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In memory of the 85th birthday of Yakov S. Lebedev (Moscow), who died in 1996, we start this Review on soft-glass matrix effects in donor–acceptor complexes with an appreciation of his pioneering work on high-field EPR spectroscopy on tribochemically generated donor–acceptor complexes. The mechanochemical activation of polycrystalline mixtures of porphyrins (and other donors) and quinone acceptors was found to produce large concentrations of triplet donor molecules and donor–acceptor radical pairs with unusual stability. The Review is continued with reporting on W-band high-field EPR and fast-laser studies on disaccharide matrix effects on structure and dynamics of donor–acceptor protein complexes related to photosynthesis, including the non-oxygenic bacterial reaction center (RC) and the oxygenic RCs Photosystem I (PS I) and Photosystem II (PS II, preliminary results). Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, in particular trehalose and sucrose. Trehalose is most effective in protecting biostructures, both in vivo and in vitro. To clarify the molecular mechanisms of disaccharide bioprotection, structure and dynamics of sucrose and trehalose matrices at different controlled hydration levels were probed by perdeuterated nitroxide spin labels and native cofactor intermediates in their charge-separated states. Trehalose forms a homogeneous amorphous phase in which the hosted molecules are uniformly distributed. Notably, their rotational mobility at room temperature is dramatically impaired by thetrehalose H-bonding network confinement to an extent that in normal protein–matrix systems is only observed at low temperatures around 150K. From the experimental results, formation of an extended H-bonding network of trehalose with protein molecules is inferred, involving both bulk and local water molecules. The H-bond network extends homogeneously over the whole matrix integrating and immobilizing the hosted protein. Taken together, these observations suggest that in photosystems, such as bacterial RCs and PS I complexes, of different size and complexity regarding subunit composition and oligomeric organization, the molecular configuration of the cofactors involved in the primary processes of charge separation is not significantly distorted by incorporation into trehalose glass, even under extensive dehydration. By means of pulsed W-band high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, in conjunction with using isotope labeled water (D2O and H217O), the biologically important issue of sensing and quantification of local water in proteins is addressed. The bacterial RC embedded into the trehalose glass matrix is used as model system. The two native radical cofactor ions of the primary electron donor and acceptor as well as an artificial nitroxide spin label site-specifically attached to the protein surface are studied in the experiments. The three paramagnetic reporter groups probe distinctly different local environments. They sense water molecules via their magnetic hyperfine and quadrupole interactions with either deuterons or 17O nuclei. It is shown that by using oxygen-17 labeled water, quantitative conclusions can be drawn differentiating between local and bulk water. It is concluded that dry trehalose operates as anhydrobiotic protein stabilizer by means of selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Such changes usually have an impact on the global function of a biological system. Finally, preliminary results of optical and W-band EPR experiments on the extremolytes ectoine and its derivative hydroxyectoine are reported; these compounds appear to share several stress-protecting properties with trehalose in terms of stabilizing protein matrices. For instance, they display remarkable stabilizing capabilities towards sensitive proteins and enzymes with respect to freeze-thawing, heat-treatment, and freeze-drying procedures. Moreover, hydroxyectoine is a good glass-forming compound and exhibits a remarkable bioprotective effect against desiccation and heat denaturation of functional protein complexes.

The kinetics of flash-induced re-reduction of the Photosystem II (PS II) primary electron donor P680 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 YZ to P680 +. The minor slower components were due to charge recombination between the primary plastoquinone acceptor QA − and P680 +. Incorporation of the PS II complex into the trehalose glassy matrix and its successive dehydration caused a progressive increase in the lifetime of all kinetic phases, accompanied by an increase of the amplitudes of the slower phases at the expense of the faster phases. At 63% relative humidity the fast components contribution dropped to ~50%. A further dehydration of the trehalose glass did not change the lifetimes and contribution of the kinetic components. This effect was ascribed to the decrease of conformational mobility of the protein domain between YZ and P680, which resulted in the inhibition of YZ → P680 + electron transfer in about half of the PS II population, wherein the recombination between QA − and P680 + occurred. The data indicate that PS II binds a larger number of water molecules as compared to PS I complexes. We conclude that our data disprove the “water replacement” hypothesis of trehalose matrix biopreservation.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K4[Fe(CN)6], DPC, TMPD, Fe2+, and Mn2+) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment–protein complexes.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus exploit the simultaneous presence in the cultural medium of the toxic oxyanion tellurite (TeO32-) and the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) by reducing tellurite to metal Te0 nanoprecipitates (TeNPs) outside the cells. Here we have studied the mechanism by which lawsone interacts with metabolically active cells and analysed both structure and composition of the TeNPs collected from the growth medium of phototrophycally grown R. capsulatus. High Resolution Transmission Electron Microscopy (HR-TEM) images and Energy-Dispersive X-ray (EDX) microanalysis of TeNPs showed a central core of polycrystalline tellurium interspersed in an organic matrix with a predominant protein-based composition. The main proteins from Te0 nanostructures were identified by Liquid Chromatography tandem-Mass Spectrometry and were all correlated with the cell outer membrane composition. The interaction of reduced lawsone with tellurite and with the bacterial cells was probed by Cyclic Voltammetry and Scanning ElectroChemical Microscopy (SECM). We concluded that lawsone is required for the reduction of tellurite to metal Te0 in a reaction mechanism dependent on reducing equivalents deriving from the cell photosynthetic metabolism. SECM experiments demonstrate that lawsone, by diffusing inside the bacterial cells, is effectively available at the membrane site of the photosynthetic electron transport chain.

Using isotope labeled water (D2O and H217O) and pulsed W-band (94 GHz) high- field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, the biologically important question of detection and quantification of local water in proteins is addressed. A bacterial reaction center (bRC) from Rhodobacter sphaeroides R26 embedded into a trehalose glass matrix is used as a model system. The bRC hosts the two native radical cofactor ions Image ID:c7cp03942e-t1.gif (primary electron donor) and Image ID:c7cp03942e-t2.gif (primary electron acceptor) as well as an artificial nitroxide spin label site-specifically attached to the surface of the H-protein domain. The three paramagnetic reporter groups have distinctly different local environments. They serve as local probes to detect water molecules via magnetic interactions (electron–nuclear hyperfine and quadrupole) with either deuterons or 17O nuclei. bRCs were equilibrated in an atmosphere of different relative humidities allowing us to control precisely the hydration levels of the protein. We show that by using oxygen-17 labeled water quantitative conclusions can be made in contrast to using D2O which suffers from proton–deuterium exchange processes in the protein. From the experiments we also conclude that dry trehalose operates as an anhydrobiotic protein stabilizer in line with the “anchorage hypothesis” of bio-protection. It predicts selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Changes in hydrogen-bonding patterns usually have an impact on the global function of a biological system.

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. While in wet trehalose matrices (down to approximately one water per trehalose molecule) P+QA- recombination kinetics are essentially coincident in wt and R26 RCs, more extensive dehydration leads to two-times faster and more distributed kinetics in the carotenoid-containing RC, indicating a stronger inhibition of the internal protein dynamics in the wt RC. Coarse-grained Brownian dynamics simulations performed on the two RC structures reveal a markedly larger flexibility of the R26 RC, showing that a rigid core of residues, close to the quinone acceptors, is specifically softened in the absence of the carotenoid. These experimental and computational results concur to indicate that removal of the carotenoid molecule has long-range effects on protein dynamics and that the structural/dynamical coupling between the protein and the glassy matrix depends strongly upon the local mechanical properties of the protein interior. The data also suggest that the conformational change stabilizing P+QA- is localized around the QA binding pocket.

Matrix effects on long-range electron transfer were studied in cyanobacterial Photosystem I (PS I) complexes, embedded into trehalose glasses at different hydration levels. W-band EPR studies demonstrated, via nitroxide spin probes, structural homogeneity of the dry PS I-trehalose matrix and no alteration of cofactors' distance and relative orientation under temperature and matrix variation. In dry trehalose glasses at room temperature (RT), PS I was stable for months. Flash-induced charge recombination kinetics were examined by high-field time-resolved EPR and optical spectroscopies. The kinetics in hydrated PS I-trehalose glasses mostly reflected the reduction of the photooxidized primary donor P700•+ by the reduced terminal iron-sulfur clusters. Upon dehydration, the P700•+ decay accelerated and became more distributed. Continuous distributions of lifetimes τ were extracted from the kinetics by two numerical approaches: a maximum entropy method (MemExp program) and a constrained regularization method (CONTIN program). Both analyses revealed that upon dehydration the contribution of the two slowest components (lifetimes τ ~ 300 ms and ~ 60 ms), attributed to P700•+[FA/FB] − recombination, decreased in parallel with the increase of the fastest component (τ ~ 150 μs), and of additional distributed phases with intermediate lifetimes. Dehydration at RT mimicked the effects of freezing water-glycerol PS I systems, suggesting an impairment of PS I protein dynamics in the dry trehalose glass. Similar effects were observed previously in bacterial reaction centers. The work presented for PS I provides new insights into the crucial issue of protein-matrix interactions for protein functionality as controlled by hydrogen-bond networks of the hydration shell.

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 photo-synthetic 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 (Q(A)(-)) to the photoxidized primary donor (P+) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s. A more severe inhibition of dynamics is found in RC trehalose glasses than in RC films: only in the latter system does a complete relaxation to the light-adapted conformation occur even at extreme dehydration, although strongly retarded. To gain insight into the large scale RC dynamics up to the time scale of days, the kinetics of thermal denaturation have been studied at 44 degrees C by spectral analysis of the Q(x) and Q(y) bands of the RC bacteriochlorin cofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 10(4). Upon increasing m, denaturation is slowed progressively, and above m similar to 500 the RC is stable at least for several days. The stronger retardation of RC relaxation and dynamics induced by trehalose is discussed in the light of a recent molecular dynamics simulation study performed in matrixes of the model protein lysozyme with and without trehalose. We suggest that the efficiency of trehalose in retarding RC dynamics and preventing thermal denaturation stems mainly from its propensity to form and stabilize extended networks of hydrogen bonds involving sugar, residual water, and surface residues of the RC complex and from its ability of reducing the free volume fraction of protein alone matrixes.

Following light-induced electron transfer between the primary donor (P) and quinone acceptor (QA) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P+QA− . Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P+QA− recombination kinetics [Malferrari et al., J. Phys. Chem. B 115 (2011) 14732-14750]. To clarify the structural basis of the conformational relaxations and the involvement of bound water molecules, we analyzed light-induced P+QA− /PQA difference FTIR spectra of RC films at two hydration levels (relative humidity r = 76% and r = 11%). Dehydration reduced the amplitude of bands in the 3700-3550 cm−1 region, attributed to water molecules hydrogen bonded to the RC, previously proposed to stabilize the charge separation by dielectric screening [Iwata et al., Biochemistry 48 (2009) 1220-1229]. Other features of the FTIR difference spectrum were affected by partial depletion of the hydration shell (r = 11%), including contributions from modes of P (9-keto groups), and from NH or OH stretching modes of amino acidic residues, absorbing in the 3550-3150 cm−1 range, a region so far not examined in detail for bacterial RCs. To probe in parallel the effects of dehydration on the RC conformational relaxations, we analyzed by optical absorption spectroscopy the kinetics of P+QA− recombination following the same photoexcitation used in FTIR measurements (20 s continuous illumination). The results suggest a correlation between the observed FTIR spectral changes and the conformational rearrangements which, in the hydrated system, strongly stabilize the P+QA− charge separated state over the second time scale.

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. Similarly accelerated kinetics was observed also in PVA at a water-to-RC molar ratio 10-fold lower than in trehalose. Despite the matrix dependence of the ET kinetics, continuous-wave (cw) EPR and electron spin echo (ESE) analyses of the photogenerated P865•+ and QA•− radical ions and P865•+QA•− radical pairs do not reveal significant matrix effects, at either 290 or 150 K, indicating no change in the molecular radical-pair configuration of the P865•+ and QA•− cofactors. Furthermore, the field dependences of the transverse relaxation times T2 of QA•− essentially coincide in trehalose and PVA at 290 K. T2 is similar in these two matrixes and in the glycerol/water system at 150 K, implying that the librational dynamics of QA•− are also unaffected by the matrix. We infer that the relative geometry of the primary donor and acceptor, as well as the local dynamics and hydrogen bonding of QA in its binding pocket, are not involved in the stabilization of P865•+QA•−. We suggest that the RC relaxation occurs rather by changes throughout the protein/solvent system. The control of the RC dynamics and ET by the environment is discussed, particularly with respect to the extraordinary efficacy of trehalose matrixes in restricting the RC motional degrees of freedom at elevated temperatures.

The photosynthetic reaction center (RC) of Rb. sphaeroides catalyzes light-induced electron transfer events which are connected to the conformational dynamics of the protein. The light-induced charge separation between the primary donor (P) and the quinone acceptor (QA) is stabilized by solvent/protein conformational rearrangements. After a laser pulse P+QA recombination, which occurs with a lifetime t~100 ms in room temperature solutions, is accelerated (t~20 ms) at cryogenic temperatures [1] and in dehydrated glassy matrices at room temperature [2]. After prolonged photoexcitation, a slow phase of recombination (t~250 s) is observed, attributed to additional conformational changes [3]. Differential FTIR bands of water associated with the QA/QA transition have been observed upon continuous illumination, leading to propose that weakly bound water molecules plays a role in P+QA stabilization [4]. By controlling the hydration level of RC-detergent films, through equilibration at given relative humidities (r), a strong inhibition of the P+QA conformational stabilization has been observed at low hydration [5]. We compared FTIR light-minus-dark (P+QA/ PQA) differential spectra in hydrated (r=76%) and dehydrated (r=11%) RC films over the 4000- 1000 cm-1. The spectra differ significantly in the 3750-3550 cm-1 range, the band attributed to weakly hydrogen bonded water molecules [5] being strongly reduced in the dried film. Dehydration also affects the 1800-1200 cm-1 range, which includes contributions from P, the quinones and the peptide. Optical absorption measurements performed under the same photoexcitation regime reveal a slow (t~5 s) kinetic component of P+QA recombination which disappears in the dehydrated sample, indicating at low r a destabilization of the charge separated state. As a whole the data suggest a correlation between the hydration shell dynamics and the conformational RC dynamics which stabilize the charge separated state.

The interplay between protein dynamics and electron transfer (ET) has been extensively investigated in the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides by hampering RC internal motions at low temperatures [1]. Alternatively, the RC dynamics can be inhibited at room temperature by incorporating the RC into dehydrated trehalose matrices [2]. In the glasses the recombination kinetics of the charge separated state P+QA are accelerated and distributed in rate as compared to solution, mimicking at room temperature the effects observed at 10 K in water-glycerol. This is taken to indicate inhibition of the RC relaxation from the dark- to the light-adapted conformation, as well as of the RC thermal fluctuations [1,2]. We proposed that the inhibition is mediated by residual water molecules of the RC hydration shell which bridge protein surface groups with trehalose molecules of the matrix by forming a network of multiple hydrogen bonds [3]. Consistently, similar effects have been observed also in RC films dehydrated in the absence of sugar [4]. However, striking differences are found between RC-trehalose glasses and RC-films: (a) The thermal stability of the RC is tremendously enhanced in the trehalose matrix. (b) In RC-trehalose matrices, the P+QA recombination after a few seconds of continuous photoexcitation is only partially decelerated as compared to the one recorded after a laser flash, whereas in dried RC films a comparable period of continuous illumination leads to a total recovery of the kinetics observed in the hydrated system. These data indicate that in films the protein dynamics can be easily regained, in contrast to trehalose glasses, which reveal much stronger structural constraints. We are extending these studies to a series of RC mutants characterized by a widely altered P+/P midpoint potential relative to wild-type [5]. In these mutants, the acceleration of P+QA recombination induced by cooling to 10 K in the dark decreased with increasing midpoint potential [6]. Interestingly, incorporation into dehydrated trehalose matrices causes instead acceleration of the kinetics by the same factor for all P+/P midpoint potential values.