On the structure of the manganese complex of photosystem II: extended-range EXAFS data and specific atomic-resolution models for four S-states

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 16.6A(-1)). The EXAFS suggests the presence of two Mn-Mn distances close to 2.7A (per Mn4Ca complex); the existence of a third Mn-Mn distance below 2.9A is at least uncertain. Interestingly, a distance of 3.7A 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(mu-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.     

Specific loss of the extrinsic 18 KDa protein from Photosystem II upon heating to 47 °C causes inactivation of oxygen evolution likely due to Ca release from the Mn-complex

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

Considerations on the mechanism of photosynthetic water oxidation – dual role of oxo-bridges between Mn ions in (i) redox-potential maintenance and (ii) proton abstraction from substrate water

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.     

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.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 A resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q(B). The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O(2) evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q(A) and Q(B) were found in the crystallized PSII. We propose that the extra quinones are located in the Q(B) cavity and serve as a PSII intrinsic pool of electron acceptors.     

Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti.

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.     

Stepwise Transition of the Tetra-Manganese Complex of Photosystem II to a Binuclear Mn2(μ-O)2 Complex in Response to a Temperature Jump: A Time-Resolved Structural Investigation Employing X-Ray Absorption Spectroscopy

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25°C to 47°C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y_D^ox radical and of released Mn²⁺ by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y_D^ox occur simultaneously (k₁ = 1.0 min⁻¹). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by ~3.3 Å, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by ~2.7 Å in the native complex are reduced to Mn(II) and released (k₂ = 0.18 min⁻¹). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di-μ-oxo bridged Mn(III)₂ complex. (3) Finally, the tightly-bound Mn₂(μ-O)₂ unit is slowly reduced and released (k₃ = 0.014 min⁻¹).     

A new interactive computerized data base and retrieval system.

A new computerized data base and retrieval system for a minicomputer has been devised for storing and retrieving clinical data. It features interactive terminal input and output using a pagination scheme as well as a subset mapping scheme for interactive retrieval. The pagination system, file structuring and retrieval system are discussed.     

Proton release from water oxidation by photosystem II: similar stoichiometries are stabilized in thylakoids and PSII core particles by glycerol

During the four-stepped catalytic cycle of water oxidation by photosystem II (PSII) molecular oxygen is released in only one of the four reaction steps whereas the release of four protons is distributed over all steps. In principle, the pattern of proton production could be taken as indicative of the partial reactions with bound water. In thylakoids the extent and rate of proton release varies as function of the redox transition and of the pH without concomitant variations of the redox pattern. The variation has allowed to discriminate between deprotonation events of peripheral amino acids (Bohr effects) as opposed to the chemical deprotonation of a particular redox cofactor, and of water. In contrast, in thylakoids grown under intermittent light, as well as in PSII core particles the pattern of proton release is flat and independent of the pH. This has been attributed to the lack in these materials of the chlorophyll a,b-binding (CAB) proteins. We now found that a thylakoid-like, oscillatory pattern of proton release was restored simply by the addition of glycerol which modifies the protein–protein interaction. Being a further proof for the electrostatic origin of the greater portion of proton release, this effect will serve as an important tool in further studies of water oxidation.