Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Variability of photosynthetic capacity and water relations of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. in the field

Measurements of dependence of photosynthetic electron transport on irradiance and analyses of stable isotope ratios (δ¹⁸O, δ¹³C, δ¹⁵N) were performed on 4 to 6-year-old pine trees (Pinus sylvestris L.) in the primeval forest reserve of Białowieża and on 21-year-old pine trees of a plantation of different provenances at the Sękocin Forest Station near Warsaw, Poland. Small differences in maximum photosynthetic electron transport rates, ETR_max were related to growth. Stable isotope analyses suggest that water relations play an important role for the performance of P. sylvestris at the sites studied. The intraspecific comparisons showed a very high variability of photosynthetic capacity between needles of given trees and between individual trees under similar conditions. Differences between specific provenances were also observed. This is relevant for ecological niche occupation in a wide geographical growth range, where P. sylvestris is actually occurring. The high physiological plasticity demonstrated reveals a conspicuous trait of this tree species.     

Mechanisms of hydration effects on the structural-dynamic and functional characteristics of photosynthetic membranes in various purple bacteria

NMR spectra and T₁, T₂ relaxation times for ¹H, ¹³C and ³¹P nuclei in membranes of R. rubrum and Rb. sphaeroides recorded at different relative humidity, as well as hydration curves and electron transfer efficiency of these membranes and membranes of E. shaposhnikovii, reveal complicated relations between structural-dynamic and functional characteristics. A number of sites of the electron transfer chain are shown to be under the control of structural-dynamic mechanisms. Different parameters characterizing these membranes at low humidity and during hydration have been established. These findings and analysis of the data from model systems reveal four different stages of hydration. Each of them is associated with specific changes in structure, dynamics, and function of photosynthetic membranes and their components. In the first stage the hydration of some polar groups leads to local changes in the dynamics of the protein component and this influences the recombination between photoactive pigment P and intermediate acceptor Q_A. The second stage is induced by incorporation of water molecules into the hydrogen bonds between the polar head groups of the lipids and within macromolecules. This results in changes of the dynamics of the membranes, the efficiency of the electron transfer between the quinones and the efficiency of photooxidation of cytochrome c. In the third stage all polar groups are hydrated owing to the appearance of free water with a high dielectric constant. This makes possible lateral mobility of membrane components and changes in distances between the interacting macromolecular components. Therefore, the regulation of photosynthetic processes can be mediated with the participation of mobile carriers. Finally, in the fourth stage, complete humidification provides conditions for regulation of photosynthesis at the cell level. The mechanisms influencing these processes and the efficiency and regulation of electron transfer in various parts of the photosynthetic chain are discussed. Received: 1 August 1996 / Accepted: 4 July 1997     

Proton extrusion by leaf discs ofVicia faba L.: light- and ion-stimulated H(su+) release†

Previous electrophysiological and tracer kinetic studies indicated that the uptake of neutral amino acids took place by means of the proton cotransport mechanism in the leaf tissue of broad bean plants. The present investigations were designed to characterize the origin of the driving force for this process, and the proton pumping activity of leaf cells ofVicia. This activity is known to be revealed when peeled broad been leaf discs, floated on a bathing solution in the light or in darkness acidify the medium. White light caused the strongest acidification. The presence of K⁺ and Na⁺ in the external solution increased the H⁺ secretion significantly, whereas addition of Ca⁺⁺caused only an insignificant enhancement of proton extrusion. The inhibitors of photosynthetic electron transport DCMTJ (50 μM) and nitrofen (50 μM) eliminated the light-enhanced H⁺ release indicating the dependence on photosynthesis. The involvement of a proton pump was evidenced by the effects of the uneoupler CCCP, the SH reagent HgCl₂ and the ATPase inhibitor orthovanadate. The experimental results support the conclusion that H⁺ extrusion byVicia leaf cells is an active electrogenic process requiring metabolic energy. In the light this energy requirement is suppliedvia photosynthetic electron transport. Dedicated to Prof. Dr. F. Jacob on the occasion of his 60th birthday     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Mechanistic Insight into the H<Subscript>2</Subscript>O/D<Subscript>2</Subscript>O Isotope Effect in the Proton Transport of the Influenza Virus M2 Protein

The M2 proton channel is essential for the replication of the flu virus and is a known drug target. The functional mechanism of channel activation and conductance is key to both the basic biology of viral replication and the design of drugs that can withstand mutations. A quantitative model was previously developed for calculating the rate of proton transport through the M2 channel. The permeant proton was assumed to diffuse to the pore, obligatorily bind to the His37 tetrad, and then dissociate and be released to either side of the tetrad. Here the model is used to calculate the effect of a change in solvent from H₂O to D₂O on the rate of proton transport. The solvent substitution affects two parameters in the model: the proton diffusion constant and the pK _a for proton binding to the His37 tetrad. When the known effects on these two parameters are included, the deuterium isotope effect calculated from the model is in quantitatively agreement with experimental results. This strict test of the theoretical model provides strong support for the hypothesis that the permeant proton obligatorily binds to and then unbinds from the His37 tetrad. This putatively essential role of the His37 tetrad in the functional mechanism of the M2 channel makes it a promising target for designing mutation-tolerant drugs.     

Synthesis and physicochemical characterization of a novel precursor for covalently bound macromolecular MRI contrast agents

 The ligand DOTASA was designed and synthesized in the aim of obtaining a kinetically and thermodynamically stable Gd(III) chelate which, through its uncoordinated carboxylate function, will provide an efficient pathway to couple the complex to bio- or macromolecules without affecting the coordination pattern of DOTA. Furthermore, it allows us to study the influence of an extra carboxylate arm on the parameters determining proton relaxivity in comparison to the commercial agent [Gd(DOTA)(H₂O)]^–. A combined variable-temperature ¹⁷O NMR, EPR and nuclear magnetic relaxation dispersion study on the Gd(III) chelate resulted in k ²⁹⁸ _ex=(6.3±0.2)×10⁶ s^–1 for the water exchange rate and τ²⁹⁸ _R=125±2 ps for the rotational correlation time. The slight increase in both k ²⁹⁸ _ex and τ²⁹⁸ _R, as compared to those for [Gd(DOTA)(H₂O)]^–, is attributed to the presence of the extra negative charge. The longer rotational correlation time results in a proton relaxivity of 5.03 mM^–1 s^–1 for [Gd(DOTASA)(H₂O)]^2–, which is approximately 30% higher than that for [Gd(DOTA)(H₂O)]^–. The increased water exchange rate of [Gd(DOTASA)(H₂O)]^2– has no consequence for proton relaxivity since this latter is exclusively limited by fast rotation for both complexes. However, for slowly rotating macromolecular agents, which contain a covalently coupled DOTASA unit instead of a coupled DOTA, this increased exchange rate will have a significant positive effect. Received: 31 December 1998 / Accepted: 4 March 1999     

Isotope effect evidence for the zinc hydroxide mechanism of carbonic anhydrase catalysis

The carbon kinetic isotope effect on the enzymatic dehydration of HCO3- ion is k12/k13 = 1.011 and is independent, within experimental error, of the addition of sucrose, substitution of D2O for H2O, and substitution of enzyme-bound Zn2+ by Co2+. These results are consistent with a ping-pong mechanism in which proton transfer between enzyme and solvent is separated from HCO3- dehydration. For the dehydration half-reaction, diffusional processes are severalfold faster than dehydration, and the rate-determining step is the dehydration itself. The intrinsic isotope effect is approximately 1.011, indicating that hydration of CO2 occurs by reaction of zinc-bound OH-, rather than zinc-bound H2O.     

Modification of protein hydrogen bonds influences the efficiency of picosecond electron transfer in bacterial photosynthetic reaction centers

Picosecond absorption spectroscopy was used to monitor laser-induced oxidation-reductions of reaction center (RC) bacteriochlorophyll (P) and bacteriopheophytin (I) in Rhodopseudomonas sphaeroides RC preparations on exposure to different chemicals. The D₂O isotope substitution of H₂O or partial substitution of water by organic solvents (ethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide) causes the appearance of a fast, nanosecond component of P⁺ reduction, the result of an increased probability of recombination of the primary ion-radical products P⁺I⁻ → PI. The effect is accompanied by a noticeable slowing down of electron transfer from photoreduced bacteriopheophytin to the primary quinone acceptor Q_A. The effect of the organic solvents, known as cryoprotectors, is correlated with their degree of hydrophobicity, i.e. the ability to penetrate the RC protein and interact with bound water and protein hydrogen bonds. The conclusion drawn from the data is that the dielectric relaxation processes through which the intermediate energy levels of the carriers in the PIQ_A system are lowered to levels necessary for the stabilization of the photochemically separated charges proceed with the involvement of protons of the nearest water-protein surrounding of the RC pigments and electron transport cofactors.     

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> during dehydration and rehydration

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO₂ assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO₂ assimilation decline was influenced by stomatal closure. Further lowering of net CO₂ assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S₂Q_A⁻ takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.     

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T₂) of 10^?3—10^?2 s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T₂ of 10^?4 s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.     

Electrochromism: a useful probe to study algal photosynthesis

In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ _H ⁺ ), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ _H ⁺ has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo.     

Co-regulation of dark and light reactions in three biochemical subtypes of C<Subscript>4</Subscript> species

Regulation of light harvesting in response to changes in light intensity, CO₂ and O₂ concentration was studied in C₄ species representing three different metabolic subtypes: Sorghum bicolor (NADP-malic enzyme), Amaranthus edulis (NAD-malic enzyme), and Panicum texanum (PEP-carboxykinase). Several photosynthetic parameters were measured on the intact leaf level including CO₂ assimilation rates, O₂ evolution, photosystem II activities, thylakoid proton circuit and dissipation of excitation energy. Gross rates of O₂ evolution ( J_\textO2 J_{{{\text{O}}_{2} }} , measured by analysis of chlorophyll fluorescence), net rates of O₂ evolution and CO₂ assimilation responded in parallel to changes in light and CO₂ levels. The C₄ subtypes had similar energy requirements for photosynthesis since there were no significant differences in maximal quantum efficiencies for gross rates of O₂ evolution (average value = 0.072 O₂/quanta absorbed, ~14 quanta per O₂ evolved). At saturating actinic light intensities, when photosynthesis was suppressed by decreasing CO₂, ATP synthase proton conductivity (g _H ⁺) responded strongly to changes in electron flow, decreasing linearly with J_\textO2 J_{{{\text{O}}_{2} }} , which was previously observed in C₃ plants. It is proposed that g _H ⁺ is controlled at the substrate level by inorganic phosphate availability. The results suggest development of nonphotochemical quenching in C₄ plants is controlled by a decrease in g _H ⁺, which causes an increase in proton motive force by restricting proton efflux from the lumen, rather than by cyclic or pseudocyclic electron flow.     

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of ¹⁴C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.Abbreviations 9-AA 9-aminoacridine - J _e flux of photosynthetic electron transport - PC photosynthetic control - pH₁ H⁺ concentration in the thylakoid lumen - pmf proton motive force - _P potential quantum yield of photochemistry of photosystem II (with open reaction centers) - Q _A primary quinone-type electron acceptor of photosystem II - q _Q photochemical quenching of chlorophyll fluorescence - q _E energy-dependent quenching of chlorophyll fluorescence - q _AA light-induced quenching of 9-amino-acridine fluorescence     

Enantiodifferent Proton Exchange in Alanine and Asparagine in the Presence of H 2 17 O

Using Time Domain ¹H Nuclear Magnetic Resonance with H₂¹⁷O (H₂¹⁷O-TD-¹HNMR), we found [H₂¹⁷O]- and pH-controlled chiral differences in proton exchange properties in alanine (Ala) and asparagine (Asn). To minimize and equalize chemical impurities, Asn enantiomers were purified by crystallization from racemic solution. At <0.1 M H₂¹⁷O, a shift in isoelectric pH (pI) occurred, ~1.14 kJ mol⁻¹ l-d-Asn ΔΔG ^o′ in the 5.91–6.42 pH range. One potential source for this asymmetry is the enantio-different magnetic moments (lμ↑ ≠ dμ↓) produced by neutral ring currents in the chiral center, leading to enantio-different nuclear spin organization and charge distribution in the amino group. At ≥pI, dissimilar interactions may occur in the hydration of the amino group with H₂¹⁷O (NH₂/H₂¹⁷O ≠ NH₂/H₂¹⁶O; NH₃ ⁺/H₂¹⁷O ≠ NH₂/H₂¹⁷O; l-*C-NH₂/H₂¹⁷O ≠ d-*C-NH₂/H₂¹⁷O). As lμ↑ ≠ dμ↓, the l-*C-amino and the d-*C-amino groups are diastereo spin-isomers. The nuclear spin of ¹⁷O may be parallel or antiparallel with the ortho-¹H¹H pair; hence two ortho-H₂¹⁷O molecules exist, also diastereo spin-isomers. As the pK of H₂¹⁷O is different from H₂¹⁶O, dissimilarities between l-*C- and d-*C-amino groups are converted into proton exchange differences. During H₂¹⁷O-TD-¹HNMR, the H₂¹⁷O molecule is a “probe” of the state of the amino group. Regarding prebiotic evolution: prebiotic chirality may not require stochastic symmetry breaking or preexisting chiral conditions; chemical chiral effects due to lμ↑ ≠ dμ↓ are small and need chiral amplification to generate an enantiomeric excess significant for prebiotic evolution; and prebiotic symmetry breaking was homochiral because the effect of lμ↑ and dμ↓ on the amino group should be similar in all alpha amino acids.     

Photosynthetic and biochemical activities in flag leaves of a newly developed superhigh-yield hybrid rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) and its parents during the reproductive stage

Responses of net photosynthetic rates to intercellular CO₂ concentration (P _n/C _i curves) and photochemical characteristics were investigated in flag leaves of newly developed superhigh-yield hybrid rice (Oryza sativa L.) LiangYouPeiJiu (LYPJ) and its maternal PeiAi64S (PA64S) and paternal WuMang9311 (WM9311) lines grown in the field during the reproductive stage. The results showed that photosynthetic functions, such as the electron transport activities of photosystems and photophosphorylation, assessed in vivo from P _n/C _i curves under field conditions declined more or earlier than those obtained in vitro. The degradation of polypeptides of thylakoid membranes was slower than those for P _Ca=360 (light-saturated net photosynthetic rate measured at 360 μmol mol⁻¹) and CE (carboxylation efficiency, obtained from the initial slope of the P _n/C _i curve). The initial inhibition of the PSII electron transport and oxygen-evolving activity induced by senescence occurred before the degradation of the oxygen-evolving complex. In comparison, LYPJ had intermediate photosynthetic functions in the early stage of leaf development, but greater photochemical activities in the mid and late stages. WM9311 showed a similar pattern of changes but lower values, and PA64S had higher values in the early stage but showed a faster rate of senescence than LYPJ. These findings implied that the hybrid LYPJ demonstrated intermediate photosynthetic activities between its parents in the early stage of leaf development, whereas it had higher photosynthetic activities than its parents in the mid and late stages, which may be responsible for its high yield.     

Photosynthetic carbon acquisition in <Emphasis Type="Italic">Sargassum henslowianum</Emphasis> (Fucales,Phaeophyta), with special reference to the comparison between the vegetative and reproductive tissues

The photosynthetic oxygen evolution characteristics were examined in both vegetative (blade) and sexual reproductive (receptacle) tissues of Sargassum henslowianum (Fucales, Phaeophyta) from the Shenao bay of Nanao Island, China, to establish the mechanism of photosynthetic acquisition of inorganic carbon (Ci) in this species. In natural seawater (pH 8.1, ca. 2.2 mM Ci), irradiance-saturated net photosynthetic rate (NPR) was greater by 25.3% in blade than receptacle, whereas dark respiratory rate (DR) was 2-fold higher in receptacle than blade. NPR at pH 8.1 was nearly saturated with the 2.2 mM Ci for both blade and receptacle. However, the values of the half-saturation constant for Ci were sharply increased at pH 9.0. NPR was significantly affected, but DR was remained unchanged, with the variation of the pH values in seawater. The data from the final pH value derived from the pH-drift experiments and the comparison between the measured and theoretically estimated photosynthetic rates suggested that both blade and receptacle were capable of acquiring HCO₃ ⁻ in seawater. The inhibitors experiments showed that a HCO₃ ⁻ dehydration mechanism mediated by external carbonic anhydrase activity occurred in both the blade and receptacle tissues of S. henslowianum. The proton buffer TRIS had no inhibitory effect on NPR at normal pH value in natural seawater (pH 8.1), but it significantly depressed NPR at pH 9.0. This suggested that proton transport occurred at the outside of the plasma membrane facilitated the operation of the carbon acquisition at pH 9.0. It was proposed that the strategy of photosynthetic carbon acquisition at higher pH would prevent the alga from the damage of over-excitation and photoinhibition in case of sunshine and calm water. We concluded that the blade and receptacle tissues of S. henslowianum have similar mechanism of acquisition of exogenous Ci from seawater to drive photosynthesis; yet they are differentiated more or less with the photosynthetic properties.     

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase

The propionate groups of heme a and a₃ in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a₃, as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H₂O–D₂O isotope substitution experiments combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).     

Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D₂O with respect to H₂O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E° occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.