray absorption spectroscopy on PSII membrane particles. An extended range for collection of extended X-ray absorption fine-structure (EXAFS) data was used (up to 16.6 Å −1 ). The EXAFS suggests the presence of two Mn–Mn distances close to 2.7 Å (per Mn 4 Ca complex); the existence of a third Mn–Mn distance below 2.9 Å is at least uncertain. Interestingly, a distance of 3.7 Å is clearly resolved in the extended-range data and tentatively assigned to a Mn–Mn distance. Taking into account the above EXAFS results (inter alia), we present a model for the structure of the PSII manganese complex, which differs from previous atomic-resolution models. Emphasizing the hypothetical character, we propose for all semi-stable S-states: (i) a structure of the Mn 4 Ca(μ-O) n core, (ii) a model of the amino acid environment, and (iii) assignments of distinct Mn oxidation states to all the individual Mn ions. This specific working model may permit discussion, verification and invalidation of its various features in comparison with experimental and theoretical findings.

Photosynthetic water oxidation provides the dioxygen of the atmosphere. Its partial reactions proceed at a Mn4Ca complex bound to photosystem II of plants and cyanobacteria. Understanding the mechanism of this biological oxidation of water molecules to O2 is one of the major challenges in life sciences. We have developed and employed X‐ray absorption Spectroscopy (XAS) techniques facilitating measurements on metalloenzymes at room temperature. By these techniques, we were able to resolve structural changes at the Mn ions, to follow oxidation‐state changes in the microseconds time domain, and to detect a novel and likely crucial intermediate in the oxygen‐evolving step of the catalytic cycle of the Mn complex. Based on the obtained results, we replace the classic S‐state model of the catalytic cycle by a more elaborated reaction scheme which solves apparent inconsistencies of earlier models, explains a large body of experimental results, and provides a fresh twist in photosynthesis research.

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.

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 Mn 4 Ca complex of photosystem II. Recently, an intermediate of the 02-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 O 2 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 (S 2 *) with bound peroxide and its slow conversion into the normal S 2 state by the release of peroxide.

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem II (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N(3)O(3) in 1, N(2)O(4) in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(II)--Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)--Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(III). In 2: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)--Mn(III)-(mu-OR)(mu-OCO)(2)-Mn(III)--Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(IV). In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at approximately 1V, but the mu-O(H) bridge is observed at the Mn(2)(III,III) level in 1 and at the Mn(III,IV) level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential.

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.