Cytochrome b 559 content in isolated photosystem II reaction center preparations Inmaculada Yruela 1 , Francisca Miota 1 , Elena Torrado 1 , Michael Seibert 2 and Rafael Picorel 1 1 Estacio ´ n Experimental de Aula Dei (CSIC), Zaragoza, Spain; 2 National Renewable Energy Laboratory, Basic Sciences Center, Golden, CO, USA The cytochrome b 559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b 559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer compo- sition and detergent concentration. Two different types of cytochrome b 559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized- minus-reduced difference extinction coefficient of cyto- chrome b 559 at 559 nm. The relative amount of D1 and cytochrome b 559 a-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b 559 heme content in photo- system II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b 559 a-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their a-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b 559 a-subunit per 1.0 D1 polypeptide in photo- system II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes. Keywords: chromatography; cytochrome b 559 ; detergent; immunoblot; photosystem II. Cytochrome (Cyt) b 559 is a hemoprotein component of the photosystem II (PSII) reaction center (RC) complex [1], and it is an integral component of the minimal isolated RC complex still capable of performing primary charge separ- ation. It is composed of two small polypeptides, the a (9 kDa) and b (4.5 kDa) subunits, encoded by the psbE and psbF genes, respectively. Each polypeptide has a single transmembrane a-helical domain [2,3]. The heme iron is bound to a single histidine residue on each subunit [4], and it is located close to the stromal surface of the membrane [2,3,5–7]. However, a location for Cyt b 559 heme on the lumenal side of the PSII membrane has also been proposed, suggesting that two hemes and two copies each of the two subunits are present in the thylakoid membrane [8,9]. Despite numerous studies [8,10,11], the exact function of Cyt b 559 is still unclear but the following are possibilities: (a) involvement in the electron transfer reactions on the oxidizing side of PSII [12,13]; (b) participation in the assembly of the water-splitting system [14]; and (c) protec- tion of PSII against photoinhibition [15–19]. It is well known that Cyt b 559 can exist in a number of different redox forms. At pH 6.0–6.5, PSII complexes, surrounded by their natural membrane environments, as in chloroplasts, thyla- koids and PSII membrane fragments, Cyt b 559 exhibits midpoint redox potentials (E¢ m )of+400mV[thehigh potential (HP) form], +200–150 mV [the intermediate potential (IP) form], and +70–60 mV [the low potential (LP) form] [1,20–22]. The HP form dominates in thyla- koids and PSII membranes with an intact water-oxidizing complex. A longstanding issue has been the number of Cyt b 559 per PSII complex. Shuvalov and coworkers argued that PSII core complex from spinach with high O 2 -evolution activity contains two Cyt b 559 per PSII [8]. However, the currently accepted value in isolated PSII RCs, based mainly on absorption spectroscopy techniques [1,23–26], is one heme per RC. Recent data based on the crystal structure of the PSII core from Synecochoccus elongatus [2] and Thermo- synechococcus vulcanus [3] are in agreement with this proposal. But a second cytochrome might have been lost during the preparation of the core material. Thus the question of one or two Cyt b 559 per PSII RC remains unresolved because the stoichiometry might depend on the isolation procedure used, the type of PSII preparation and/ or the organism examined. Correspondence to R. Picorel, Estacio ´ n Experimental de Aula Dei (CSIC), Ctra. Montan ˜ ana 1005, Zaragoza E-50080, Spain. Fax: + 34 976 716145; Tel.: + 34 976 716053; E-mail: picorel@eead.csic.es Abbreviations: Cyt, cytochrome; D1/D2 HD, heterodimer made by crosslinking of D1 and D2 polypeptides; DM, n-dodecyl b- D -malto- side; HP, high potential; IMAC, immobilized metal affinity chroma- tography; IP, intermidiate potential; LP, low potential; Mes, 2-(N-morpholino) ethane-sulfonic acid; PS, photosystem; RC, reaction center. Enzymes: glucose oxidase (EC 1.1.3.4); catalase (EC 1.11.1.6). (Received 23 January 2003, revised 21 March 2003, accepted 26 March 2003) Eur. J. Biochem. 270, 2268–2273 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03597.x To address this issue, we have determined the D1 and Cyt b 559 contents of various PSII RC preparations obtained from market spinach and chamber-grown sugar beets. The cytochrome content was assayed by both spectrophoto- metric and immunological methods. The former measures the heme content and the latter quantitates the amount of protein present. Note that it is not the aim of this work to define the quality or performance of the different RC preparations, but rather to examine the effect of several isolation procedures on the Cyt b 559 content of these preparations. The results presented in this work strongly suggest that the Cyt b 559 heme content and Cyt b 559 a-subunit/D1 ratio are highly dependent on the RC isolation procedure used; this ratio is close to one in all PSII-enriched membranes tested. Materials and methods Biological material Sugar beet (Beta vulgaris L. cv. Monohill) was grown hydroponically in a growth chamber on half-Hoagland solution, under the following conditions: 325 lEinsteinsÆ m )2 Æs )1 cool fluorescent light (16 h light period), 25 °C, and 80% humidity. Spinach was purchased from the local market at different times during the year. Preparation of PSII membranes PSII-enriched membrane fragments were isolated according to [27] with some modifications [25]. Samples were suspen- ded in 0.4 M sucrose, 15 m M NaCl, 5 m M MgCl 2 and 50 m M 2-(N-morpholino) ethane-sulfonic acid (Mes)/ NaOH (pH 6.0), frozen in liquid nitrogen and stored at )80 °C until use. Preparation of D1-D2-Cyt b 559 complexes Four RC preparations with about six chlorophyll (Chl) a per two pheophytins (Pheo) and one preparation with about five Chl per two Pheo were isolated from PSII-enriched mem- brane fragments using modifications to the standard proce- dure [23]. This method solubilizes PSII-enriched membranes at1mgChlÆmL )1 with 4% (w/v) Triton X-100 for 1 h. After centrifugation the resultant supernatant is loaded onto a weak anion-exchange Toyopearl TSK DEAE-650(S) col- umn pre-equilibrated with 50 m M Tris/HCl (pH 7.2) and 0.05% (w/v) Triton X-100 buffer. The column was washed extensively with the same buffer until the optical density of the 417 nm peak (main Soret Pheo peak) was much higher than that at 435 nm (main Soret Chl a peak). The material was then eluted with a 50–200 m M NaCl linear gradient in the same buffer. Hereafter, we will call this preparation RC1 and consider it the standard control material. Variations of this procedure were performed as follows: RC2-Strong anion-exchange Q-Sepharose Fast-Flow column and 0.1% (w/v) Triton X-100 in 50 m M Mes/NaOH (pH 6.5) washing buffer [28]; RC3-Toyopearl TSK DEAE-650(S) column and 1% (w/v) Triton X-100 and 1.5% (w/v) taurine in 50 m M Tris/HCl (pH 7.2) washing buffer [29]; and RC4-Toyopearl TSK DEAE-650(S) column and 1% (w/v) Triton X-100 in 50 m M Tris/HCl washing buffer [30]. In each case, after detergent exchange with 0.1% (w/v) n-dodecyl-b- D -malto- side (DM) to stabilize the RC [31], the RC complexes were eluted with a 60–300 m M linear salt gradient in 50 m M Mes/ NaOH (pH 6.5) and 0.1% (w/v) DM, and the fractions were collected at 1 mLÆmin )1 . In some cases, the preparations obtained as indicated above were exposed to an additional chromatography step using a copper immobilized metal affinity chromatographic [Cu(II)-IMAC] column. To pre- pare the Cu(II)-IMAC column, 100 mL of 0.1 M CuSO 4 in distilled water were passed through a Chelating-Sepharose Fast-Flow column (Amersham-Pharmacia, 1 · 10 cm). Excess copper was eliminated by washing with 100 mL distilled water. The column was equilibrated with 50 mL of 50 m M Na 2 HPO 4 (pH 6.5), 50 m M NaCl, 0.2% (w/v) Triton X-100 and 1.2 m M DM. Then the RC samples were diluted sixfold in 50 m M Mes/NaOH (pH 6.5), loaded onto the Cu(II)-IMAC column and washed with 50 m M Mes/NaOH (pH 6.5), 50 m M NaCl, 0.2% (w/v) Triton X-100 and 1.2 m M DM buffer. The samples were then eluted with 1–10 m M imidazole linear gradient in 50 m M Mes/NaOH (pH 6.5) and 0.1% (w/v) DM. RC5, a preparation contain- ing about five Chl per two Pheo, was isolated from PSII- enriched membranes following the method described in [32] using the Cu(II)-IMAC column described above. All the isolation steps were done at 4 °C in the dark. The pigment composition of the isolated RC complexes was determined as described in [33]. Spectroscopy The RC Cyt b 559 heme content was measured spectro- photometrically. To measure the dithionite-reduced minus ferricyanide-oxidized absorption spectra in the 510– 600 nm region, the RC samples were diluted to an optical density of 0.6–1.2 absorption units at the red maximum peak at around 675.5 nm with a buffer containing 50 m M Mes/NaOH (pH 6.5) and 0.1% (w/v) DM (this buffer yields more transparent D1-D2-Cyt b 559 complex suspensions than Tris/HCl buffers at higher pH). A differential extinc- tion coefficient of 21 m M )1 Æcm )1 at the maximun at 559 nm minus the minimum at around 570 nm [1] was used to determine the heme content of the different preparations. Difference absorption spectra were recorded using 1 cm optical pathlength cuvettes at 10 °CwithaBeckmanDU 640 spectrophotometer. Constant temperature was main- tained using a circulating bath (MultiTempII, Amersham- Pharmacia). Samples were oxidized with 2 m M ferricyanide and then reduced by adding 1 lL of a saturated solution of sodium dithionite prepared in 10 m M Tris/HCl, pH 7.5 (at this pH the dithionite is more stable than at lower pH) and maintained in an ice-pocket. The addition of another lLof saturated solution did not further increase the absorption at 559 nm, demonstrating that the Cyt b 559 content was completely reduced with the first dithionite addition. It should be noticed that the same results were obtained without the addition of ferricyanide because the Cyt b 559 from D1-D2-Cyt b 559 complex preparations as obtained from the chromatography column is always in the oxidized state. All of the measurements were carried out under anaerobic conditions maintained by adding 0.23 mgÆmL )1 glucose oxidase (Sigma, EC 1.1.3.4), 80 lgÆmL )1 catalase (Sigma, EC 1.11.1.6) and 10 m M glucosetothesample[31]. Ó FEBS 2003 Cytochrome content of different D1-D2-Cyt b559 complex preparations (Eur. J. Biochem. 270) 2269 SDS/PAGE and immunoblot Electrophoresis was carried out as in [34] using a 4% (w/v) acrylamide stacking gel and a 12–20% (w/v) acrylamide linear gradient resolving gel containing 6 M urea. To avoid the interference of lipids and detergents during the electro- phoresis, the RC samples were diluted 10-fold in 1 : 1 ethanol/acetone (v/v), incubated for 1 h at 20 °C, and centrifuged at 9000 g for 10 min at 4 °C [35]. The pellet containing the protein was resuspended in 50 m M Mes/ NaOH (pH 6.5). The samples were diluted 1 : 1 in 2% (w/v) SDS, 2 M urea, 40 m M dithiothreitol, and 50 m M Mes/ NaOH (pH 6.5), and then denaturated at room temperature for50min.Replicategelswererununderthesame conditions at the same time, and the proteins were transferred onto nitrocellulose membranes for immuno- detection using a Bio-Rad Mini Trans-Blot Cell. The transfer buffer was 25 m M Tris/HCl (pH 7.5), 192 m M glycine and 20% methanol. After protein transfer, one of the blots was probed with rabbit antibodies raised against a synthetic peptide homologous to the N-terminus of the spinach PSII D1 protein and the other with rabbit antibodies raised against the spinach a-subunit of Cyt b 559 [36]. The standard peroxidase development procedure with 4-chloro- 1-naphthol as the substrate was used to visualize the blots. The gels and blots were scanned with a Studio Scan II Si (AGFA), and the intensity of the bands was quantified by densitometry using US National Institute of Health Software ( NIH IMAGE ) available at http://www.ncbi.nih.gov. Results and discussion We examined the Cyt b 559 contentoffivedifferentD1-D2- Cyt b 559 PSII RC preparations containing either five or six Chl per RC. Dithionite-reduced minus ferricyanide-oxidized absorption spectroscopy shows that the absolute Cyt b 559 heme stoichiometry varied between 0.91 and 1.41 hemes per two Pheo (Table 1). This method is commonly used to determine the Cyt b 559 content in PSII RC preparations [1] and shows that heme content can vary depending on the specific RC isolation procedure. In RC1, the standard preparation for the purpose of this paper, we measured slightly more than six Chl per two Pheo (Table 1). The PSII RC is known to contain two Pheo [2,3]. RC2 contained a little less Chl (6.18) but more Cyt b 559 heme (1.11) per two Pheo compared to RC1. RC3 contained less Chl (5.80) but more Cyt b 559 heme (1.19) than RC1 and RC2. RC4 contained 6.05 Chl and even more Cyt b 559 heme (1.41). RC5 had about five Chl per two Pheo as expected and 1.12 Cyt b 559 heme per two Pheo. The only significant difference between the RC1 through the RC4 procedures was the concentration of Triton X-100 used during RC isolation wash steps, and the presence of 1.5% (w/v) taurine in RC3. Despite the different Cyt b 559 contents all the preparations showed ÔnormalÕ room temperature absorption spectra, i.e. the six Chl preparations with maxima at 675.5 nm and the five Chl preparation with a maximum at around 677 nm. This indicates that the cytochrome content has nothing to do with the spectral quality of the preparations. In order to compare the actual Cyt b 559 protein content of each preparation rather than the heme content as above, we used polyclonal antibodies against D1 and the a-subunit of Cyt b 559 to assess changes in the ratio of the polypeptide levels in the different RC preparations. Figure 1 shows immunoblots using antibodies against the D1 polypeptide (upper box) and the a-subunit (lower box). No D1 degradation product, little D1/D2 heterodimer and no Cyt b 559 aggregate formation were detected in any of our preparations. Table 2 shows the relative Cyt b 559 a-subunit/ D1 integrated densities of the two respective bands in the blot (Fig. 1). RC1 and RC2 show similar ratios, but RC3 and especially RC4 and RC5 showed much higher ratios. The results of Tables 1 and 2 demonstrate that increasing the concentration of Triton X-100 during RC washing procedure leads to a higher Cyt/D1 ratio both on a heme and a protein basis. However, the variation of the Cyt b 559 a-subunit to D1 ratio with the detergent concentration was more dramatic in the case of the immunoblot data (Table 2) compared to the spectrophotometric data (Table 1). This indicates that free a-subunit polypeptide (with no heme) from degraded cytochrome must bind preferentially to the column and is coeluted with the native RC complex. Note Table 1. Pigment and cytochrome b 559 (heme) content determined spectrophotometrically for various D1-D2-Cyt b 559 PSII RC complexes purified using different procedures, as described in Materials and methods. Values represent means ± SE (n ¼ 4). Values in parentheses represent the Triton X-100 concentration used during the isolation washing procedure. Variable RC1 (0.05%) RC2 (0.1%) RC3 (1%) RC4 (1%) RC5 (0.2%) Chl a 6.41 ± 0.15 6.18 ± 0.20 5.80 ± 0.13 6.05 ± 0.21 5.12 ± 0.13 Pheo a 22222 Cyt b 559 a 0.91 ± 0.18 1.11 ± 0.13 1.19 ± 0.09 1.41 ± 0.15 1.12 ± 0.08 a This row also represents the Cyt b 559 heme/RC ratio. Fig. 1. Immunoblots of several isolated D1-D2-Cyt b 559 PSII RC preparations using anti-D1 (upper box) and anti-Cyt b 559 a-subunit (lower box) serum. Lane 1, RC1; lane 2, RC2 ; lane 3, RC3 ; lane 4, RC4; lane 5, RC5. 2270 I. Yruela et al.(Eur. J. Biochem. 270) Ó FEBS 2003 that the difference spectroscopy employed only detects the heme in native Cyt b 559 , but the antibody blots detect all a-subunit polypeptide present. Taurine used to prepare RC3 is a chaotropic agent that is sometimes used to strip off loosely bound contaminants to give cleaner protein pre- parations, and thus RC3 exhibits lower Cyt b 559 protein content than RC4 (prepared the same way, but without taurine). In the case of the IMAC procedure (RC5), the heme content was a little higher than in RC1, but the a-subunit/D1 ratio was even higher than that in RC4. This result occurs despite the lower Triton X-100 concentration used (0.2% [w/v] in RC5 compared to 1% [w/v] in RC4). The simplest explanation for the much higher level of a-subunit polypeptide content compared to heme content in RC5 results from a consideration of the chemistry of the Cu(II)-IMAC column. The Cu(II), linked to the column matrix, binds to any available histidine residue on the surface of a protein. As Cyt b 559 is on the surface of the D1-D2-Cyt b 559 complex [2,3,6], the two histidines that bind the heme are prone to be attack by Cu(II). As a consequence the heme is displaced and washed out of the column, but the free polypeptide subunits remain bound to the Cu(II)- IMAC column by the histidine residues until coeluted with the native RC complex after the application of the imidazole elution step. In order to examine this hypothesis, RC2 and RC3 were passed through a Cu(II)-IMAC column, and the results are shown in Fig. 2. Both preparations exhibit a higher a-subunit/D1 ratio after passing through the Cu(II)- IMAC column, i.e. the RC2 and RC3 showed a densito- metric ratio of 1.02 and 1.32, respectively, after the column compared to 0.62 and 1.09 before the column. The results reported here indicate that the Cyt b 559 content in the PSII RC complex can be altered by the purification procedure. In order to confirm that these variations did not result from variations in Cyt b 559 content in the starting PSII membranes, we analysed several batches of market spinach harvested during different times of the year and sugar beets grown under controlled environmental conditions in a growth chamber. Figure 3 shows the blots using antibodies against D1 (upper box) and Cyt b 559 a-subunit (lower box). All of the PSII membrane batches had similar Cyt b 559 /D1 ratios, independent of the harvest time of the year, growth conditions or plant species (the spectrophotometric method also confirmed the presence of similar concentrations of Cyt b 559 in all PSII membranes, data not shown). The absolute Cyt b 559 /D1 ratio in the membranes was calculated using a calibration curve gener- ated with different amounts of RC1 control material. Figure 4A shows the blot from the gel containing different amounts of RC1 control sample that was used to generate the standard correlation curve represented in Fig. 4B. The gel also contains a duplicate of amounts of PSII membranes (Fig. 4A) corresponding to the Cyt b 559 and D1 content that fits within the correlation curve. The densitometric values obtained from the blot for the a-subunit and D1 from the membranes were introduced in the generated calibration curve to calculate the absolute ratio of these polypeptide content present in membranes considering that ratio 1 : 1 in RC1 control sample. This absolute ratio resulted in 1.25 Cyt b 559 a-subunit per D1 polypeptide. Assuming that RC1 control sample, obtained using the lowest Triton X-100 concentration (0.05%, w/v), contains one Cyt per D1, we can conclude that: (a) PSII-enriched membranes from higher plants contain a little more than one but certainly less than two Cyt b 559 a-subunits per D1 polypeptide; (b) both the Cyt b 559 heme and a-subunit contents of D1-D2-Cyt b 559 complex depend on the puri- fication procedure used to obtain the preparations; (c) a Table 2. Relative cytochrome b 559 a-subunit/D1 integrated density ratios of the Western blot bands in Fig. 1. All the ratios were normalized to the value of RC1 (the standard control preparation) in Table 1. The absolute densitometric band ratio of RC1 was 0.81. Values represent means ± SE (n ¼ 4). Variable RC1 RC2 RC3 RC4 RC5 a-Subunit/D1 0.91 ± 0.12 0.84 ± 0.18 1.30 ± 0.15 2.11 ± 0.12 2.27 ± 0.21 Triton conc. (w/v) 0.05% 0.1% 1% a 1% 0.2% a Contained Taurine. Fig. 2. Immunoblots of RC2 and RC3 preparations before and after passing through a Cu(II)-IMAC column. Lane 1, RC2; lane 2, RC2 after Cu(II)-IMAC column; lane 3, RC3; lane 4, RC3 after Cu(II)- IMAC column. Fig. 3. Immunoblots of PSII membrane preparations from market spinach obtained in autumn (lane 1), winter (lane 2), spring (lane 3), and from sugar beets (lane 4) grown in a growth chamber under controlled environmental conditions. Upper box: immunodetection with antibody against D1 protein; lower box: immunodetection with antibody against Cyt b 559 a-subunit. Ó FEBS 2003 Cytochrome content of different D1-D2-Cyt b559 complex preparations (Eur. J. Biochem. 270) 2271 high Triton X-100 concentration during the chromato- graphic washing steps clearly increases both the heme and the a-subunit content per RC; and (d) RC preparations using Cu(II)-IMAC exhibit a very high a-subunit poly- peptide compared to their heme content. Acknowledgements The authors thank M. V. Ramiro for her helpful technical assistance. We are indebted to Drs A. K. Matto and R. Barbato for their kind gifts of antibodies against the D1 and Cyt b 559 polypeptides, respectively. E. T. was recipient of a predoctoral fellowship from the CONSI + D (Diputacio ´ n General de Arago ´ n). This work was supported by the Ministry of Science and Technology of Spain (Grant PB98-1632 and BMC2002-00031) (RP) and by the Division of Energy Biosciences, Office of Science, U.S. Department of Energy (under Contract #DE-AC36–99G010337) (MS). References 1. Stewart, D.H. & Brudvig, G.W. (1998) Cytochrome b559 of photosystem II. Biochim. Biophys. Acta 1367, 63–87. 2. Zouni, A., Witt, H T., Kern, J., Fromme, P., Kraube, N., Saen- ger, W. & Orth, P. (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A ˚ resolution. Nature 409, 739–743. 3. Kamiya, N. & Shen, J R. 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Immunoblots of different amounts of RC1-control sample to generate the calibration curve and PSII membrane proteins. A(upper box): immunodetection with antibody against D1 protein; A (lower box): immunodetection with antibody against Cyt b 559 a-subunit. Left lane: molecular mass standards; lanes 1, 2, 3, 4 and 5: increasing amounts of RC1 complex (5–43 l M reaction centers); lanes 6 and 7: a duplicate of PSII membranes whose amounts of a-subunit and D1 fit within the calibration curve. Increasing amounts of RC1 complex were added to obtain a calibration curve to calculate the absolute Cyt a-subunit/D1 ratio of PSII membranes assuming a Cyt b 559 a-subunit/ D1 ratio of 1 : 1 in the RC1 control sample. 2272 I. Yruela et al.(Eur. J. Biochem. 270) Ó FEBS 2003 24. Gounaris, K., Chapman, D.J., Booth, P., Crystall, B., Giorgi, L.B., Klug, D.R., Porter, G. & Barber, J. (1990) Comparation of the D1–D2-Cyt b559 reaction center complex of photosystem two isolated by different methods. 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