The plastid terminal oxidase PTOX controls the oxidation level of the plastoquinone pool in the thylakoid membrane and acts as a safety valve upon abiotic stress, but detailed characterization of its role in protecting the photosynthetic apparatus is limited. Here we used PTOX mutants in two model plants Arabidopsis thaliana and Marchantia polymorpha. In Arabidopsis, lack of PTOX leads to a severe defect in pigmentation, a so-called variegated phenotype, when plants are grown at standard light intensities. We created a green Arabidopsis PTOX mutant expressing the bacterial carotenoid desaturase CRTI and a double mutant in Marchantia lacking both PTOX isoforms, the plant-type and the alga-type PTOX. In both species, lack of PTOX affected the redox state of the plastoquinone pool. Exposure of plants to high light intensity showed in the absence of PTOX higher susceptibility of photosystem I to light-induced damage while photosystem II was more stable compared with the wild type demonstrating that PTOX plays both, a pro-oxidant and an anti-oxidant role in vivo. Our results shed new light on the function of PTOX in the protection of photosystem I and II.

An intramolecular Diels-Alder (IMDA) reaction efficiently accelerated by Schreiner's thiourea is reported, to build a functionalized cytochalasin scaffold (periconiasin series) for biological purposes. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines while actin polymerization is preserved.

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was −255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies.

PsbV2 is a c ‐type cytochrome present in a very low abundance in the thermophilic cyanobacterium Thermosynechococcus elongatus . We purified this cytochrome and solved its crystal structure at a resolution of 1.5 Å. The protein existed as a dimer in the crystal, and has an overall structure similar to other c ‐type cytochromes like Cyt c 6 and Cyt c 550 , for example. However, the 5th and 6th heme iron axial ligands were found to be His51 and Cys101, respectively, in contrast to the more common bis‐His or His/Met ligands found in most cytochromes. Although a few other c ‐type cytochromes were suggested to have this axial coordination, this is the first crystal structure reported for a c ‐type heme with this unusual His/Cys axial coordination. Previous spectroscopic characterizations of PsbV2 are discussed in relation to its structural properties.

The redox potential of Q(A) in Photosystem II (PSII) from Thermosynechococcus elongatus was titrated monitoring chlorophyll fluorescence. A high potential form (E-m = +60 +/- 25 mV) was found in the absence of Mn4Ca, the active site for water oxidation. The low potential form (E-m = -60 +/- 48 mV), which is difficult to measure in conventional titration experiments, could be "locked in" by cross-linking the active enzyme. This indicates that the presence of Mn4Ca is relayed to the quinone site by significant structural changes in the protein. The presence of high and low potential forms agrees with what has been seen in plants, algae from our lab and in T. elongatus (Shibamoto et al., Biochemistry 48 (2009) 10682-10684). In the latter work, the potentials of Q(A) were shifted to lower potentials compared to other measurements. The redox potential of Q(A) in Mn-depleted PSII from spinach was titrated in the presence of redox mediators and the midpoint potential was shifted by 80 mV towards a more negative value compared to titrations without mediators. The lower values of the midpoint potential of the (Q(A)/Q(A)(-)) redox couple in the literature could be due to a perturbation due to a specific mediator. (C) 2011 Elsevier B.V. All rights reserved.

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA(3) gene over the psbA(1) gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at <10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q(x) band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo(D1) Q(x) band induced by Q(A)(-) (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t(1/2)>50 ms) Q(A)(-) yield was approximately 95% in D1:3, and approximately 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q(x) band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13(1)-keto of Pheo(D1), which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P(D1), in particular Ser153Ala. Photo-induced Q(A)(-) formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+.

The active site for water oxidation in photosystem II (PSII) consists of a Mn 4 Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S 0 to S 4 ) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn 4 cluster interacting with TyrZ, can be trapped by illumination of the S 0 , S 1 and S 2 states at cryogenic temperatures. The EPR signals reported were attributed to S 0 TyrZ, S 1 TyrZ and S 2 TyrZ, respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn 4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD, PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ interacting with the Mn cluster in S 0 , S 1 , S 2 and also probably the S 3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 Å units away from the Ca site. This could relate to the earlier reported modulation of the potential of Q A by the occupancy of the Ca site. It is also shown that in the S 3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.

Bromide anomalous X-ray diffraction analyses have been used to locate chloride binding sites in the vicinity of the water splitting/oxygen evolving centre (OEC) of Photosystem II. Three-dimensional crystals of PSII from Thermosynechococcus elongatus were grown from (i) isolated PSII crystals infiltrated with bromide or (ii) PSII obtained from cells cultured in a medium in which the chloride content was totally replaced by bromide. In either case, the anomalous diffraction yielded the same result, the existence of two bromide binding sites in the vicinity of the OEC. Neither are in the first coordination sphere of the Mn and Ca ions which form the catalytic centre of the OEC, being about 6 to 7 angstrom from the metal-cluster. Site 1 is located close to the side chain nitrogen of D2-K317 and the backbone nitrogen of D1-Glu333 while Site 2 is adjacent to backbone nitrogens of CP43-Glu354 and D1-Asn338. Their positioning close to postulated hydrophilic channels may suggest a role in proton removal from, or substrate access to, the OEC.

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). Cl- is also required for enzyme activity. To study the role of Ca2+ and Cl- in PSII, these ions were biosynthetically substituted by Sr2+ and Br-, respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O2 polarography, and thermoluminescence spectroscopy. The effect of the Ca2+/Sr2+ and Cl-/Br- exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O2 release was similar to that of the S3TyrZ. to S0TyrZ transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being ≈6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S3 state as manifest by thermoluminescence. These observations indicate that Cl- is involved in the water oxidation mechanism. The possibility that Cl- is close to the active site is discussed in terms of recent structural models.