Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation.

The effect of fatty acid desaturation on lipid fluidity in thylakoid membranes isolated from the STR7 mutant was investigated by electron paramagnetic resonance (EPR) using spin label probes. The spectra of both 5- and 16-n-doxylstearic acid probes were measured as a function of the temperature between 10 and 305 K and compared to those of the wild type. This complete thermal evolution provides a wider picture of the dynamics. The spectra of the 5-n-doxylstearic acid probe as well as their temperature evolution were identical in both STR7 mutant and wild type thylakoids. However, differences were found with the 16-n-doxylstearic acid probe at temperatures between 230 and 305 K. The differences in the thermal evolution of the EPR spectra can be interpreted as a 5-10 K shift toward higher temperatures of the probe motional rates in the STR7 mutant as compared with that in the wild type. At temperatures below 230 K no differences were observed. The results indicated that the lipid motion in the outermost region of the thylakoids is the same in the STR7 mutant as in the wild type while the fluidity in the inner region of the STR7 mutant membrane decreases. Our data point out a picture of the STR7 thylakoid membrane in which the lipid motion is slower most probably as a consequence of fatty acid desaturation deficiency.

Pigment stoichiometry of a newly isolated D1-D2-Cyt b559 complex from the higher plant Beta vulgaris L.

Two D1-D2-Cyt b559 complexes with different pigment stoichiometry were isolated from the higher plant B. vulgaris. The procedures for isolating both complexes only differed in the washing time of the DEAE column with 50 mM Tris-HCl, pH 7.2, 0.05% Triton X-100 and 30 mM NaCl. When the column was washed until the eluate had an absorbance of 0.01 at 670 nm, the isolated D1-D2-Cyt b559 complex presented a pigment stoichiometry of 6 chlorophyll a, 2 beta-carotene, and 1 cytochrome b559 per 2 pheophytin a. In contrast, when the column was exhaustively washed until the eluate reached an absorbance of 0.005 at 670 nm, the complex had a stoichiometry of 4 chlorophyll a, 1 beta-carotene, and 1 cytochrome b559 per 2 pheophytin a. We think that the former stoichiometry corresponds to that of the native D1-D2-Cyt b559 complex. Moreover, both preparations showed 2 mol of pheophytin a per 1 mol of reaction center protein.

Bicarbonate binding to the water-oxidizing complex in the photosystem II. A Fourier transform infrared spectroscopy study.

The light-induced Fourier transform infrared difference (FT-IR) spectrum originating from the donor side of O2-evolving photosystem (PS) II was obtained in non-depleted and CO2-depleted PSII membrane preparations. The observed spectrum free of contributions from the acceptor side signals was achieved by employing 2 mM/18 mM ferri-/ferrocyanide as a redox couple. This spectrum showed main positive bands at 1589 and 1365 cm(-1) and negative bands at 1560, 1541, 1522 and 1507 cm(-1). CO-depleted PSII preparations showed a quite different spectrum. The main positive and negative bands disappeared after depletion of bicarbonate. The addition of bicarbonate partially restored those bands again. Comparison between difference FT-IR spectra of untreated and bicarbonate-depleted PSII membranes indicated that the positive bands at 1589 and 1365 cm(-1) can be assigned to COO- stretching modes from bicarbonate. The higher frequency corresponds to u[as] (COO-) and the lower frequency to u[s] (COO-). 13C-Labeling FT-IR measurements confirmed these findings and also suggested that the negative band at 1560 cm(-1) can be ascribed to u[as] (COO-). The data are discussed in the framework of the suggestion that bicarbonate can be a ligand to the Mn-containing water-oxidizing complex of PSII.

Characterization of a D1-D2-cyt b-559 complex containing 4 chlorophyll a/2 pheophytin a isolated with the use of MgSO4.

A D1-D2-cyt b-559 complex containing 4 chlorophyll alpha, 1 beta-carotene and 1 cytochrome b-559 per 2 pheophytin a has been isolated from spinach with 30% yield using a Q-Sepharose Fast-Flow anion-exchange column equilibrated with 0.1% Triton X-100, 10 mM MgSO4 and 50 mM Tris-HCl (pH 7.2). The preparation was then stabilized with 0.1% dodecyl-beta-D-maltoside. This method gave a yield 10 times higher than that using a Fractogel TSK-DEAE 650(S) column equilibrated with 0.1% Triton X-100, 30 mM NaCl and 50 mM Tris-HCl (pH 7.2). The PS II RC complex was characterized using absorption and fluorescence spectroscopy at 277 and 77 K. A selective reversible bleaching under reducing conditions with maximum at 682 nm, associated with pheophytin a reduction, and light-induced absorption differences with a lifetime of 1.0 ms, ascribed to the triplet state of P680 were measured and indicated that the isolated D1-D2-cyt b-559 complex is active in charge separation. The results are compared with the data obtained for a PS II RC preparation containing 6 chlorophyll alpha, 2 beta-carotene and 1 cyt b-559 per 2 pheophytin a.

Light-induced absorption spectra of the D1-D2-cytochrome b 559 complex of Photosystem II: Effect of methyl viologen concentration.

The light-induced difference absorption spectra associated to the photo-accumulation of reduced pheophytin a were studied in the isolated D1-D2-Cyt b559 complex in the presence of variable methyl viologen concentrations and different illumination conditions under anaerobiosis. Depending on the methyl viologen/reaction centre ratio, the relative intensities of the spectral bands at 681.5+/-0.5, 667.0+/-0.5 and 542.5+/-0.5 nm were modified. The reduced pheophytin a located at the D1-branch of the complex absorbs at 681.7+/-0.5 nm, and at least two additional pigment species contribute to the Q(y) band of the difference absorption spectra with maxima at 667.0+/-0.5 and 680.5+/-0.5 nm. We propose the additional species correspond to a peripheral chlorophyll a and the pheophytin a located at the D2-branch of the complex, respectively. The blue absorbing chlorophyll at 667 nm is susceptible to chemical redox changes with a midpoint reduction potential of +470 mV. The Q(x) absorption bands of both pheophytins localised at the D2- and D1-branch of the D1-D2-Cyt b559 complex were at 540.7+/-0.5 and 542.9+/-0.5, respectively. The results indicated that the two pheophytin molecules can be photoreduced in the D1-D2-Cyt b559 complex in certain experimental conditions.

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants.

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-QA-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, Ki, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the QB niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.

Pigment assignment in the absorption spectrum of the photosystem II reaction center by site-selection fluorescence spectroscopy.

The steady state fluorescence properties of the photosystem II reaction center (D1-D2-cyt-b559 complex, PSII-RC) have been investigated by site-selection spectroscopy. The pattern of the vibronic bands in the emission spectra is used to identify the fluorescing species that have their absorption maxima on the red edge of the spectrum (at around 682 nm). At 10 K, even samples with a low content of red absorbing chlorophyll a (Chl) show pure Chl emission upon excitation at 685 nm, whereas at 77 K the fluorescence of the PSII-RCs is contributed to by Chl and pheophytin a (Pheo) in a ratio of roughly 8:2. These results allow an unequivocal distinction between two different spectral decompositions that were recently suggested for the absorption spectrum of the PSII-RC [Konermann, L., & Holzwarth, A. R. (1996) Biochemistry 35, 829]. Only one of these decompositions is compatible with the experimental data presented here according to which the absorption on the red edge of the spectrum is dominated by an accessory Chl.

Cu(II)-inhibitory effect on photosystem II from higher plants. A picosecond time-resolved fluorescence study.

The influence of Cu(II) inhibition on the primary reactions of photosystem II (PSII) electron transport was studied by picosecond time-resolved fluorescence on isolated PSII membranes. The fluorescence decay from Cu(II)-inhibited PSII centers showed a dominant amplitude of a fast phase (100-300 ps) similar to PSII centers in the uninhibited "open state" and minor contributions of components around 600 ps and 2.6 ns. These data indicate efficient primary charge separation in PSII membranes incubated with Cu(II). The quantum yield of primary reactions in the inhibited PSII centers was similar to that of "open" PSII centers. Kinetic analysis of the decay curves in the framework of the exciton/radical pair equilibrium model showed no significant changes in the rate constants associated with the charge separation/recombination equilibrium. However, in closed centers (QA reduced), a decrease in the rate constant K23, associated with the back-reaction of a relaxed radical pair, by a factor of 4 was calculated. The free energy losses upon primary charge separation (delta G1) and during subsequent radical pair relaxation (delta G2) were also determined in Cu(II)-inhibited centers and were compared with uninhibited centers. No changes in the delta G1 values and a significant decrease in the delta G2 values were found as compared with those of control PSII centers in the "closed" state. These data indicate that Cu(II) does not affect primary radical pair formation, but strongly affects the formation of a relaxed radical pair, by neutralizing the negative charge on QA- and eliminating the repulsive interaction between Pheo- and QA- and/or by modifying the general dielectric properties of the protein region, surrounding these cofactors. Moreover, a close attractive interaction between Pheo-, QA-, and Cu2+ can be proposed. Our results are in good agreement with very recent EPR results indicating an additional effect of Cu2+ on the acceptor side [Jegerschöld et al. (1995) Biochemistry 34, 12747-12758].

Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site.

Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU but became even more sensitive to Cu(II) and o-phenanthroline than nontreated thylakoids; however, trypsinized thylakoids were insensitive to inhibitors in the presence of silicomolybdate. We conclude that Cu(II) impaired the photosystem II electron transfer before the QB niche, most probably at the pheophytin-QA-Fe domain.

Precise location of the Cu(II)-inhibitory binding site in higher plant and bacterial photosynthetic reaction centers as probed by light-induced absorption changes.

Light-dependent absorption change at 325 nm, ascribed to QA activity, was strongly reduced in the presence of Cu(II) in oxygen-evolving core complex. This change was much less affected in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that the Cu(II)-binding site is different from that of the DCMU and that Cu(II) blocks QA reduction. Cu(II) did not eliminate the absorption change at 545 nm, ascribed to pheophytin reduction, in Na2S2O4-treated oxygen-evolving core and D1-D2-cytochrome b559 complexes. This indicates that Cu(II) does not affect the electron transport between P680 and pheophytin. Moreover, the activity of the bacterial reaction center probed by the absorption change at 790 nm was inhibited by Cu(II), but the signal at 530 nm, associated to the reduction of bacteriopheophytin in Na2S2O4-treated reaction center, was not inhibited. We conclude that Cu(II) impaired the photosynthetic electron transport between pheophytin and QA in both higher plants and photosynthetic bacteria. Cu(II) would bind to an amino acid(s) highly conserved in non-oxygenic and oxygenic reaction centers, which is(are) necessary for the electron transfer between pheophytin and QA. Based on the atomic structure of the bacterial reaction center several schemes of possible Cu(II) binding are shown.

Photoinhibition of photosystem II from higher plants. Effect of copper inhibition.

Strong illumination of Cu(II)-inhibited photosystem II membranes resulted in a faster loss of oxygen evolution activity compared with that of the intact samples. The phenomenon was oxygen- and temperature-dependent. However, D1 protein degradation rate was similar in both preparations and slower than that found in non-oxygen evolving PSII particles (i.e. Mn-depleted photosystem II). These results seem to indicate that during illumination Cu(II)-inhibited samples do not behave as a typical non-oxygen evolving photosystem II. Cytochrome b559 was functional in the presence of Cu(II). The effect of Cu(II) inhibition decreased the amount of photoreduced cytochrome b559 and slowed down the rate of its photoreduction. The presence of Cu(II) during illumination seems to protect P680 against photodamage as occurs in photosystem II reaction centers when the acceptor side is protected. The data were consistent with the finding that production of singlet oxygen was highly reduced in the preparations treated with Cu(II). EPR spin trapping experiments showed that inactivation of Cu(II)-treated samples was dominated by hydroxyl radical, and the loss of oxygen evolution activity was diminished by the presence of superoxide dismutase and catalase. These results indicate that the rapid loss of oxygen evolution activity in the presence of Cu(II) is mainly due to the formation of .OH radicals from superoxide ion via a Cu(II)-catalyzed Haber-Weiss mechanism. Considering that this inactivation process was oxygen-dependent, we propose that the formation of superoxide occurs in the acceptor side of photosystem II by interaction of molecular oxygen with reduced electron acceptor species, and thus, the primarily Cu(II)-inhibitory site in photosystem II is on the acceptor side.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II.

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0-8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3- is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of approximately 2.5 microM and 20-34 microM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20-34 microM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. cell cultures deficient in fatty acid desaturation.

The unusual tolerance to heat stress of STR7, an atrazine-resistant mutant isolated from photosynthetic cell-suspension cultures of soybean (Glycine max L. Merr. cv. Corsoy) and characterized previously [M. Alfonso et al. (1996) Plant Physiol 112:1499-1508] has been studied. The STR7 mutant maintained normal growth and fluorescence parameters at higher temperatures than the wild type (WT). The temperature for 50% inactivation of the oxygen-evolving activity of STR7 thylakoids was 13 degrees C higher than in the WT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with specific antibodies revealed that the integrity of photosystem II in the STR7 mutant was maintained at higher temperatures than in the WT. This unusual intrinsic tolerance to high temperatures contrasted with the higher sensitivity to heat stress reported as a feature linked to the triazine-resistance trait. The chloroplast membrane of STR7 accumulated an unusually high content of saturated C16:0 and reduced levels of C16:1 and C18:3 unsaturated fatty acids compared with the WT. Among all the lipid classes, chloroplastic lipids synthesized via the prokaryotic pathway (mono-galactosyl-diacyl-glycerol, phosphatidylglycerol and di-galactosyl-diacyl-glycerol), which represented more than 75% of the total lipid classes, showed the most substantial differences in C16:0 and C18:3 levels. In addition, changes in the physicochemical properties of the thylakoid membrane and chloroplast ultrastructure were also detected.

Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre.

The detergent effect on Cytochrome b559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1-D2-Cyt b559 complex preparations. Various n-dodecyl-beta-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1-D2-Cyt b559 complexes. Low spin heme EPR spectra were obtained but the g(z) feature positions changed depending on the detergent conditions Redox potentiometric titrations showed a unique redox potential cytochrome b559 form (E'm = + 123-150 mV) in all the D1-D2-Cyt b559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome b559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.