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 K1 ). 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(m-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 and O₂ formation are catalyzed by a Mn₄Ca complex bound to the proteins of photosystem II (PSII). The catalytic site, including the inorganic Mn₄CaO(n)H(x) 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 1.9Å has been achieved by XRD. Most likely however, all XRD data on the Mn₄CaO(n)H(x) 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 O−O bond formation chemistry are discussed. This article is part of a Special Issue entitled: Photosystem II.

The atmospheric dioxygen (O2) 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 (S3fS0؉O2) 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. manganese complex ͉ photosystem II ͉ X-ray spectroscopy ͉ bioinorganic chemistry ͉ oxygen pressure I n oxygenic photosynthesis, plants, algae, and cyanobacteria facilitate the primary biomass formation by exploiting solar energy for driving the conversion of water (H 2 O) and carbon dioxide (CO 2 ) into carbohydrates (C n H 2n O n ). Dioxygen (O 2 ) is formed as product of the water oxidation chemistry of the photosystem II (PSII) protein complex (1-5). Photosynthetic CO 2 fixation and O 2 formation have shaped the Earth's atmosphere (6) by (i) lowering the CO 2 level to Ͻ0.04% and (ii) creation of a maximal O 2 concentration of 35% (7), Ϸ300 million years before the present and a current level of Ϸ20%.In photosynthetic organisms, the low level of atmospheric CO 2 can limit photosynthesis, related to the activity of CO 2 -fixing ribulose bisphosphate carboxylase (8, 9). It is less clear whether water oxidation by PSII is directly affected by the concentration of O 2 , the immediate product of light-driven water splitting. This question was only recently addressed experimentally by Clausen and Junge (10). They exposed PSII from cyanobacteria to oxygen partial pressures (pO 2 ) ranging from 0.2 to 30 bar and, analyzing flash-induced near-UV absorption changes, discovered a pO 2 effect with a half-saturation pressure of only Ϸ2 bar (10). Subsequently, in measurements of delayed Chl fluorescence, together with Clausen and Junge we confirmed that also the donor side of the plant PSII in its native membrane environment is affected by elevated pO 2 (11). The surprisingly low halfpressure of only 2-3 bar was taken as an indication that the efficiency of the O 2 formation step could be reduced significantly by product inhibition. Such a limitation of the water oxidation chemistry may have imposed restraints on the evolution of oxygenic photosynthesis (10). Notably, the pO 2 inside of photosynthetic cells in microbial mats and bioreactors can be severalfold higher th...

The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O 2 activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2 red ) showed an isotropic six-line EPR signal at g ϳ 2 of the Mn ( Ribonucleotide reductases (RNRs) 3 are the only enzymes that, in all organisms, catalyze the reduction of ribonucleotides to their deoxy forms essential for DNA synthesis (1-3). RNRs also are important targets in cancer and antiviral therapy (4, 5).Class I RNRs found in eukaryotes and microorganisms (6) are heterotetrameric enzymes of R1 2 R2 2 organization. The R1 protein contains the nucleotide binding site and R2 houses a dinuclear metal center, which is the site of dioxygen (O 2 ) activation and, in conventional RNRs, is of the Fe-Fe type (7).Extensive investigations on Fe-Fe RNRs from, e.g. Escherichia coli, Saccharomyces cerevisiae, Mus musculus, and Homo sapiens have established that the catalytic reactions involve activation of an O 2 molecule at the di-metal cluster to generate a high potential site, which oxidizes a nearby tyrosine residue to a tyrosyl radical, Y ⅐ (8 -10). In E. coli R2 this Tyr-122 is at ϳ6 Å distance to the nearest iron (11, 12). Subsequent proton-coupled electron transfer (13) leads to the re-reduction of Y ⅐ and to the oxidation of a cysteine at the substrate binding site in R1 to a radical (C ⅐ ) (14, 15). C ⅐ initiates ribonucleotide reduction involving disulfide formation by two additional cysteines (16). Regeneration of reduced cysteines requires electron input from external thio-or glutaredoxins and ultimately from NADPH (17).At least the Fe(II) 2 , Fe(III) 2 , Fe(IV)Fe(III), and Fe(IV) 2 oxidation states of the metal center seem to be involved in the electron transfer reactions (18, 19) of classic RNRs. The Fe(III)-Fe(IV) state, termed "intermediate X" (20,21), is crucial because it oxidizes the tyrosine to Y ⅐ , leaving the di-iron site in the Fe(III) 2 state. Y ⅐ usually survives a large number of catalytic cycles, but when it is lost, the inactive Fe(III) 2 -Met form remains (22). Reactivation of the enzyme first requires reduction of the metal site to Fe(II) 2 , which then must react with O 2 , leading to the cleavage of the O-O bond and again to the formation of Fe(III) 2 and Y ⅐ (22, 23).According to the above reaction sequences, a tyrosine radical and the Fe(IV)Fe(III) state (X) have been anticipated to be decisive for RNR function. This view has been challenged recently because R2 proteins of RNRs in several species have been discovered (24, 25), containing a redox-inert phenylalanine instead of the tyrosine. One enzyme is found in the important human pathogenic bacterium Chlamydia trachomatis (Ct) (25,26). It is a fully functional RNR and the only RNR encoded in the genome of this organism (27,28). ...

Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn(4)Ca complex of PSII in its dark-stable S(1) state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl(-)-depleted and Br(-)-substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br(-). EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of approximately 5 A to Br(-). The native Cl(-) ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S(1) state.