Extrinsic Polypeptides Research Articles

Decline in photosynthetic electron transport activity and changes in thylakoid protein pattern in field grown iron deficient Peach ( Prunus persica L.)

In this work we have studied the influence of lime-induced iron deficiency on some features of the thylakoids from peach trees grown in calcareous soils. Changes in photosynthetic pigments, soluble proteins, photosynthetic activities and thylakoid polypeptides were investigated. The level of total chlorophyll (Chl) and soluble proteins, on a unit fresh weight basis, showed a progressive decrease upon increase in iron deficient condition. When various photosynthetic activities were followed in isolated thylakoids, iron deficiency caused inhibition of whole chain and PS II activity. Smaller inhibition of PS I activity was observed even in severely chlorotic leaves. The artificial exogenous electron donor, Mn 2+ failed to restore PS II activity in both mildly and severely chlorotic leaves, while DPC and NH 2OH significantly restored the PS II activity in severely chlorotic leaves. The same results were obtained when F v/ F m was evaluated by Chl fluorescence measurements. The marked loss of PS II activity in thylakoids isolated from severely chlorotic leaves was due to the loss of D1 protein, 33, 23, 17 kDa extrinsic polypeptides, 28-25 kDa LHCP II polypeptides and 10 kDa polypeptide.

Mutation Val235Ala weakens binding of the 33-kDa manganese stabilizing protein of photosystem II to one of two sites.

The 33-kDa protein of the photosynthetic O2-evolving complex, also known as manganese stabilizing protein, contributes to the structural stability of the photosystem II tetranuclear Mn cluster and stimulates the water-oxidizing activity of this cluster. Quantification of extrinsic polypeptides in photosystem II has yielded data that support stoichiometries of either one or two copies of each protein per photosystem II reaction center. We recently described the cold-sensitive assembly of a mutant 33-kDa protein with a single amino acid replacement (Val235Ala) [Betts, S. D., Ross, J. R., Pichersky, E., & Yocum, C. F. (1996) Biochemistry 35, 6302-6307]. We have extended the characterization of this mutation. When photosystem II membranes depleted of the 33 kDa extrinsic protein are exposed to mixtures of wild type and Val235Ala manganese stabilizing protein, binding of the wild type protein is strongly preferred. If, however, protein containing the Val235Ala mutation is first bound to photosystem II only half of this protein (about 1 mol/mol of photosystem II reaction centers) is susceptible to displacement by the wild type protein, even after multiple exposures to the latter. These results support the conclusion that 2 mol of manganese stabilizing protein are bound per reaction center. Our data show as well that the mutant 33-kDa protein competes with the wild type protein for at least one of two binding sites on photosystem II and that the mutant protein binds tightly to only one of two sites. These results demonstrate that the two binding sites on photosystem II for the 33-kDa protein have different properties with respect to recognition and binding of this protein.

Changes of the photosystem 2 activity and thylakoid proteins in spruce seedlings during water stress

We subjected spruce [Picea glauca (Moench) Voss × P. engelmanni Parry hybrid complex] seedlings to a severe water stress (shoot water potential ≤-3.5 MPa) to permit assessment of stress effects on photosystem 2 (PS2) in isolated photosynthetic membranes. The thylakoids and Triton-treated membranes isolated from stressed seedlings showed declines in O2-evolving capacity (H2O → 2,6-dichloro-p-benzoquinone, DCBQ) and electron transport activity (H2O → 2,6-dichlorophenol indophenol, DCIP). A partial restoration of O2-evolution by adding CaCl2 suggested an effect of water stress on the oxygen-enhancing extrinsic (OEE) polypeptides. Water stress had an additional impact on the reaction centre, shown by the inability of 1,5-diphenylcarbazide (DPC) to restore the electron transport (DPC → DCIP) to the levels seen in control membranes. Quantification of specific photosynthetic membrane proteins by immunoblots strengthened the above suggestions: after drought stress, concentrations of OEE1 and OEE2 declined by 40 %, and amount of the reaction centre protein D1, ATP synthetase, and cytochrome f also declined. The specific effect of stress on these proteins was confirmed by the fact that the amount of chlorophyll-protein complex CP2 was unchanged in membranes isolated from drought-stressed seedlings.

Effect of TCA-depletion of extrinsic polypeptides on electron transport on oxidizing side of PS II

Effect of TCA-depletion of extrinsic polypeptides on electron transport on oxidizing side of PS II

DEGRADATION AND RELEASE FROM THE THYLAKOID MEMBRANE OF PHOTOSYSTEM II SUBUNITS AFTER UV-B IRRADATION OF THE LIVERWORT CONOCEPHALUM CONICUM

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.

X-ray Absorption Spectroscopy of Calcium-Substituted Derivatives of the Oxygen-Evolving Complex of Phostosytem II

X-ray absorption spectroscopy (XAS) has been used to characterize the structural consequences of Ca{sup 2+} replacement in the reaction center complex of the photosynthetic oxygen-evolving complex (OEC). EPR and activity measurements demonstrate that, in the absence of the 17 and 23 kDa extrinsic polypeptides, it is not necessary to use either low pH or Ca chelators to effect complete replacement of the active site Ca{sup 2+} by Sr{sup 2+}, Dy{sup 3+}, or La{sup 3+}. The extended X-ray absorption fine structure (EXAFS) spectra for the OEC show evidence for a Mn...Mn interaction at ca. 3.3 A that could arise either from Mn...Mn scattering within the Mn cluster or Mn...Ca scattering between the Mn cluster and the inorganic Ca{sup 2+} cofactor. There is no significant change in either the amplitude or the phase of this feature when Ca{sup 2+} is replaced by Sr{sup 2+} or Dy{sup 3+}, thus demonstrating that there is no EXAFS-detectable Mn...Ca contribution at ca. 3.3 A in these samples. The only significant consequence of Ca{sup 2+} replacement is a small change in the ca. 2.7 A Mn...Mn distance. The average Mn...Mn distance decreases 0.014 A when Ca{sup 2+} is replaced by Sr{sup 2+} and increases 0.012 A whenmore » Ca{sup 2+} is replaced by Dy{sup 3+}. 75 refs., 10 figs., 5 tabs.« less

95/06648 Direct determination of sulfur in coal by X-ray fluorescence analysis

In chloroplast photosystem II, the extrinsic polypeptide of 33 kDa is involved in the stabilization the Mn cluster in charge of water splitting and in the fulfillment of the Ca2+-cofactor requirement for oxygen evolution. The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures. The FTIR spectroscopic results showed that the isolated polypeptide is characterized by a major proportion β-sheet conformation (36%) with 27% α-helix, 24% turn, and 13% β-antiparallel structures.

The Sequential Release of Three Extrinsic Polypeptides in the PSII Particles by High Concentrations of Trichloroacetate

The Sequential Release of Three Extrinsic Polypeptides in the PSII Particles by High Concentrations of Trichloroacetate

The sequential release of three extrinsic polypeptides in the PSII particles by high concentrations of trichloroacetate

of DDC or DDC disulfide, and the shoulder spectra characteristic of SeT was observed in the wavelength region longer than 300nm. In the FAB-MS spectra of the isolated selenotrisulfide, molecular ion peak, ( M + I ) +, was observed at m/z = 377. Thus, almost pure yellow crystalline SeT was obtained by a very simple preparation method without further purification. Molar ratio of DDC : selenious acid in the reaction under the investigated conditions was 2 : 1 on analysis by the continous variation method and the molar ratio method. A balanced equation for the reaction may be written as follows:

Granal photosystem II complexes contain only the high redox potential form of cytochrome b-559 which is stabilised by the ligation of calcium

Photosystem-II-enriched membrane fragments obtained by detergent solubilisation of thylakoid membranes were found to contain almost exclusively the high redox potential form of cytochrome b-559 ( E m,6.0 = + 353 mV), provided that the haem was maintained in the reduced state during the isolation procedure. A reducing potential was required during the isolation due to the instability of the oxidised form of the high-potential couple. Additional detergent treatment of such preparations converted all of the cytochrome to a low potential form ( E m,6.0 of around + 100 mV). Dissociation of the 23 kDa extrinsic polypeptide, bound at the lumenal side of Photosystem II, had no effect on the redox state of the cytochrome provided that calcium remained in association with the Photosystem II complex. Removal of the 33 kDa extrinsic protein, in addition to the 23 kDa, resulted in the conversion of the haem to an intermediate ( E m,6.0 = + 169 mV) redox form, independent of the presence of calcium. Considering that these preparations are derived from the granal regions of the thylakoid membranes, the data suggest that, in vivo, Photosystem II complexes in these regions contain only the high redox potential form of the cytochrome. The data further suggest that, in addition to the 33 kDa protein, ligation of calcium rather than the 23 kDa polypeptide is required for the stabilisation of this form of cytochrome b-559.

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+.

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative secondary structure analysis of the 33 kDa extrinsic polypeptide of photosystem II by FTIR spectroscopy

In chloroplast photosystem II, the extrinsic polypeptide of 33 kDa is involved in the stabilization the Mn cluster in charge of water splitting and in the fulfillment of the Ca 2+-cofactor requirement for oxygen evolution. The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures. The FTIR spectroscopic results showed that the isolated polypeptide is characterized by a major proportion β-sheet conformation (36%) with 27% α-helix, 24% turn, and 13% β-antiparallel structures.

Europium- and Dysprosium-Ions as Probes for the Study of Calcium Binding Sites in Photosystem II

Abstract Trivalent lanthanide cations are suitable probes for Ca2+-binding sites in photosystem II (PS II). PS II membranes prepared from Nicoticinci tabacum, intact and depleted of the extrinsic polypeptides were exposed to lanthanide ions (Dy3+ and Eu3+). Small concentrations of dysprosium and europium ions enhance oxygen evolution under short saturating flashes. Higher concentrations of the rare earth cations cause the release of the three extrinsic peptides (17, 23 and 33 kDa) and reduce O2 yield. The reactivation of the PS II membranes, thus depleted of the 33 kDa subunit, by Ca2+ ions is not possible. Comparing Eu3+ with Dy3+ in this effect shows that Eu3+ is more effective than Dy3+, because a lower Eu3+-concentration in comparison to that of Dy3+ inactivates O2-evolution. The differences between europium and dysprosium can be explained by their different ionic radius. Our results suggest the existence of two Ca-binding regions: one with a low affinity for calcium would be located on the contact surface of the 23 and 33 kDa proteins and the second one with a high affinity, should be located close to the Mn-cluster and to tyrosine-161 (Z ). The more tightly-bound calcium would be responsible for the activity of the PS II system.

The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II

Mercury (Hg 2+), a sulfhydryl group reactant, wasused to probe structure-function relationships in photosystem II (PSII). In the present work, we investigated the impact of mercury on the polypeptide composition of PSII submembrane preparations. Electrophoretic analysis revealed that the incubation of the membranes in the presence of mercury produces the depletion of a polypeptide of molecular weight of 33 kDa. This polypeptide corresponds to the extrinsic protein EP33 of the oxygen evolving complex removed following urea treatment. However, the two closely related extrinsic polypeptides of 16 and 23 kDa, usually removed concomitantly after urea treatment, remained unaffected after the mercury treatment. These data demonstrated the existence of an intrinsic binding site for EP23. The molecular mode of action of mercury in the oxygen evolving complex of PSII is discussed.

INHIBITION OF OXYGEN EVOLUTION IN CHLOROPLAST PHOTOSYSTEM II BY THE PROTEIN‐MODIFYING AGENT TETRANITROMETHANE

Abstract— The protein‐modifying agent tetranitromethane (TNM) reacts with tyrosine residues and ‐SH groups. It was found to inhibit photo synthetic electron transport on the water splitting side of photosystem II (P. V. Sane and U. Johanningmeier, Z. Naturforsch. 35c, 293–297, 1979). In the present work the inhibition by TNM is studied in detail using photosystem II submembrane fractions. It is shown that the action of TNM with membrane‐bound proteins could imply the modification of tyrosine residues. At concentrations below 30 μM and with short incubation periods (<2 min), TNM produces the release of the extrinsic polypeptides involved in the stabilization of the water‐splitting complex, this being correlated with inhibition of electron transport at a site prior to H2O2 electron donation even though the inhibition cannot be prevented by the addition of Cl or Ca2+, which are known cofactors for oxygen evolution. As the incubation period or the concentration of TNM is increased, photosynthetic pigments are bleached, starting with aggregates absorbing at relatively long wavelengths. The inhibition by low concentrations of TNM differs from the effect of most of the previously reported inhibitors acting at the oxygen‐evolving complex of photosystem II.

The PsaC Protein Is Necessary for the Stable Association of the PsaD, PsaE, and PsaL Proteins in the Photosystem I Complex: Analysis of a Cyanobacterial Mutant Strain

The PsaC protein binds two 4Fe-4S centers, FA and FB, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of β-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex.

Molecular mechanism of action of Pb 2+ and Zn 2+ on water oxidizing complex of photosystem II

Pb 2+ and Zn 2+ inhibition of photosystem II (PSII) activity was reported to be mediated via displacement of native inorganic cofactors (Cl −, Ca 2+ and Mn 2+) from the oxygen evolving complex, OEC [Rashid and Popovic (1990) FEBS Lett. 271, 181–184; Rashid et al. (1991) Photosynth. Res. 30, 123–130]. Since the binding sites of these cofactors are protected by a shield of three extrinsic polypeptides (17, 23 and 33 kDa), we investigated whether these metal ions affect the extrinsic polypeptide shield of OEC. By immunoblotting with antibodies recognizing the 23 and 33 kDa polypeptides, we showed that both the metal ions significantly dissociated the 23 kDa (+17 kDa) polypeptide, and partially dissociated the 33 kDa. Ca 2+, one of the important inorganic cofactors of oxygen evolution, strongly prevented the dissociating action of Pb 2+ but did not prevent the action of Zn 2+. The probable molecular mechanism of action of Pb 2+ and Zn 2+ on PSII OEC is discussed.

The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 μM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 μM. 3.) The photoproduction of O2 (-) is coupled with H(+)-uptake. This effect is diminished by the addition of the O2 (-)-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2 (-) in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.

Effect of excess Cu on the photosynthetic apparatus of runner bean leaves treated at two different growth stages

Runner bean plants (Phaseolus coccineus L., cv Piekny Jas) were treated with excess Cu (20 mg l−1 in the form of CuSO4.5H2O) at different stages of growth to investigate, 10 days after the element treatment, the effect of Cu on acyl lipid and polypeptide composition of the thylakoid membranes and their PSII photochemistry. The plants treated with Cu in the initial stage of leaf growth showed a strong reduction in the area and fresh weight of the primary leaves. The concentration of chlorophyll and acyl lipids slightly increased when calculated on leaf area or fresh weight basis. The decrease in individual acyl lipid classes expressed on chlorophyll basis was accompanied by lower accumulation of some extrinsic polypeptides of the oxygen evolving complex and decrease in PSll activity (80% of control). Chlorophyll a fluorescence measurements suggest an inhibitory effect of Cu on the acceptor side of PSII due to induced inhibition of the Calvin cycle and down‐regulation of electron transport. However, plants treated with Cu by the end of the intensive growth stage of the primary leaves showed chlorosis and almost unchanged leaf area. Moreover, significant changes in acyl lipid content as well as a distinct loss of core antenna PSII polypeptides and oxygen evolving complex subunits were observed. Changes in chlorophyll a fluorescence parameters of the thylakoid membranes suggest that low PSII activity (50% of control) may result from an alteration both in the acceptor and donor sides of PSII and its reaction centre. The growth stage of plants in which Cu was applied to plants and the duration of Cu action appears to be of great importance for the interpretation of experimental data.

The effects of calcium depletion on the O 2-evolving complex in spinach PS II: The S∗ 1, S∗ 2 and S∗ 3 states and the role of the 17 kDa and 23 kDa extrinsic polypeptides

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples depleted of calcium by various NaCl treatments which inhibit oxygen evolution without displacement of managanese. Changes in the pre-edge feature due to 1s → 3d transitions and shifts in the edge position of samples in the S∗ 1, S∗ 2 and S∗ 3 states indicate managanese oxidation for the S∗ 1 → S∗ 2 and S∗ 2 → S∗ 3 transitions. Analysis of the EXAFS shows changes on NaCl treatment compared to native PS II membranes which are further modified by the chelator, EGTA. The intensity of the Fourier transform peak at about 1.8 Å, assigned to oxygen, increases with increasing S-state in agreement with oxidation state changes, although the average distance for this first shell remains constant. Each of the inhibitor-treated S-states have a short average Mn-O bond length, showing the retention of the μ-oxo bridges postulated to occur in native samples. The Mn-Mn shell, found at 2.7 Å in native PS II membranes is split in NaCl-treated samples to give a 2.7 Å Mn-Mn and 3.0 Å Mn-X interaction (X = Mn,C/O/N). Splitting of the 2.7 Å shell is most apparent in the higher S-states, S∗ 3 > S∗ 2 > S∗ 1 . Although the scatterers at 3.0 Å could not be uniquely identified, the intensity favours heavy scatterers, Mn/Ca, over light scatterers, C/O/N. The cluster appears to contain at least two inequivalent Mn-Mn pairs or shows multiple scattering from a ligand such as tyrosine/histidine. NaCl treatment results in a smaller 3.3 3.6 Å intensity compared to untreated PS II samples which could be due to replacement of calcium scatterers at this distance and/or a structural rearrangement. EGTA addition results in an S∗ 2 state with a modified EPR spectrum but has only a small effect on the XAS. The changes on removal of the 17 and 23 kDa extrinsic polypeptides are small compared to the effect of the calcium depletion/NaCl treatment, indicating a minor role for these polypeptides on the structure of the cluster. Changes in the electron spin lattice relaxation time, T 1 of the dark stable tyrosine radical Y D have also been studied using pulsed EPR. The T 1 relaxation times decreased with increasing modified S-state S∗ 1 > S∗ 2 > S∗ 3 , indicating oxidation occurring at or near the manganese cluster.