Use of bacterial photosynthetic vesicles to evaluate the effect of ionic liquids on the permeability of biological membranes.

Ionic liquids (ILs) are salts composed of a combination of organic or inorganic cations and anions characterized by a low melting point, often below 100 °C. This property, together with an extremely low vapor pressure, low flammability and high thermal stability, makes them suitable for replacing canonical organic solvents, with a reduction of industrial activities impact on the environment. Although in the last decades the eco-compatibility of ILs has been extensively verified through toxicological tests performed on model organisms, a detailed understanding of the interaction of these compounds with biological membranes is far from being exhaustive.

A Specific Host/Microbial Signature of Plasma-Derived Extracellular Vesicles Is Associated to Thrombosis and Marrow Fibrosis in Polycythemia Vera.

Polycythemia vera is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. However, no disease-specific risk factors have been identified so far. Circulating extracellular vesicles (EVs) are mostly of megakaryocyte (MK-EVs) and platelet (PLT-EVs) origin and, along with phosphatidylethanolamine (PE)-EVs, play a role in cancer and thrombosis.

Trehalose matrix effects on electron transfer in Mn-depleted protein-pigment complexes of Photosystem II.

The kinetics of flash-induced re-reduction of the Photosystem II (PS II) primary electron donor P was studied in solution and in trehalose glassy matrices at different relative humidity. In solution, and in the re-dissolved glass, kinetics were dominated by two fast components with lifetimes in the range of 2-7 μs, which accounted for >85% of the decay. These components were ascribed to the direct electron transfer from the redox-active tyrosine Y to P.

Dansyl acetyl trehalose: a novel tool to investigate the cellular fate of trehalose.

A fluorescent derivative of trehalose with two dansyl groups (DAT) has been synthesized. It is characterised by a large Stokes shift, good permeability in human living cells and a well detectable fluorescent signal within the cells. Notably, in intestinal cells DAT is sequestered in vesicles induced by trehalose pre-treatment and colocalizes with lipid droplets.

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii.

Background: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg.

Methods: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity.

The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Q site of bacterial cytochrome bc.

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c, which form two catalytic domains, the Q (hydroquinone (QH) oxidation) and Q (quinone (Q) reduction) sites. At the Q site, the pathways of bifurcated electron transfers emanating from QH oxidation are known, but the associated proton release routes are not well defined.

Ionic liquids effects on the permeability of photosynthetic membranes probed by the electrochromic shift of endogenous carotenoids.

Ionic liquids (ILs) are promising materials exploited as solvents and media in many innovative applications, some already used at the industrial scale. The chemical structure and physicochemical properties of ILs can differ significantly according to the specific applications for which they have been synthesized. As a consequence, their interaction with biological entities and toxicity can vary substantially.

Retardation of Protein Dynamics by Trehalose in Dehydrated Systems of Photosynthetic Reaction Centers. Insights from Electron Transfer and Thermal Denaturation Kinetics.

Conformational protein dynamics is known to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presence of glass forming disaccharides, like trehalose, resulting in enhanced protein thermal stability. To shed light on such matrix effects, we have compared the retardation of protein dynamics in photosynthetic bacterial reaction centers (RC) dehydrated at controlled relative humidity in the absence (RC films) or in the presence of trehalose (RC-trehalose glasses). Small scale RC dynamics, associated with the relaxation from the dark-adapted to the light-adapted conformation, have been probed up to the second time scale by analyzing the kinetics of electron transfer from the photoreduced quinone acceptor (QA(-)) to the photoxidized primary donor (P(+)) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s.

Dehydration affects the electronic structure of the primary electron donor in bacterial photosynthetic reaction centers: evidence from visible-NIR and light-induced difference FTIR spectroscopy.

The photosynthetic reaction center (RC) is a membrane pigment-protein complex that catalyzes the initial charge separation reactions of photosynthesis. Following photoexcitation, the RC undergoes conformational relaxations which stabilize the charge-separated state. Dehydration of the complex inhibits its conformational dynamics, providing a useful tool to gain insights into the relaxational processes.

Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus.

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te(IV)) and selenite (Se(IV)) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R. capsulatus cells via acetate permease and it is reduced to Te(0) in the cytoplasm in the form of splinter-like black intracellular deposits no clear mechanism was described for Se(0) precipitation.

Redox regulation of the Calvin-Benson cycle: something old, something new.

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin-Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process.

Trehalose preserves the integrity of lyophilized phycoerythrin-antihuman CD8 antibody conjugates and enhances their thermal stability in flow cytometric assays.

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation.

Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy.

Following light-induced electron transfer between the primary donor (P) and quinone acceptor (Q(A)) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P(+)Q(A)(-). Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P(+)Q(A)(-) recombination kinetics [Malferrari et al., J.

Coupling between electron transfer and protein-solvent dynamics: FTIR and laser-flash spectroscopy studies in photosynthetic reaction center films at different hydration levels.

We report on the relationship between electron transfer, conformational dynamics, and hydration in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides. The kinetics of electron transfer from the photoreduced quinone acceptor (Q(A)(-)) to the photo-oxidized primary donor (P(+)), a charge recombination process sensitive to the conformational dynamics of the RC, has been analyzed at room temperature in dehydrated RC-detergent films as a function of the residual water content under controlled relative humidity (r). The hydration level was evaluated by FTIR spectroscopy from the area of the combination band of water (5155 cm(-1)).

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site.

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function.

Bacterial photosynthetic reaction centers in trehalose glasses: coupling between protein conformational dynamics and electron-transfer kinetics as studied by laser-flash and high-field EPR spectroscopies.

The coupling between electron transfer (ET) and the conformational dynamics of the cofactor−protein complex in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides in water/glycerol solutions or embedded in dehydrated poly(vinyl alcohol) (PVA) films or trehalose glasses is reported. Matrix effects were studied by time-resolved 95 GHz high-field electron paramagnetic resonance (EPR) spectroscopy at room (290 K) and low (150 K) temperature. ET from the photoreduced quinone acceptor (QA•−) to the photo-oxidized donor (P865•+) is strongly matrix-dependent at room temperature: In the trehalose glasses, the recombination kinetics of P865•+QA•−, probed by EPR and optical spectroscopies, is faster and broadly distributed as compared to that of RCs in solution, reflecting the inhibition of the RC relaxation from the dark- to the light-adapted conformational substate and the hindrance of substate interconversion.

X-ray absorption studies of Zn(2+)-binding sites in Escherichia coli transhydrogenase and its betaH91K mutant.

Transhydrogenase couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. The binding of Zn(2+) to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn(2+) bound to Escherichia coli transhydrogenase.

Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins.

In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple-scattering EXAFS analysis performed within the rigid-body refinement scheme, non-muffin-tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye-Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography.

Charge recombination kinetics and protein dynamics in wild type and carotenoid-less bacterial reaction centers: studies in trehalose glasses.

The coupling between electron transfer and protein dynamics has been investigated in reaction centers (RCs) from the wild type (wt) and the carotenoid-less strain R26 of the photosynthetic bacterium Rhodobacter sphaeroides. Recombination kinetics between the primary photoreduced quinone acceptor (QA-) and photoxidized donor (P+) have been analyzed at room temperature in RCs incorporated into glassy trehalose matrices of different water/sugar ratios. As previously found in R26 RCs, also in the wt RC, upon matrix dehydration, P+QA- recombination accelerates and becomes broadly distributed, reflecting the inhibition of protein relaxation from the dark-adapted to the light-adapted conformation and the hindrance of interconversion between conformational substates.

Protein-matrix coupling/uncoupling in "dry" systems of photosynthetic reaction center embedded in trehalose/sucrose: the origin of trehalose peculiarity.

Trehalose is a nonreducing disaccharide of glucose found in organisms, which can survive adverse conditions such as extreme drought and high temperatures. Furthermore, isolated structures, as enzymes or liposomes, embedded in trehalose are preserved against stressing conditions [see, e.g.

Water Activity Regulates the Q(A)(-) to Q(B) electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides.

We report on the effects of water activity and surrounding viscosity on electron transfer reactions taking place within a membrane protein: the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. We measured the kinetics of charge recombination between the primary photoxidized donor (P(+)) and the reduced quinone acceptors. Water activity (aW) and viscosity (eta) have been tuned by changing the concentration of cosolutes (trehalose, sucrose, glucose, and glycerol) and the temperature.

The fe2+ site of photosynthetic reaction centers probed by multiple scattering x-ray absorption fine structure spectroscopy: improving structure resolution in dry matrices.

We report on the x-ray absorption fine structure of the Fe(2+) site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe(2+) is ligated with four N(epsilon) atoms from four histidine (His) residues and two O(epsilon) atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N(epsilon) distances, and two distinct Fe-O(epsilon) bond lengths resolved.

Confinement of cardiolipin and ubiquinone in reaction-center core complexes purified from the photosynthetic bacterium Rhodobacter sphaeroides.

The core complex formed by the reaction center and the light harvesting complex 1 (RC-LH1) was purified from the photosynthetic bacterium Rhodobacter sphaeroides. We analyzed the lipid and ubiquinone (UQ) complements copurifying with the RC-LH1 complex and with the peripheral antenna (LH2). In RC-LH1 UQ was almost ten times more concentrated than in the LH2 and in the native membranes from which the complexes were extracted.

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase.

The metal content of bovine NADH-Q oxidoreductase determined by inductively-coupled plasma atomic-emission spectroscopy reveals the presence of about one atom of zinc per molecule of flavin mononucleotide. We applied Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) to investigate the local structure of the bound zinc ion and to identify the nature of the coordinating residues. The EXAFS spectrum is consistent with a structured zinc binding site.