Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions

COPPER EFFECT ON CYTOCHROME b559 OF PHOTOSYSTEM II UNDER PHOTOINHIBITORY CONDITIONS by María Bernal1, Mercedes Roncel2, Jose María Ortega2, Rafael Picorel1 and Inmaculada Yruela1,* 1Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), Apdo. 202, E-50080 Zaragoza, Spain. 2Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas (CSIC), Americo Vespucio s/n, E-41092 Sevilla, Spain. ABSTRACT Toxic Cu(II) effect on Cytochrome b559 under aerobic photoinhibitory conditions was examined in two different PSII membrane preparations active in oxygen evolution. The preparations differ in the content of Cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu(II) induced the oxidation of the high-potential form of Cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidised high-potential form of Cytochrome b559 present in Cu(II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of Cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu(II) effect on Cytochrome b559 as compared to the rapid loss of oxygen evolution activity in the same conditions. This result indicates that Cytochrome b559 is affected after PSII centers are photoinhibited. The high-potential form was more sensitive to toxic Cu(II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of Cytochrome b559 was completely lost, however the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. Results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of Cytochrome b559, respectively. Keywords: cytochrome b559, copper, photosystem II, photoinhibition, redox potential Abbreviations: Cyt, cytochrome; DCBQ, dichlorobenzoquinone ; D1, polypeptide of the photosystem II reaction centre ; HP, high potential; LP, low potential; MES, 2-(N-morpholino) ethane-sulfonic acid; PAGE, polyacrilamide gel electrophoresis; PS, photosystem; RC, reaction center; SDS, sodium dodecyl sulphate ; Tris, Tris(hydroxymethyl)aminomethane. 2 INTRODUCTION Several heavy metals are essential for plant growth and development, but their excess can easily lead to toxic effects. Among them Cu(II) is known to be toxic at high concentrations for photosynthetic organisms (Clijsters and van Asche, 1985; Maksymiec, 1997). Extensive in vitro studies have shown that photosystem II (PSII) is more susceptible to copper toxicity (for review see Droppa and Horváth, 1990; Barón et al. 1995) than photosystem I (PSI) (Ouzounidou et al. 1997). Both acceptor and donor sides of PSII have been proposed as copperinhibitory sites. At the acceptor side, the QB binding site (Mohanty et al. 1989) and the Pheo-Fe-QA domain (Yruela et al. 1991, 1992, 1993, 1996a) have been reported. Evidences that copper ion impairs the function of the oxidising side has been reported (Cedeño-Maldonado et al. 1972; Vierke and Struckmeier, 1977; Shioi et al. 1978a,b; Bohner et al. 1980; Samuelsson and Öquist, 1980). It has been shown that the electron flow from Tyrz to P680+ is blocked by Cu(II) (Schröder et al. 1994; Arellano et al. 1995). Indeed, Králova et al. (1994) and Sersen et al. (1997) have proposed that Cu(II) interacts not only with Tyrz but also with TyrD on D2 protein. A possible direct interaction between copper and calcium at the oxidising side of PSII was also shown both in vitro (Sabat, 1996) and in vivo (Maksymiec and Baszynski, 1999). Additional effects of Cu(II) toxicity on both donor and acceptor sides have been reported using higher copper concentrations. In such conditions the Mn-cluster and the extrinsic proteins on the donor side can be affected. Interaction of copper with the non-heme Fe2+ and cytochrome (Cyt) b559 on the acceptor side has been also reported. (Renger et al. 1993; Sersen et al. 1997; Jegerschöld et al. 1995, 1999). 3 The photosynthetic activity decreases when oxygenic organisms are exposed to prolonged illumination with high light intensities. This process which includes the functional impairment of PSII electron transport and the structural damage of the PSII reaction centre is known as photoinhibition (Aro el al. 1993). The influence of Cu(II) toxicity on photoinhibition has also been investigated (Yruela et al. 1996b; Pätsikkä et al. 1998) demonstrating that Cu(II) enhances the adverse effects of excess light on PSII. Over the past 10 years the participation of Cyt b559 in a redox mechanism to protect PSII against donor and acceptor side photoinhibition has been proposed (for review see Stewart and Brudvig, 1998). Evidence that Cyt b559 prevents the overreduction of PSII acceptor side have been reported (Nedbal et al. 1992; Poulson, 1995). On the other hand, it was postulated that the role of this heme protein is to act as an auxiliary electron donor delivering electrons to oxidised chlorophylls in the PSII reaction centre (Buser et al. 1992; Faller et al. 2001). Cytochrome b559 can exhibit several redox potential forms, a labile highpotential (HP) form with a midpoint redox potential (E’m) around +400 mV, a intermediate potential (IP) form ranging from +200 to +150 mV and a low-potential (LP) form with an E’m value from +70 to +20 mV (Stewart and Brudvig, 1998; Kaminskaya et al. 1999; Mizusawa et al. 1999; Roncel et al. 2001). The IP form has been proposed to be a protonated LP form (Roncel et al. 2001). The HP form dominates in thylakoids and PSII membranes with an intact water-oxidizing complex. Several authors have reported that photoinhibition induces changes in the redox properties of Cyt b559 (Styring et al. 1990; Allakhverdiev et al. 1997; Ortega et al. 1999). The conversion of the HP to the LP form has been observed during photoinhibitory illumination in native PSII membranes (Ortega et al. 1999). 4 Cu(II) interaction with Cyt b559 has been reported earlier (Jegerschöld et al. 1995; Yruela et al. 1996b), however no data on its specific action during photoinhibition have been shown. The aim of this work is to investigate the changes of Cyt b559 caused by photoinhibition under low toxic Cu(II) conditions. The results show that HP Cyt b559 form is very sensitive to toxic Cu(II) action during photoinhibitory illumination whereas LP Cyt b559 form is not affected under the same experimental conditions. MATERIALS AND METHODS Biological material.- Beta vulgaris cv Monohill was grown hydroponically in a growth chamber in half-Hoagland nutrient solution under 325 μmol quanta.m-2.s-1 from fluorescent and incandescent lamps at 25 ºC, 80% humidity and 16-h photoperiod. Spinach was obtained from local market. Photosystem II membrane isolation.- Highly enriched PSII membranes with a rate of oxygen evolution activity of c.a. 500 μmol O2 mg Chl-1 h -1, using dichlorobenzoquinone (DCBQ) as artificial electron acceptor, were prepared from market spinach and sugar beet according to Berthold et al. (1981). Samples were suspended in 0.4 M sucrose, 15 mM NaCl, 5 mM MgCl2 and 50 mM 2-(N- morpholino) ethanesulphonic acid (Mes-NaOH), pH 6.0, frozen in liquid nitrogen and stored at –80 ºC until use. Photoinhibition.- Photoinhibitory treatment was done using intact and Cu(II)- treated oxygen-evolving PSII membranes preparations. PSII membranes at 200 5 μg Chl ml-1 resuspended in 3.5 ml buffer containing 0.4 M sucrose, 15 mM NaCl and 50 mM Mes-NaOH, pH 5.0-6.0 or 50 mM Tris(hydroxymethyl)aminomethane (Tris-HCl), pH 7.8, were incubated with 125 μM CuCl2 (equivalent to a Cu(II)/PSII ratio of 137). To calculate the Cu(II) per PSII unit ratio in PSII membranes a content of 250 Chl per RC was assumed (Berthold et al. 1981). Incubations with Cu(II) were done at 23 ºC for 1 min under constant stirring in the dark before exposing the samples to heat-filtered white light (3,000 μmol quanta m-2s-1) to induce photoinhibitory conditions. The temperature was maintained at 23 ºC by circulating water from a temperature-controlled waterbath around the photoinhibition cell. The temperature increment inside of reaction cell was less than 1 ºC after 20 min illumination. Control samples were kept in the dark under identical conditions to the illuminated samples in order to monitor dark inactivation processes unrelated to photoinhibition. After treatment all of the samples were washed twice with 15 mM NaCl and 50 mM Mes-NaOH, pH 5.0-6.0 or 50 mM Tris-HCl, pH 7.8. The pellets obtained by centrifugation at 23,000 x g were resuspended in the same buffer plus 0.3 M sucrose and then, oxygen-evolution activity was measured. Oxygen evolution activity.- Oxygen evolution activity was measured with a Clark-type oxygen electrode in the presence of 0.5 mM DCBQ as artificial electron acceptor. The light intensity on the surface of the cuvette was 3,000 μmol quanta m-2s-1. Samples were diluted in 3 ml buffer containing 25 mM Mes-NaOH, pH 6.5, 10 mM NaCl and 0.3 M sucrose. The Chl concentration was 10 μgml-1. 6 Gel Electrophoresis and Immunoblotting.- The electrophoretic separation of PSII proteins was performed by SDS-PAGE on 12.5% (w/v) or 12-20% (w/v) acrylamide linear gradient gels containing 6 M urea according basically to Laemmli (1970). The gels were either stained with Coomassie B-Blue 250 or electroblotted to identify D1 and α-subunit of Cyt b559 proteins, respectively. After transferring to a nitrocellulose membrane, proteins were detected with a rabbit anti-D1 or anti-Cyt b559 α-subunit. To do that, the nitrocellulose membrane was cut across in two parts. Upper and lower parts of the membrane revealed the D1 and α-subunit of Cyt b559, respectively, using goat anti-rabbit IgG coupled to horseradish peroxidase as a secondary antibody. To obtain extrinsic protein markers the spinach PSII membranes at 0.5 mg mL-1 were suspended in 0.8 M Tris-HCl (pH 9.1), 5 mM EDTA and incubated for two hours in darkness. Then, the membranes were treated with 1.5 M NaCl for 30 min in darkness, centrifuged at 35,000 x g for 20 min and washed twice with 20 mM NaCl, 0.4 M sucrose and 50 mM Mes-NaOH (pH 6.5). The supernatants containing the extrinsic proteins were concentrated with Centripep tubes (Amicon, cut-off 10,000 kDa) and used as marker in electrophoresis assays (Mizusawa et al. 1999). Optical measurements.- Difference absorption spectra were recorded using 1- cm optical pathlength cuvettes at 10ºC with a Beckman DU 640 spectrophotometer. The content of HP and LP forms of Cyt b559 was determined from the difference absorption spectra in the 510-600 nm region. A differential extinction coefficient of 21 mM-1.cm-1 at the maximum at 559 nm minus the minimum at around 570 nm (Stewart and Brudvig, 1998) was used. Samples at 50 μg Chl ml-1 in 25 mM Mes-NaOH, pH 6.5, 10 mM NaCl and 0.3 M sucrose were 7 oxidised with 2 mM ferricyanide (HPred) and then reduced with 4 mM hydroquinone (HPox) and 2 mM sodium dithionite (LPox). Sodium dithionite solution was freshly prepared in 0.5 M Tricine, pH 7.5. Potentiometric redox titrations of PSII membrane suspensions were carried out at 20ºC under argon by following the absorbance changes at 559 nm minus 570 nm in the difference absorption spectra obtained by the sequential addition of aliquots of 0.1 M sodium dithionite. Spectra were recorded in an Aminco DW-2000 UV-Vis spectrophotometer. Samples were previously oxidized with 25 μM potassium ferricyanide. The redox potential in the reaction cell was simultaneously measured with a potentiometer (Methrom Herisau, Switzerland) provided with a combined Pt-Ag/AgCl microelectrode (Crison Instruments, Allela, Spain) previously calibrated against a saturated solution of quinhydrone (E’m, pH7, +280 mV at 20ºC). Redox mediators used and midpoint redox potential determinations were as described in Roncel et al. (2001). 8 RESULTS Effect of Cu(II) on Cytochrome b559 in the dark. Firstly, Cyt b559 was examined in control PSII-enriched membranes active in oxygen evolution from spinach and sugar beet plants. The amounts of HP and LP forms of Cyt b559 under aerobic conditions were obtained from reduced minus oxidised difference absorption spectra in the 510-600 nm region (for details see Materials and Methods). Upon addition of ferricyanide to a dark incubated sample the initially reduced HP form becomes oxidised (Fig. 1). Then, it can be reduced by hydroquinone. A further addition of dithionite resulted in the reduction of the oxidised LP form present. Data show that PSII membranes from spinach and sugar beet differed in the content of Cyt b559 redox potential forms. Thus, spinach PSII membrane preparations contain around 20-50% reduced HP form and 80- 50% oxidised LP form of Cyt b559 depending on the pH. It is worth mentioning that LP form assignment is given to all the Cyt b559 species that cannot be reduced by hydroquinone. The values are similar to that reported by others in these kinds of preparations (Ortega et al. 1999; Gadjieva et al. 1999). A marked difference is observed in PSII membranes from sugar beet where only the LP form of Cyt b559 was present at any pH value investigated. The absence of the HP form of Cyt b559 in oxygen-evolving PSII membranes was also found by others in preparations from pea (Roncel et al. 2001). Reductive potentiometric redox titrations at pH 6.0 showed clearly the existence of two different components with E’m of +397 mV and +206 mV in spinach PSII membranes; the named HP and LP potential forms (Fig. 2). In the case of sugar beet preparations, only one redox component with an E’m of +162 mV was found. 9 by Cu(II) is difficult to explain precisely. It should be mentioned that a direct oxidation of the HP Cyt b559 form by Cu(II) is thermodynamically unfavourable because the midpoint redox potential of Cu(I)/Cu(II) (E’m,7 = +167 mV) is markedly lower than that of HP Cyt b559 form (E’m,7 = +350 - +400 mV). This phenomenon could only be possible if i) Em of Cyt b559 is not any longer in HP form; ii) Em of copper redox couple is increased; iii) a non-direct oxidation mechanism takes place. The former is discarded based on our results (Fig. 2); the second one in thermodynamic terms might be if the oxidizing agent were the redox couple Cu(I)/Cu (E’m,7 = +521 mV). Cu(II) can be reduced via oxygen radical species (O2-) to Cu(I) under aerobic conditions. However, our results do not support this possibility since we do not observed that the redox potential of the medium to become more positive than +400 mV by addition of CuI(II). Thus, another oxidation mechanism should be considered. Recently, we have obtained information about the structure and the electronic distribution of Cyt b559 heme group in PSII reaction center preparations based on CW-EPR and HYSCORE experiments (Yruela et al. 2003; García-Rubio et al. 2003). Our results indicate that the exchangeable electron is localized in a confined iron orbital with a negligible mixture of nitrogen p-orbitals in the heme group of Cyt b559. This makes very unlikely that any substrate or reactant can be located close enough to the active site for a direct one-step electron exchange. The transfer mechanism could be better understood if it were a multistage complex process where the iron acts as a final reservoir. Our investigations suggest that redox processes will depend in a complex way on the whole protein conformation. Interestingly, we observed the rapid loss of oxygen evolution activity as compared to slower kinetics of Cu(II) effect on Cyt b559 suggesting the Cu(II)- 16 induced effects on Cyt b559 do not contribute to the fast rate of photoinhibition. It is also worth mentioning that at pH 5.0 the loss of oxygen evolution was faster than at pH 6.0 whereas the contrary was observed with respect to the Cyt b559 in the Cu(II)-treated samples during illumination. These findings indicate that Cyt b559 is affected after PSII centers are photoinhibited. The mechanisms underlying the Cu(II)-induced effect on oxygen evolution and Cyt b559 should be independent. It has been proposed that the protective role of Cyt b559 against photoinhibition is based on a conversion mechanism involving different redox components of this heme protein (Gadjieva et al. 1999; Ortega et al. 1999). In this sense our results show similar rate of oxygen evolution during photoinhibition in the two kinds of PSII preparations examined either in the absence or presence of Cu(II). Therefore the participation of Cyt b559 preventing photoinhibition process is not clear in our experiments. On the other hand, since any loss of Cyt b559 heme content was observed by addition of Cu(II) in the dark as well as in control conditions under illumination the results indicate that Cu(II) is probably acting directly on Cyt b559 in the light inducing the destabilisation of heme group. The data show a pH-dependent behaviour of Cyt b559 during illumination in the presence of Cu(II). At pH 5.0, no Cu(II) effect on total Cyt b559 content was observed, however at pH 6.0-7.8 total Cyt b559 was rapidly affected (Fig. 4). This pH-dependence suggests that Cu(II) interaction with Cyt b559 takes place at a protonatable amino acid residue. It is well established that the His residue has a high affinity to bind copper, and evidence that Cu(II) inhibits photosynthetic complexes from purple bacteria by binding to His residues have been reported (Utschig et al. 2000, 2001; Rao et al. 2000). It has been suggested that in the HP form one of the two His ligands of heme group is protonated and forms a 17 hydrogen bond with a carbonyl group of the protein backbone providing a highly hydrophobic environment surrounding the heme group. Such ambience is considered essential for the maintenance of the unstable HP form of Cyt b559 (Berthomieu et al. 1992; Roncel et al. 2001). These authors also proposed that His residues in the LP form of Cyt b559 were unprotonated. Since the protonation of His weakens the bond between the heme iron and imidazole Nδ compared to the case of unprotonated His, the destabilisation of the heme group coordination by Cu(II) can be favoured in the HP form. Recently, Burda et al. (2003) have proposed that HP Cyt b559 oxidation in the dark probably occurs by deprotonation of His ligands. Such oxidation could make the HP form more sensitive to photoinhibition. However, we observed the HP Cyt b559 damage takes place independently of its redox state. Interestingly, at pH 6.0-7.8 the HP form of Cyt b559 was more sensitive to toxic Cu(II) action than the LP form and irreversible damage to the HP form took place during photoinhibition contrary to what occurs in control PSII membranes where a conversion of the HP into the LP form was observed. Similar results were reported by others in fresh preparations (Styring et al. 1990; Mor et al. 1997), although the molecular mechanism involved in the conversion between the Cyt b559 forms is not clear at present. These findings indicate that Cu(II) interaction with Cyt b559 could impair the conversion process to the more stable LP form. The LP form was not affected (spinach and sugar beet preparations) in both control and Cu(II)-treated preparations during illumination at pH 5.0-6.0. Surprisingly, at pH 5.0 the extent of Cu(II) effect on the HP form was less than at pH 6.0 although this finding could be due to a protein conformational change at this low pH preventing the Cu(II)-induced damaging effect. In view of these considerations, 18 we suggest that the higher sensitivity of the HP form of Cyt b559 to photoinhibition in the presence of Cu(II) could be due to blocking of its conversion to the LP form. The destabilisation of the heme group did not involve cytochrome protein degradation. 19 ACKNOWLEDGEMENTS The authors are grateful to Maria V. Ramiro for skilful technical assistance. We are indebted to Drs. A.K. Matto and R. Barbato for their kind gifts of antibody against the D1 and α-subunit of Cyt b559 polypeptides of PSII, respectively. M. Bernal was recipient of an I3P Programme fellowship from Consejo Superior de Investigaciones Científicas. This work was supported by the Dirección General de Investigación (Grant BMC2002-00031) to R.P. and Gobierno de Aragón (Grant P015/2001) to I.Y., and it has been done within GC DGA 2002 Program of Gobierno de Aragón. 20 REFERENCES Allakhverdiev AI, Klimov VV, Carpentier R (1997) Evidence for the involvement of cyclic electron transport in the protection of photosystem II against photoinhibition: influence of a new phenolic compound. 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Biochemistry. 34: 10932-10938 Rao SBK, Tyryshkin AM, Roberts AG, Bowman MK, Kramer DM (2000) Inhibitory copper binding site on the spinach cytochrome b6f complex: Implications for Qo site catalysis. Biochemistry 39: 3285-3296 25 6.0 and 7.8 in control spinach and sugar beet PSII membranes, respectively. 100% activity corresponded to 223, 230 and 36 μmol O2.mg Chl-1.h-1 and 280, 187 and 23 at pH 5.0, 6.0 and 7.8 in Cu(II)- treated spinach and sugar beet PSII membranes, respectively. Figure 4.- Time course of Cyt b559 content in oxygen-evolving PSII membranes from spinach ({,z) and sugar beet (Ì,S) during photoinhibitory treatment in the absence ({,Ì) and presence (z,S) of 125 μM CuCl2 [Cu(II) per PSII unit = 135] at 23 ºC. (A) pH 5.0; (B) pH 6.0; (C) pH 7.8. Other experimental conditions as in Fig. 3. Figure 5.- Time course of Cyt b559 content in oxygen-evolving PSII membranes from spinach during photoinhibitory illumination treatment in the absence ({,Ì) and presence (z,S) of 125 μM CuCl2 [Cu(II) per PSII unit = 135] at 23 ºC. (A) pH 5.0; (B) pH 6.0. S,Ì content of HP form; z,{ content of total Cyt b559. Other experimental conditions as in Fig. 3. Figure 6.- A) SDS-PAGE analysis of PSII membrane preparations from spinach incubated with 125 μM CuCl2 [Cu(II) per PSII unit = 135] after photoinhibitory illumination treatment at pH 6.0. Lanes 1-4: control PSII membranes illuminated for 0, 5, 10, 20 min; lanes 5-8: Cu(II)-treated PSII membranes illuminated for 0, 5, 10, 20 min. B) Immunoblots with serum anti-D1 subunit and anti-α-subunit of Cyt b559 of PSII membrane 32 preparation from spinach incubated with 125 μM CuCl2 [Cu(II) per PSII unit = 135] at 23 ºC after photoinhibitory illumination treatment at pH 6.0. Lanes 1-4: control PSII membranes illuminated for 0, 5, 10, 20 min; lanes 5-8: Cu(II)-treated PSII membranes illuminated for 0, 5, 10, 20 min. 33 34 35 36 37 38 39