A noble metal-free proton-exchange membrane fuel cell based on bio-inspired molecular catalysts.

Hydrogen is a promising energy vector for storing renewable energies: obtained from water-splitting, in electrolysers or photoelectrochemical cells, it can be turned back to electricity on demand in fuel cells (FCs). Proton exchange membrane (PEM) devices with low internal resistance, high compactness and stability are an attractive technology optimized over decades, affording fast start-up times and low operating temperatures. However, they rely on the powerful catalytic properties of noble metals such as platinum, while lower cost, more abundant materials would be needed for economic viability. Replacing these noble metals at both electrodes has long proven to be a difficult task, so far incompatible with PEM technologies. Here we take advantage of newly developed bio-inspired molecular H2 oxidation catalysts and noble metal-free O2-reducing materials, to fabricate a noble metal-free PEMFC, with an 0.74 V open circuit voltage and a 23 µW cm-2 output power under technologically relevant conditions. X-ray absorption spectroscopy measurements confirm that the catalysts are stable and retain their structure during turnover.

Cobalt-oxo core of a water-oxidizing catalyst film.

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.

Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes--implications for photosynthetic water-oxidation.

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.

Photosynthetic O2 formation tracked by time-resolved x-ray experiments.

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.

Structural and oxidation state changes of the photosystem II manganese complex in four transitions of the water oxidation cycle (S0 --> S1, S1 --> S2, S2 --> S3, and S3,4 --> S0) characterized by X-ray absorption spectroscopy at 20 K and room temperature.

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.

Non-standard structures of the Ni-Fe cofactor in the regulatory and the NAD-reducing hydrogenases from Ralstonia eutropha.

Spectroscopy on two oxygen-insensitive Ni-Fe hydrogenases from Ralstonia eutropha (NAD-reducing, soluble hydrogenase; hydrogen sensor, regulatory hydrogenase) reveals non-standard catalytic behaviour and unique structures of their Ni-Fe cofactors. Possible mechanistic implications are briefly discussed.

A fluorometric method for the differentiation of algal populations in vivo and in situ.

Fingerprints of excitation spectra of chlorophyll (Chl) fluorescence can be used to differentiate 'spectral groups' of microalgae in vivo and in situ in, for example, vertical profiles within a few seconds. The investigated spectral groups of algae (green group, Chlorophyta; blue, Cyanobacteria; brown, Heterokontophyta, Haptophyta, Dinophyta; mixed, Cryptophyta) are each characterised by a specific composition of photosynthetic antenna pigments and, consequently, by a specific excitation spectrum of the Chl fluorescence. Particularly relevant are Chl a, Chl c, phycocyanobilin, phycoerythrobilin, fucoxanthin and peridinin. A laboratory-based instrument and a submersible instrument were constructed containing light-emitting diodes to excite Chl fluorescence in five distinct wavelength ranges. Norm spectra were determined for the four spectral algal groups (several species per group). Using these norm spectra and the actual five-point excitation spectrum of a water sample, a separate estimate of the respective Chl concentration is rapidly obtained for each algal group. The results of dilution experiments are presented. In vivo and in situ measurements are compared with results obtained by HPLC analysis. Depth profiles of the distribution of spectral algal groups taken over a time period of few seconds are shown. The method for algae differentiation described here opens up new research areas, monitoring and supervision tasks related to photosynthetic primary production in aquatic environments.

The tetra-manganese complex of photosystem II during its redox cycle - X-ray absorption results and mechanistic implications.

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S(1)-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S(3)-state; (f) XAS-based working model for the S(2)-S(3) transition; (g) XAS-based working model for the S(0)-S(1) transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S(3)-(S(4))-S(0) transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.

Preparation protocols for high-activity photosystem II membrane particles of green algae and higher plants, pH dependence of oxygen evolution and comparison of the S2-state multiline signal by X-band EPR spectroscopy.

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.

Water and bromide in the active center of vanadate-dependent haloperoxidases.

Two aqua-oxovanadium complexes, viz. [A-VO(H2O)(sal-L-Leu)] (1) and [VO(H2O)2(5-Br-sal-Gly)] x H2O(2 x H2O), containing the water ligands in cis- and trans-positions to the oxo group at V-OH2 distances ranging from 2.008 to 2.228 A, have been structurally characterized in order to model the apical electron density feature found in the structures of fungal and algal vanadate-dependent peroxidases. Br K-edge XAS of bromide-treated bromoperoxidase from Ascophyllum nodosum and model compounds (including 2 x H2O) has been used to show that the substrate bromide does not bind to active site vanadium but to a light atom, possibly carbon, in its vicinity.

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Does the structure of the water-oxidizing photosystem II-manganese complex at room temperature differ from its low-temperature structure? A comparative X-ray absorption study.

Detailed information on room-temperature structure and oxidation state of the Photosystem II (PS II) manganese complex is needed to put mechanistic considerations on solid grounds. Because previously this information had not been available, the tetranuclear manganese complex was investigated by X-ray absorption spectroscopy (XAS) on PS II membrane particles at 290 K. Due to methodical progress (collection of XAS spectra within 10 s or less), significant X-ray radiation damage can be avoided; room-temperature XAS investigations on the PS II in its native membrane environment become feasible. Thus, the ambiguity with respect to the mechanistic relevance of low-temperature XAS results is avoidable. At 290 K as well as at 18 K, the manganese complex in its dark-stable state (S(1)-state) seemingly is a Mn(III)(2)Mn(IV)(2) complex comprising two di-mu(2)-oxo bridged binuclear manganese units characterized by the same Mn-Mn distance of 2.71-2.72 A at both temperatures. Most likely, manganese oxidation states and the protonation state of the bridging oxides are fully temperature independent. Remarkably, at room-temperature manganese-ligand distances of 3.10 and 3.65 A are clearly discernible in the EXAFS spectra. The type of bridging assumed to result in Mn-Mn or Mn-Ca distances around 3.1 A is, possibly, temperature-dependent as suggested by distance lengthening upon cooling by 0.13 A. However, mechanistic proposals on photosynthetic water oxidation, which involve the dimer-of-dimers model [Yachandra, V. K., et al. (1993) Science 260, 675-679] are not invalidated by the presented results.

Chlorophyll fluorescence transients of Photosystem II membrane particles as a tool for studying photosynthetic oxygen evolution.

The rise of the chlorophyll fluorescence yield of Photosystem II (PS II) membranes as induced by high-intensity actinic light comprises only two distinct phases: (1) the initial O-J increase and (2) the subsequent J-P increase. Partial inhibition of the PS II donor side by heating or washing procedures which remove peripheral PS II proteins or cofactors of the oxygen-evolving complex results in decrease of magnitude and rate of the J-P phase. The rate constant of the J-P increase is directly proportional to the steady-state rate of oxygen evolution; complete suppression of the J-P phase corresponds to full inhibition. A characteristic dip after J-level is observed only in Tris-washed or severely heated PS II membranes; manganese release correlates with appearance of the dip after J-level as verified by EPR spectroscopy. Presence of stabilizing cosolutes (glycine betaine, sucrose) or addition of donor-side cofactors (bicarbonate, chloride, calcium) to PS II membranes before heating (47 degrees C, 5 min) diminishes J-P phase suppression and prevents dip appearance, whereas the addition after heating is without effect. In conclusion, analysis of chlorophyll fluorescence transients of PS II membranes is a potentially useful tool for investigations on photosynthetic oxygen evolution. A decreased rate of the J-P phase can be employed as a convenient indicator for partial inhibition of oxygen-evolution activity; the appearance of a dip after J-level is suggestive of manganese release.

Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum.

Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.

X-ray absorption spectroscopy on layered photosystem II membrane particles suggests manganese-centered oxidation of the oxygen-evolving complex for the S0-S1, S1-S2, and S2-S3 transitions of the water oxidation cycle.

By application of microsecond light flashes the oxygen-evolving complex (OEC) was driven through its functional cycle, the S-state cycle. The S-state population distribution obtained by the application of n flashes (n = 0. 6) was determined by analysis of EPR spectra; Mn K-edge X-ray absorption spectra were collected. Taking into consideration the likely statistical error in the data and the variability stemming from the use of three different approaches for the determination of edge positions, we obtained an upshift of the edge position by 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the S0-S1, S1-S2, and S2-S3 transitions, respectively, and a downshift by 2.3-3.1 eV for the S3-S0 transition. These results are highly suggestive of Mn oxidation state changes for all four S-state transitions. In the S0-state spectrum, a clearly resolved shoulder in the X-ray spectrum around 6555 eV points toward the presence of Mn(II). We propose that photosynthetic oxygen evolution involves cycling of the photosystem II manganese complex through four distinct oxidation states of this tetranuclear complex: Mn(II)-Mn(III)-Mn(IV)2 in the S0-state, Mn(III)2-Mn(IV)2 in the S1-state, Mn(III)1-Mn(IV)3 in the S2-state, and Mn(IV)4 in the S3-state.

Structure and orientation of the oxygen-evolving manganese complex of green algae and higher plants investigated by X-ray absorption linear dichroism spectroscopy on oriented photosystem II membrane particles.

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.

Light-harvesting in Acaryochloris marina--spectroscopic characterization of a chlorophyll d-dominated photosynthetic antenna system.

Oxygenic photosynthesis of the prokaryote Acaryochloris marina involves chlorophyll d (Chl d) as the major pigment [Miyashita et al. (1996) Nature 383, 402]. Four spectral forms of Chl d (peak wavelengths: 694, 714, 726 and 740 nm) are resolvable by low-temperature absorption spectroscopy on intact cells. Based on fluorescence spectra (at 290 K and 77 K) and on analysis of fluorescence induction curves we conclude: (1) excitation energy is efficiently transferred between the various spectral forms of Chl d and the PS II reaction center; (2) Chl d serves as a light-harvesting pigment for both, Photosystem II (PS II) and PS I; (3) excitation energy transfer between PS II units occurs.

On the relation between absorption and fluorescence emission spectra of photosystems: Derivation of a Stepanov relation for pigment culsters.

Stepanov (1957a, Soviet Physics-Doklady 2: 81-84) obtained an equation which relates the absorption spectrum and the fluorescence emission spectrum of a single dye molecule. Here, a similar equation is derived for a cluster of interacting pigments, e.g. the antenna pigments of a photosystem. This relation can be used to assess the possibility of occurrence of rapid exciton equilibration (Dau and Sauer, 1996, Biochim. Biophys. Acta, 1273: 175-190). The excited state potential of a pigment cluster is discussed and compared to the excited state potential of a single pigment.

Structural consequences of ammonia binding to the manganese center of the photosynthetic oxygen-evolving complex: an X-ray absorption spectroscopy study of isotropic and oriented photosystem II particles.

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.

Correlation between structure and magnetic spin state of the manganese cluster in the oxygen-evolving complex of photosystem II in the S2 state: determination by X-ray absorption spectroscopy.

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.