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

DETERGENT EFFECT ON CYTOCHROME b559 ELECTRON PARAMAGNETIC RESONANCE SIGNALS IN THE PHOTOSYSTEM II REACTION CENTRE. Yruela, I.1*, García-Rubio, I.2, Roncel, M.3, Martínez, J.I.2, Ramiro, M.V.1, Ortega, J.M.3, Alonso, P.J.2 and Picorel, R1. 1 Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo. 202, E-50080-Zaragoza, Spain. 2 Instituto de Ciencia de Materiales de Aragón, Consejo Superior de Investigaciones Científicas-Universidad de Zaragoza, Plaza San Francisco s/n, E-50009-Zaragoza, Spain. 3 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Americo Vespucio s/n, E-41092-Sevilla, Spain. Corresponding author: I. Yruela; [email protected], Fax: +34 976 716145, phone: +34 976 716058 Keywords: cytochrome b559, detergent, electron paramagnetic resonance, photosystem II, reaction centre, redox potential. 1 ABSTRACT 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-β-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 gz feature positions changed depending on 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. 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. 2 1. INTRODUCTION Cytochrome (Cyt) b559 is an integral component of photosystem (PS) II reaction centre (RC). Its presence is critical for the biogenesis and stable assembly of the PSII RC, but it is not involved in the primary electron transport in PSII. D1-D2-Cyt b559 complexes with the minimum polypeptide composition that are able to perform efficient light-induced primary charge separation still bound Cyt b559.1 Harsh treatments are required to separate Cyt b559 from this complex 2,3 and the tight relationship with the D1/D2 heterodimer that binds the essential cofactors for PSII primary photochemistry suggests an essential role of Cyt b559. However, despite many studies performed during the last decades4-6 the exact function(s) of this hemoprotein is still unclear. Cytochrome b559 consists of two small polypeptides, the α (9 kDa) and β (4.5 kDa) subunits with a transmembrane α-helical domain and heterodimeric structure.6,7 Two histidine residues within the hydrophobic domain of each polypeptide act as ligands of heme iron. One of the most intriguing properties of Cyt b559 is the remarkable variability of the midpoint redox potential (E’m) of the heme group. In its natural membrane environment it exhibits a labile high-potential (HP, E’m ≈ +380 mV) and a stable lowpotential (LP, E’m ≈ +20 - +200 mV) form. An intermediate potential (IP) was also reported in chloroplasts, thylakoids and intact PSII membranes.6,8-12 Heme iron of Cyt b559 in PSII RC is known to display the typical low-spin electron paramagnetic resonance (EPR) signals gx ≈ 1.5, gy ≈ 2.3 and gz ≈ 3.0. However, slightly different gz values for Cyt b559 have been observed depending on the preparation and extent of purification of samples.6 These changes in EPR spectra were connected in the past with changes between different redox forms of Cyt b559. Indeed, it has been suggested that HP and LP forms of Cyt b559 can be distinguished by their EPR spectra. The HP form of Cyt b559 was associated to a gz around 3.01-3.08 based on EPR measurements in chloroplasts and PSII membranes with high content of this form. On the other hand, LP Cyt b559 form was related to a gz in the 2.93-3.04 range. 3 The gz value of 2.93 was measured in the isolated Cyt b559.6,13-17 Nevertheless, a straightforward relationship between redox potential and EPR signal is not clear. Other factors that were suggested to influence the EPR gz position were the purification degree of the sample and the hydrophobicity of heme environment.6,14 In order to elucidate factors controlling EPR signal, we study here in detail EPR spectra of oxidised LP Cyt b559 in the D1-D2-Cyt b559 complex and in more intact PSII preparations. Our results clearly demonstrate that detergents modify the EPR spectrum of Cyt b559 and suggest that EPR signal variations and midpoint redox potential could not be directly related. The way detergents affect EPR parameters is discussed in terms of a more general concept of hydrophobicity referred in.14 4 2. MATERIALS AND METHODS Preparation of PSII membranes.- Highly enriched-PSII membranes were isolated from market spinach according to Berthold et al.18 Samples were suspended in 0.4 M sucrose, 15 mM NaCl, 5 mM MgCl2 and 50 mM 2-(N-morpholino) ethanesulfonic acid (Mes)-NaOH, pH 6.0, frozen in liquid nitrogen and stored at –80ºC until use. PSII membranes exhibited oxygen evolution rates of 520 ± 30 μmol of O2 mg Chl-1 h-1 using DCBQ as artificial electron acceptor. Preparation of D1-D2-Cyt b559 complexes.- A standard D1-D2-Cyt b559 complex preparation containing six chlorophyll (Chl) molecules per RC was isolated from highly purified oxygen-evolving PSII membranes from market spinach18 according to the procedure of Nanba and Satoh1 and modified by Montoya et al.19 This method makes use of a Toyopearl TSK-DEAE column. Samples loaded in the column were washed with 0.05% (w/v) Triton X-100 until absorbance at 417 nm was higher than that at 435 nm. Detergent was subsequently exchanged by n-dodecyl-β-D-maltoside (β-DM) at different concentrations ranging from 0 to 0.2% (w/v) or 0.15% (w/v) sucrose monocaprate. Detergent replacement was done until Triton X-100 absorbance at 280 nm was lower than 0.01. Then, D1-D2-Cyt b559 complexes were eluted with a linear salt gradient in the same buffer and fractions were collected at 1 ml/min. This method was also used with three different modifications described in the literature.20-22 The variations, concerning basically the column and washing buffer conditions are the following: i) Toyopearl TSK-DEAE column, 1% (w/v) Triton X-100; ii) Toyopearl TSK-DEAE column, 1% (w/v) Triton X-100 and 1.5% (w/v) taurine ; iii) QSepharose Fast-Flow (Pharmacia) column and 0.15% (w/v) Triton X-100. The β-DM concentration in the elution buffer was 0.1% (w/v) for these latter samples and detergent replacement was done as explained above. All isolated D1-D2-Cyt b559 complex preparations contained six Chl per RC. 5 D1-D2-Cyt b559 complexes were isolated at different pH in the 5.5-7.7 range by changing the pH of the washing buffer. For pH 5.5-6.5 and pH 7.0-7.7 Mes-NaOH and tris(hydroxymethyl)aminomethane (Tris)-HCl buffers, respectively, were used. Desalted D1-D2-Cyt b559 complex preparations were prepared by a 2 h dialysis against the salt free elution buffer free of salt using a 30,000 kDa cut-off dialysis tube (Spectrapor). All isolation procedures were done in darkness in a cooled chamber at 4 ºC. Samples were then frozen in liquid N2 and stored at –80 ºC. Pigment composition of isolated D1-D2-Cyt b559 complex preparations was determined as described in Eijckelhoff and Dekker.23 Cytochrome b559 content was calculated from the dithionitereduced minus ferricyanide-oxidised absorption difference spectra using an extinction coefficient of 21.0 mM-1cm-1 at 559 nm.1 Preparation of PSII core complexes.- PSII core complex samples were prepared following the method described in24 with some modifications. An ion exchange Toyopearl TSK-DEAE column was used. The column was washed at 2 ml/min for 2 h and subsequently the core complexes were eluted with a linear salt gradient in 50 mM Mes-NaOH, pH 6.5 with 0.03% (w/v) Triton X-100. Additionally, PSII core complexes were eluted from the column after detergent exchange with 0.1% (w/v) β-DM in the same buffer. Detergent exchange was done until Triton X-100 absorbance at 280 nm was lower than 0.01. Pigmented fractions were concentrated in Centripep (Amicon) tubes. Isolation of monomeric and dimeric PSII RC and core complexes.- . Monomeric and dimeric complexes were isolated by sucrose density gradient centrifugation. To do that, PSII RC were suspended in 50 mM Mes-NaOH, pH 6.5 or 50 mM Tris-HCl, pH 7.2 and 0.1% β-DM (w/v).25 PSII cores were suspended in 25 mM Mes-NaOH, pH 6.5, 10 mM NaCl, 5 mM CaCl2 and 10 mM NaHCO3 and incubated with β-DM to a final concentration of 1.25% (w/v).26 The solubilised PSII RC and PSII core samples were homogenised, loaded onto a freshly prepared 0.1-1.0 M sucrose gradient and 6 centrifuged at 90,000xg in a Beckman SW41 swing-out rotor overnight and 75,000xg in a Beckman SW28 swing-out rotor for 22 h, respectively. The sucrose gradient buffer composition was 10 mM NaCl, 50 mM Mes-NaOH, pH 6.5 or 50 mM Tris-HCl, pH 7.2 and 0.1% (w/v) β-DM for PSII RC samples and 10 mM NaCl, 5 mM CaCl2, 25 mM MesNaOH, pH 6.5, and 0.03% (w/v) β-DM for PSII cores. The chlorophyll-rich fractions were then removed from the sucrose gradients, frozen in liquid nitrogen and stored at - 80 ºC. Potentiometric redox titrations.- Potentiometric redox titrations were carried out under argon at 12 ºC using D1-D2-Cyt b559 complex samples (5 μM Chl) in 50 mM Mes-NaOH, pH 6.5, by following the absorbance changes at 559 minus 570 nm induced by sequential addition of aliquots of 0.1 M sodium dithionite. The measurements were performed in an Aminco DW-2000 UV-Vis spectrophotometer using the dual wavelength mode. Samples were previously oxidised with 25 μM potassium ferricyanide. The redox potential in the reaction cell were simultaneously measured with a potentiometer (Methrom Herisau, Switzerland) provided with a combined Pt-Ag/AgCl microelectrode (Crison Instruments, Spain) previously calibrated against a saturated solution of quinhydrone (E’m, pH 7, +280 mV at 20 ºC). In addition to ferricyanide (E’m, pH 7, +430 mV) the following redox mediators were used: 10 μM 1,4-benzoquinone (E’m, pH 7, +280 mV), 20 μM 2,3,5,6-tetramethyl-p-phenylendiamine (E’m, pH 7, +240 mV), 20 μM 1,2naphthoquinone (E’m, pH 7, +145 mV), 2.5 μM N-methyl-phenazonium methosulfate (E’m, pH 7, +80 mV), 10 μM N-methyl-phenazonium ethosulfate (E’m, pH 7, +55 mV) and 20 μM tetramethyl-p-benzoquinone (E’m, pH 7, +5 mV). EPR measurements.- Samples were concentrated (0.5 – 1.2 mM Chl) in Centripep-30 and Centricon-30 (Amicon) tubes for EPR measurements. Continuous wave EPR spectra were recorded with a Bruker ESP380E spectrometer working at the X-band 7 (frequency about 9.6 GHz). Typical measurements were achieved at 8 K with 1.46 μW microwave power (which ensures no saturation effects on the signal) and 1 mT of modulation amplitude. 8 3. RESULTS Effect of n-dodecylβ -D-maltoside on Cyt b559 EPR signal. The influence of β-DM on Cyt b559 EPR spectrum was observed in a standard D1-D2-Cyt b559 complex preparation containing six Chl per RC. D1-D2-Cyt b559 complexes were isolated in the presence of various β-DM concentrations ranging from 0 to 0.2% (w/v) (for details see Materials and Methods). No pigment and polypeptide composition varied among samples after detergent treatments. All samples displayed a typical low spin heme EPR signal, with principal values of the g tensor being around gz ≈ 3.0, gy ≈ 2.3 and gx ≈ 1.5.6 Although all spectra were similar, some differences were detected in their gz features, with no changes at gx and gy positions. Following Taylor’s model27 it is expected that gy value remains nearly unaffected when a small shift in gz value occurs. Besides, considering that the gx feature is very broad, it is not possible to detect small changes in it as those observed for gz. Intermediate field feature in the EPR spectra stay at gy = 2.26 for all samples and, when the high field feature is detected, a gx value of 1.53 is obtained. Figure 1 shows the detergent effect on the low field feature in EPR spectra of Cyt b559. The D1-D2-Cyt b559 complex sample suspended in the presence of 0.1% (w/v) β-DM displays a gz feature with a maximum at 2.98 and a full width at half maximum (FWHM) of 13 mT (Fig. 1A,a). On the other hand, D1-D2-Cyt b559 complex samples in the presence of lower β-DM concentration (0.03% (w/v) β-DM) display a gz feature with a maximum at 2.93 that seems to be asymmetric showing a smoother decrease towards lower fields and FWHM of 15 mT (Fig. 1A,b). An EPR signal with similar gz maximum value has been reported in the literature for D1-D2-Cyt b559 complexes isolated following the same procedure.28 The dependence of gz position on β-DM concentration is shown in Fig. 1B. The concentration at which gz peak shifts (0.03-0.06% (w/v)) is above the critical micelle concentration (c.m.c.) of β-DM.29 At c.m.c. detergent monomer molecules self9 ABBREVIATIONS Chl, chlorophyll; c.m.c, critical micelle concentration; Cyt, cytochrome; D1 and D2, core polypeptides of the photosystem II reaction centre; DCBQ, 2,6-dichlorobenzoquinone; E’m, midpoint potential; EPR, electron paramagnetic resonance; FWHM, full width at half maximum; HP, high potential; IP, intermediate potential; LP, low potential; Mes, 2- (N-morpholino)ethanesulfonic acid; PS, photosystem; RC, reaction centre; Tris, tris(hydroxymethyl)aminomethane. REFERENCES [1] O. Nanba and K. 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EPR studies of low-spin iron(III) tetraphenylporphyrins, J. Am. Chem. Soc., 1984, 106, 6888-6898. [37] R. Quinn, M. Nappa, and J.S. Valentine, New fiveand six-coordinate imidazole and imidazolate complexes of ferric tetraphenylporphyrin, J. Am. Chem. Soc., 1982, 104, 2588-2595. [38] J.C. Salerno, S. Yoshida and T.E. King, Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes, J. Biol. Chem., 1986, 261, 5480-5486. [39] B. Lanne, B.G. Malmström and T. Vänngard, The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles, Biochim. Biophys. Acta, 1979, 545, 205-214. [40] M.A. Noordermeer, G.A. Veldink and J.F.G. Vliegenthart, Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation, FEBS Lett., 2001, 489, 229-232. 20 FIGURE LEGENDS Figure 1. (A) Low field region of EPR spectra of oxidised Cyt b559 heme in D1-D2-Cyt b559 complex samples eluted from the column in the presence of a) 0.1% (w/v) of β-DM; b) 0.03% (w/v) of β-DM; c) 0.05% (w/v) of Triton X-100; d) 0.15% (w/v) sucrose monocaprate. (B) Dependence of the Cyt b559 EPR gz value on β-DM concentration. EPR conditions: temperature, 8K; microwave power, 1.46 μW. Other experimental conditions are described in Materials and Methods. Figure 2. Low field region of EPR spectra of oxidised Cyt b559 heme in D1-D2-Cyt b559 complexes: a) sample eluted from the column in the presence of 0.03% (w/v) Triton X-100; b) the same RC sample in (a) subsequently treated with 0.1% (w/v) β-DM through a DEAE-Toyopearl TSK-650S column to replace Triton X100; c) sample eluted from the column in the presence of 0.1% (w/v) β-DM after detergent exchange; d) the same RC sample in (c) after removing β-DM detergent. EPR conditions: temperature, 8K; microwave power, 1.46 μW. Other experimental conditions are described in Materials and Methods. Figure 3. Potentiometric redox titrations of Cyt b559 heme in D1-D2-Cyt b559 complex preparations at pH 6.5 in the presence of 0.03% (w/v) β-DM concentration (μ); 0.1% (w/v) β-DM (F); 0.15% (w/v) sucrose monocaprate (Ì). Experimental conditions are described in Materials and Methods. Figure 4. EPR spectra at the gz region of the oxidised Cyt b559 heme in PSII core complex sample in the presence of a) 0.03% (w/v) Triton X-100; b) 0.1% (w/v) β-DM EPR conditions: temperature, 8K; microwave power, 1.46 μW. Other experimental conditions are described in Materials and Methods. 21 22 200 220 240 260 280 b c d a Magnetic Field (mT) A 0.0 0.1 0.2 2.90 2.93 2.96 2.99 g z value % (w/v) β -DM Fig. 1 23 200 220 240 260 280 d c b a Magnetic Field (mT) Fig. 2 24 -100 0 100 200 300 0 25 50 75 100 % Reduced Cyt b559 Ambient redox potential (mV) Fig. 3 200 220 240 260 280 b a Magnetic Field (mT) Fig. 4 25