The state of manganese in the photosynthetic apparatus: 5. The chloride effect in photosynthetic oxygen evolution. Is halide coordinated to the EPR-active manganese in the O2-evolving complex? Studies of the substructure of the low-temperature multiline EPR signal

The role of chloride in the manganese-containing oxygen-evolving complex of Photosystem II has been studied by observing the amplitude of the multiline EPR signal as a function of Cl⁻ concentration or when Cl⁻ is replaced by Br⁻ or F⁻. The correlation of the multiline EPR signal intensity and O₂ activity with the concentration of Cl⁻ shows that chloride is involved in oxygen evolution at the S₂ or earlier S states, and that it is necessary for the production of an EPR-detectable S₂ state. We have developed a new method for the preparation of subchloroplast PS II particles containing Br⁻ and F⁻) and have used these particles for studying the EPR fine structure at high resolution. The fine structure shows a multiplet of 4–6 lines with 10–15 G spacing; at the resolution of our experiment there are no significant differences between the Cl⁻-and Br⁻-containing samples, suggesting that the halide is not a ligand of the EPR-active Mn. Various structural possibilities for the Mn complex, which would account for the observed fine structure of the multiline EPR spectrum are discussed.     

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI⁻ secretion, their effects on CI⁻ transport and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl⁻ sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca²⁺-sensitive fluorescent probe, fura-2AM were used to assess Cl⁻ transport and [Ca²⁺]_i, respectively. Basal Cl⁻ transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl⁻ channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na⁺/K⁺/2Cl⁻ cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I⁻ > F⁻ ≥ Cl⁻ > Br⁻. DPC inhibited I⁻ influx by ∼50%, F⁻ influx by 80%, Cl⁻ influx by 85%, and Br⁻ influx by 90%. Furosemide significantly inhibited influx of Br⁻ (84%) and Cl⁻ (81%) but not of F⁻ and I⁻. The effects of agents known to alter biological response by increasing [Ca²⁺]_i in other epithelial systems were used to stimulate Cl⁻ transport. Cl⁻ influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca²⁺ ionophore A23187 (2.05 ± 0.40). The Cl⁻ permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl⁻ influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl⁻ permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl⁻ transport by activating the Na⁺/K⁺/2Cl⁻ cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl⁻ channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc.     

The effect of chloride on the thermal inactivation of oxygen evolution

The effect of Cl depletion on the sensitivity of the oxygen-evolving complex of Photosystem II (PS II) to heat treatment was examined by a parallel study of the Hill activity (H₂O2,6-dichlorophenolindophenol), Cl^- binding (by ³⁵Cl-NMR) and Mn release (by EPR). The extent of thermal inactivation in spinach thylakoids was found to depend on the degree of Cl^- depletion in the sample. In partially Cl^--depleted thylakoids, mild heating (38°C, 3 min) was found to eliminate inflections in plots of both Hill activity versus [Cl^-] (at low light intensity) and excess ³⁵Cl-NMR linewidth versus [Cl^-] (in the dark). In PS II membranes, the same treatment reduced the differences between the linewidth maxima and minima, particularly in the region of 0.3 mM and 7.0 mM Cl^-, as compared to unheated membranes. These results indicate that mild heating affects the Cl^--binding domains within the oxygen-evolving complex, OEC, EPR measurements of the temperature dependence of Mn release from heated thylakoids show that Mn release begins to correlate with the loss of Hill activity only at higher temperatures, where the OEC is already substantially inactivated. We conclude from these studies that the Cl^--binding domains of the OEC constitute a principal site of damage by heat treatment.     

The association of functional anions with the oxygen-evolving center of chloroplasts

The association of the site of photosynthetic water oxidation with Cl⁻ and other activating anions was analyzed with Photosystem II-enriched thylakoid particles prepared by Triton X-100 treatment. On the basis of the experimental evidence it is proposed that, regardless of the presence of the extrinsic 18 and 23 kDa polypeptides, the reactivation of Cl⁻-depleted particles by added anions is contingent upon the protonation of membrane-bound buffering groups with an apparent pK_a of approx. 6. The rate of dissociation of the anion from the site of water oxidation followed the order NO₃⁻ . ClO₄⁻ . Br⁻ ≈ Cl⁻. A model is developed that takes into account the known requirement of more than 1 anion/center for maximum activity. It assumes a finite and anion-dependent capacity of the water-oxidizing site for the activating anions, and that the rate of photosynthetic oxygen evolution is proportional to the number of anions at the water-oxidizing enzyme. In accord with suggestions made earlier, and in agreement with their further elaboration by Coleman and Govindjee (Proc. 16th FEBS Congress, Moscow, VNU Science Press, Utrecht), it is proposed that the Cl⁻ requirement of Photosystem II is linked to protonation-deprotonation events associated with photosynthetic water oxidation.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

A comparative study of myoglobin stability in the presence of Hofmeister anions of ionic liquids and ionic salts

To reveal the impact of ionic liquids (ILs) on the stability of proteins, a series of ILs possessing same 1-butyl-3-methylimidazolium cation [Bmim]⁺ with a set of Hofmeister anions such as SCN⁻, HSO₄⁻, Cl⁻, Br⁻, CH₃COO⁻ and I⁻ were used and their effects on the myoglobin (Mb) structure and stability were studied. For the sake of comparison and also to explore the extent of the stabilization behavior of ILs toward Mb stability, we have chosen a set of ionic salts (I_s) of a fixed sodium cation (Na⁺) with the same series of anions such as SCN⁻, SO₄⁻², Cl⁻, Br⁻, CH₃COO⁻ and I⁻. UV–vis, fluorescence and circular dichroism (CD) spectroscopic techniques were used in order to investigate the stability behavior of Mb in ionic species (I_s and ILs). The results reveal that both I_s and ILs had a negative influence on the stability of Mb. Apparently, the flexibility in the native structure of Mb gradually increases with the increase in the concentration of I_s and ILs at pH 7.0. Therefore, a sharp decrease in the transition temperature (T_m) of the native Mb is observed in the presence of I_s and ILs.     

The role of chloride in O2 evolution by thylakoids from salt-tolerant higher plants

(1) Thylakoids isolated from leaves of two salt-tolerant higher plant species were found to require high (greater than 250 mM) concentrations of Cl⁻ for maximal rates of photosynthetic O₂ evolution and maximum variable chlorophyll a fluorescence yield. These activities were also tolerant to extremely high (2–3 M) salt concentrations. Their pH dependence was markedly different in the absence and presence of sufficient salt levels. (2) When Cl⁻ was provided as CaCl₂, as opposed to MgCl₂, KCl or NaCl, higher rates of O₂ evolution were obtained, suggesting that Ca²⁺ has an important role in Photosystem II reactions. (3) The site of Cl⁻ action was located on the electron donor side of Photosystem II. (4) O₂ evolution in the presence of optimal Cl⁻ concentrations showed a pH dependence closely matched by that of ³⁵Cl-NMR line broadening, which is indicative of Cl⁻ binding. This pH-dependent ³⁵Cl-NMR line-width broadening was not altered significantly by treatment of the thylakoids with EDTA; it was, however, abolished by heat treatment. (5) Only anions with similar ionic radii (Br⁻, NO⁻₃) were effective in replacing Cl⁻. Small anions such as F⁻ and OH⁻ were inhibitory; larger ions had no effect. The inhibition by F⁻ is due, at least in part, to displacement of Cl⁻. The selectivity is attributed to a combination of steric and ionic field effects. (6) It is proposed that Cl⁻ facilitates Photosystem II electron transport by reversible ionic binding to the O₂-evolving complex itself or to the thylakoid membrane in close proximity to it.     

Stability and oscillation properties of thermoluminescent charge pairs in the O2-evolving system depleted of Cl− or the 33 kDa extrinsic protein

The effects of Cl⁻ depletion and removal of the 33 kDa extrinsic protein on the charge stabilization in O₂-evolving Photosystem II (PS II) particles were studied by curve fitting and deconvolution of thermoluminescence bands. The following results were obtained. (1) Cl⁻ depletion reversibly decreases the redox potential of the S₂ state by 60–80 mV, and thereby elevates the recombination temperature of both S₂Q⁻_B and S₂Q⁻_A charge pairs. (2) Removal of the 33 kDa extrinsic protein specifically elevates the recombination temperature of the S₂Q⁻_A charge pair, with practically no effect on the S₂Q⁻_B pair. This was tentatively interpreted as showing that the protein removal decreases the redox potential of both S₂ and Q⁻_B, but not of Q⁻_A, and, thus, the effects are mutually cancelled for the S₂Q⁻_B pair, but are manifested for the S₂Q⁻_A pair. (3) Deconvolution of glow curves demonstrated that S₃ is not formed in Cl⁻-depleted PS II, but is formed in 33 kDa protein-depleted PS II even at a low (20 mM) Cl⁻ concentration. Analysis of thermoluminescence oscillations confirmed that Cl⁻ depletion interrupts S₂-S₃ transition, whereas the protein removal interrupts S₃-(S₄)-S₀ transition at mM Cl⁻. (4) Cl⁻ depletion by SO²⁻₄ replacement in the absence of 33 kDa protein affected thermoluminescence in a different way from that in the presence of the protein. Based on these findings, the properties of charge pairs in the Cl⁻-depleted PS II particles were discussed in relation to the role of the 33 kDa extrinsic protein.     

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

Effects of metal ions on the hydrolysis of p-nitrophenyl caproate by modified poly(ethylenimine)s

Hydrolysis of nitrophenyl caproate by modified poly(ethylenimine)s containing imidazole moieties is markedly enhanced in the presence of divalent metal ions. The order of effectiveness is Cu(II) > Co(II) > Zn(II) > Ni(II) > Mn(II), with Cu increasing the rate about 20-fold. The acceleration by the metal ion is much greater in the presence of CH₃COO^? or Cl^? as counterions than in the presence of ClO₄^?. Turnover experiments, in which moles of substrate cleaved were in substantial excess of moles of metal present, established the catalytic nature of the effects of the metals. The extent of binding of Cu(II) by the polymers was measured. Maximum accentuation in rate of hydrolysis of nitrophenyl ester was observed for the imidazole-containing polymer when the ratio of imidazole:Cu(II) was 2.75. Some possible mechanisms for the rate enhancement by metal ions are described.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Effects of phosphonium-based ionic liquids on the lipase activity evaluated by experimental results and molecular docking

In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P₄₄₄₄]⁺, [P_444(14)]⁺, [P_666(14)]⁺) and anions (Cl⁻, Br⁻, [Deca]⁻, [Phosp]⁻, [NTf₂]⁻) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]⁻ and [NTf₂]⁻ increase the BCL activity, while the remaining [P_666(14)]-based ILs with the Cl⁻, Br⁻, and [Deca]⁻ anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P_666(14)][NTf₂] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L⁻¹). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]⁻ has a higher binding affinity compared to Cl⁻ and Br⁻, and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]⁻ and [NTf₂]⁻ have high binding energies (−6.2 and −5.6 kcal·mol⁻¹, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.     

Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

Study on the photo-generation of superoxide radicals in Photosystem II with EPR spin trapping techniques

Direct EPR evidence of the photo-generation of superoxide radicals (O₂ ^–.) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O₂ ^–. was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O₂ ^–. formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O₂ ^–. were generated from PS II, not from PS I. The inhibition of O₂ ^–. formation by DCMU also suggested that O₂ ^–. were generated from the Q_Bbinding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O₂ ^–., showing that the oxygen-evolving system is involved in O₂ ^–. removal rather than production.This revised version was published online in October 2005 with corrections to the Cover Date.     

Regeneration of the high-affinity manganese-binding site in the reaction center of an oxygen-evolution deficient mutant of Scenedesmus by protease action

The O₂-evolution deficient mutant (LF-1) of Scenedesmus obliquus inserts an unprocessed D1 protein into the thylakoid membrane and binds less than half the wild type (WT) level of Mn. LF-1 photosystem II (PS II) membrane fragments lack that part of the high-affinity Mn²⁺-binding site found in WT membranes which may be associated with histidine residues on the D1 protein (Seibert et al. 1989 Biochim Biophys Acta 974: 185–191). Hsu et al. (1987 Biochim Biophys Acta 890: 89–96) purport that the high-affinity site (characterized by competitive inhibition of DPC-supported DCIP photoreduction by M concentrations of Mn²⁺) in Mn-extracted PS II membranes is also the binding site for Mn functional in O₂ evolution. Proteases (papain, subtilisin, and carboxypeptidase A) can be used to regenerate the high-affinity Mn²⁺-binding site in LF-1 PS II membranes but not in thylakoids. Experiments with the histidine modifier, DEPC, suggest that the regenerated high-affinity Mn²⁺-binding sites produced by either subtilisin or carboxypeptidase A treatments were the same sites observed in WT membranes. However, none of the protease treatments produced LF-1 PS II membranes that could be photoactivated. Reassessment of the processing studies of Taylor et al. (1988 FEBS Lett 237: 229–233) lead us to believe that their procedure also does not result in substantial photoactivation of LF-1 PS II membranes. We conclude that (1) the unprocessed carboxyl end of the D1 protein in LF-1 is located on the lumenal side of the PS II membrane, (2) the unprocessed fragment physically obstructs or perturbs that part of the high-affinity Mn²⁺-binding site undetectable in LF-1, and (3) the D1 protein must be processed at the time of insertion into the membrane for normal O₂-evolution function to result.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DEPC diethylpryocarbonate - DPC 1,5-diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - LDS-PAGE lithium dodecylsulfate polyacrylamide gel electrophoresis - LF-1 a low-fluorescent mutant of Scenedesmus obliquus - MES 4-morpholineethanesulfonic acid - PS II photosystem II - PMSF phenylmethylsulfonyl fluoride - RC photosystem II reaction center - Tris tris(hydroxymethyl)aminomethane - WT wild type Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC-02-83CH10093.     

Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule

Ionic Cl⁻ currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl⁻ conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying I-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I⁻ > Br⁻ > Cl⁻ > > glutamate. The forskolin-activated Cl⁻ conductance demonstrated a linear I-V relationship and its ion selectivity was Br⁻ > Cl⁻ > I⁻ > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl⁻ currents, and DIDS inhibited only the swelling-activated Cl⁻ current. Forskolin had no effect on the activation of the swelling-activated Cl⁻ current. In DCTb cells which exhibited swelling-activated Cl⁻ currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl⁻ currents. In the continuous presence of I⁻ which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl⁻ conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.