Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

1 Reconstitution, spectroscopy and redox properties of the photosynthetic recombinant cytochrome b559 from higher plants. María A. Lujána, Jesús I. Martínezb, Pablo J. Alonsob, Fernando Guerreroc, Mercedes Roncelc, José M. Ortegac, Inmaculada Yruelaa and Rafael Picorela*. aEstación Experimental de Aula Dei (EEAD), Consejo Superior de Investigaciones Científicas (CSIC), Carretera Montañana 1005, E-50059 Zaragoza, Spain. bInstituto Ciencia de Materiales de Aragón (CSIC-Universidad de Zaragoza), C/ Pedro Cerbuna 12, E-50009 Zaragoza, Spain. cInstituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), C/ Américo Vespucio 49, E41092 Sevilla, Spain. *Corresponding author: Rafael Picorel, Estación Experimental de Aula Dei, Carretera Montañana 1005, E-50059 Zaragoza, Spain. Phone: 34-976-716053; FAX: 34-976-716145; email:[email protected]. 2 Abstract A study of the in vitro reconstitution of sugar beet cytochrome b559 of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b559 form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b559. Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b559 subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b559-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b559-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b559. A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme. Keywords: Cytochrome b559, electron paramagnetic resonance, reconstitution, redox titration. Abbreviations Abs Absorbance BCA bicinchroninic acid Cyt cytochrome 3 β-DM n-dodecyl-β-D-maltoside DEAE diethyl aminoethyl cellulose Eh ambient redox potencial Em midpoint redox potential EPR electron paramagnetic resonance ε extinction coefficient HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HP high potencial HS high spin IP intermediate potencial IPTG isopropyl β-D-1-thiogalactopyranoside KDS potassium dodecyl sulphate LHCP light-harvesting chlorophyll-protein LP low potential LS low spin MBP maltose-binding protein MES 2-(N-Morpholino)ethanesulfonic acid OD optical density PAGE polyacrylamide gel electrophoresis PMSF phenylmethanesulfonylfluoride PS photosystem SDS sodium dodecyl sulphate TRIS tris(hydroxymethyl)aminomethane 4 Introduction Cytochrome b559 (Cyt b559) is an integral component of the photosystem II (PSII) reaction center (Stewart and Brudvig 1998). It comprises two small polypeptides, α (9 kDa) and β (4.5 kDa) subunits, encoded by psbE and psbF genes, respectively. Each subunit contains one alphahelix that spans the thylakoid membrane, with the N-terminus in the stromal side (Guskov et al. 2009). The b-type heme is coordinated to two histidines in a planar axial structure (Babcock et al. 1985; García-Rubio et al. 2003), one from each subunit, and it is located towards the stromal side (Picorel et al. 1994; Guskov et al. 2009). Despite many attempts, Cyt b559 function remains unclear. It has been demonstrated that Cyt b559 is essential for the correct assembly of the PSII (Pakrasi et al. 1989; Swiatek et al. 2003), although it is not involved in the primary electron transfer within PSII (Stewart and Brudvig 1998). One of the most accepted hypotheses put forward suggests its involvement in PSII protection against photoinhibition (Stewart and Brudvig 1998; Hung et al. 2010). Cytochrome b559 has singular redox properties among b-type cytochromes. It exhibits several midpoint redox potential (Em) forms (Cramer and Whitmarsh 1977; Ortega et al. 1988; Thompson et al. 1989; Roncel et al. 2001): a high-potential form (HP, Em ≈ +400 mV), an intermediate-potential form (IP, Em ≈ +200-150 mV), and a low-potential form (LP, Em ≈ +100 mV). The HP form is very labile as it has only been observed in intact chloroplasts and some isolated PSII preparations (Stewart and Brudvig 1998). Purified Cyt b559 displayed only the LP form (Metz et al. 1983). The molecular mechanisms responsible for these singular redox properties are mainly unknown, although several hypotheses have been proposed (Metz et al. 1983; Babcock et al. 1985; Roncel et al. 2001; Kaminskaya et al. 2007; Shibamoto et al. 2008). The degree of the heme exposure to solvents and its environmental hydrophobicity seem to modulate the Cyt b559 redox properties (Ortega et al. 1988; Ahmad et al. 1993; Kaminskaya et al. 1999; Roncel et al. 2001). 5 Cytochrome b559 is a paramagnetic species, thus electron paramagnetic resonance (EPR) spectroscopy is a suitable technique to characterize this metalloprotein. The principal values of the g-tensor are gZ ≈ 3.05-2.9, gY ≈2.26-2.15, and gX ≈ 1.5-1.4, which correspond to a low spin (LS) heme center (Babcock et al. 1985; Stewart and Brudvig 1998; Yruela et al. 2003). Some in vitro and in vivo studies have been carried out with the β subunit alone. It was found that the β subunit was able to undergo (ββ) homodimerization and bound the heme group to make a Cyt b559-like structure. It showed spectral and redox properties similar to those of native Cyt b559. In vitro reconstitutions with commercial heme and the β subunit from the cyanobacterium Synechocystis sp. PCC 6803 were described, either from chemically synthesized peptide (Francke et al. 1999) or from the inclusion bodies of Escherichia (E.) coli expressing β subunit in (Prodohl et al. 2005). Furthermore, in vivo formation of the (ββ) homodimeric Cyt b559-like structure was reported when the β subunit from Synechocystis (Prodohl et al. 2005) or Synechococcus sp. PCC 7002 (Yu et al. 2003) was expressed in E. coli. The polypeptide was integrated into the host bacterial inner membrane as a (ββ) homodimer, and then it bound bacterial endogenous heme to make a Cyt b559-like structure. In the present study, we first report the expression in E. coli of the two Cyt b559 subunits α and β from plant species, and the psbE gene from a cyanobacterium. With the recombinant subunits from sugar beet (MBP-Rsubα and MBP-Rsubβ), we studied the ability of in vitro reconstitution of αα, ββ and αβ forms, the heterodimer being more efficient. We also describe the insertion of α and β subunits from sugar beet and Synechocystis (MBP-Ssubα and MBPSsubβ), and the β subunit from maize (MBP-Msubβ) in E. coli inner cytoplasmic membrane during their expression. Only the β subunit from the cyanobacterium was able to bind heme to form a Cyt b559-like structure in vivo. The spectroscopic and redox properties of the obtained forms are described. 6 Materials and methods Cloning and expression of the cytochrome b559 subunits. The psbE and psbF genes from sugar beet and Synechocystis and psbF gene from maize were cloned and expressed in E. coli as fusion proteins using standard procedures. Genes were obtained from genomic DNA by PCR with specific primers, containing a BamHI site forward oligonucleotides and a HindIII site reverse primers. Each gene was cloned into pMAL-c2X expression vector and the sequence of each final construct was confirmed by DNA sequencing. These constructions will give fusion proteins composed of MBP (maltose-binding protein), a protein carrier, and α or β Cyt b559 subunits. Escherichia coli TB1 competent cells were transformed with the constructs for expression of each fusion protein. A pre-culture was grown overnight from a selected single colony at 37 ºC in LB broth (Miller) medium in the presence of 100 µg ml-1 ampicillin. A larger volume of LB broth with ampicillin was inoculated with the pre-culture on the next day and it was grown at 37 ºC until the OD600nm reached 0.6 units. Then 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture to induce the expression of the fusion proteins for 3 h at 37 ºC, with the exception of the MBP-Ssubα that was expressed with 1 mM IPTG for 17 h at 18 ºC. After induction, cells were harvested by centrifugation at 10,000g for 5 min and frozen at -20 ºC until use. For protein purification, the bacterial pellet was resuspended in 50 mM HEPES, pH 7.5, 10 mM EDTA, and broken by sonication (Ultrasonic Processor XL 2020 Misonix, Farmingale, NY USA) for 45 min (90 s pulses with 60 s intervals) with sample recipient on ice to avoid heating. Cell extracts were clarified by centrifugation at 15,000g for 10 min at 4 ºC, obtaining the insoluble material with the inclusion bodies as a pellet. This pellet was saved and the supernatant centrifuged again at 40,000g for 45 min at 4 ºC to sediment small cytoplasmic membrane fragments, which were resuspended in the same buffer and analyzed by UV-Vis absorption and EPR spectroscopies, and redox potentiometric titration. 7 The saved pellet from the first centrifugation containing the inclusion bodies was washed once by centrifugation (10,000g for 5 min at 4 ºC) with 50 mM HEPES, pH 7.5, 10 mM EDTA, 1% (w/v) Triton X-100, resuspended with 20 mM TRIS-HCl, pH 8.0, 50 mM sodium dodecyl sulphate (SDS) and saved to make subsequently in vitro reconstitutions. Total protein concentration was determined using the BCA reagent (Pierce Thermo Scientific, Rockford, IL USA). Proteins were separated on SDS-PAGE [12% or 20% (w/v) acrylamide and 4 M urea] and revealed by Coomassie Brilliant Blue staining. In vitro reconstitution In vitro heterodimeric Cyt b559 reconstitution was done using equimolar amounts of MBP-Rsubα and MBP-Rsubβ fusion proteins obtained as explained above. The detergent SDS of the fusion protein mixture was exchanged with 0.15 mM n-dodecyl β-D-maltoside (β-DM) by precipitating with 50 mM KCl. After incubating for 10 min on ice, the precipitated potassium dodecyl sulphate (KDS) was removed by centrifugation at 6,000g for 15 min at 4 ºC. Finally, equimolar amounts of heme from chemical hemin chloride (Fluka, Buchs, Switzerland) dissolved as described by Kroliczewski and Szczepaniak (2002), were added to the fusion protein mixture. After in vitro reconstitution, protease cleavage was performed with Factor Xa to release the Cyt b559 subunits from the MBP. Cleavage was done in 20 mM TRIS-HCl, pH 8.0, 1 mM CaCl2, 0.15 mM β-DM with 10 units of Factor Xa per mg of fusion protein at 22 ºC for 4 h. The reaction was stopped by adding 1 mM PMSF protease inhibitor. The resultant cleavage mixture was purified by weak anionic-exchange chromatography with a TSK Toyopearl DEAE 650s (TOSOH Bioscience GmbH, Stuttgart, Germany) column pre-equilibrated with 20 mM TRIS-HCl, pH 8.0, and 0.15 mM β-DM. After sample loading, the column was washed with five column volumes of the same buffer, and the material eluted with a 0-500 mM NaCl continuous gradient in the same buffer at a flow rate of 0.5 ml min-1 in 1-ml fractions. Fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining, and those containing Cyt b559 subunits were pooled and 8 concentrated using a Centriprep or Centricon 3000 NMWL filter (Millipore, Billerica, MA USA). Since some hemes could have been detached during the chromatography, commercial heme was added again in excess to the concentrated sample to ensure maximum reconstitution, and, immediately after, the mixture was passed through three consecutive desalting PD-10 columns (GE Healthcare) to remove most of free heme (Kroliczewski and Szczepaniak 2002). The final sample was concentrated ten times using a Centricon 3000 NMWL filter tube, and the degree of Cyt b559 reconstitution was determined by UV-Vis spectroscopy. In vitro (αα) and (ββ) homodimeric Cyt b559 reconstitutions were obtained in the same way but using one type of fusion protein only. Absorption spectroscopy Visible absorption spectra in the 400-600 nm range were obtained with a Beckman DU 640 spectrophotometer (Beckman Coulter, Brea, CA USA). Samples were measured either in airoxidized or reduced with 10 mM sodium dithionite. The spectra were compared at the α band at around 559 nm of the reduced minus oxidized differential absorption spectra. Two extinction coefficients (ε) were used to determine the extent of the cytochrome reconstitution using the difference absorption spectra, i.e., ε559.5nm - 577nm = 21.5 mM-1 cm-1 or ε559.5nm - isosbestic point = 17.5 mM-1 cm-1 (Stewart and Brudvig 1998). Electron paramagnetic resonance EPR measurements were recorded with a Bruker ESP380E spectrometer (Bruker, Karlsruhe, Germany) working at the X-band. For low-temperature measurements, an Oxford CF935 liquid helium continuous-flow cryostat (Oxford Instruments, Eynsham, UK) was used. Typical conditions for continuous-wave EPR (CW-EPR) measurements were: temperature, 15K; microwave frequency, 9.70 GHz; microwave power, 3.2 x 10-2 mW; modulation amplitude, 3.0 Gauss. Two-pulse echo induced EPR (2p ei-EPR) experiments were performed by using the 9 (п/2 - т - п - т) sequence, and detecting the echo intensity as a function of the applied magnetic field. The experimental conditions were 6K and 9.78 GHz. Some ei-EPR spectra showed a spurious contribution most probably from a Cu(II) species (see Results). In order to make the contributions from heme centers easier to see, a numerical subtraction of this spurious signal was performed. From a sample where the relative intensity of the heme signals was low, the Cu(II) signal in the field interval (270-360 mT) was isolated. This signal was subtracted from the measured spectra for the Cyt b 559 reconstituted samples. Additionally, a numerical “adjacent averaging” filter was used in order to improve the signal-tonoise ratio without losing the signals resolution. Potentiometric redox titrations Potentiometric redox titrations were carried out basically as described by Guerrero et al. (2011). For titrations, samples were suspended in 2.5 ml buffer containing 40 mM MES-NaOH, pH 6.5, 0.587 mM β-DM, and the following redox mediators: 10 µM 2,5-dimethyl-p-benzoquinone (E’m7 = +180 mV), 20 µM o-naphtoquinone (E’m7 = +145 mV), 2.5 µM N-methyl-phenazonium methosulfate (E’m7 = +80 mV) and 20 µM duroquinone (E’m7 = +5 mV). Experiments were done at 20 ºC under argon atmosphere and continuous stirring. Reductive titrations were performed by first oxidizing with 25 µM potassium ferricyanide and then reducing it stepwise with small aliquots of 0.1 M sodium dithionite. After addition of sodium dithionite, the absorption spectrum between 500-600 nm range and the redox potential of the solution were simultaneously recorded by using, respectively, a SLM Aminco DW2000 UV-Vis spectrophotometer and a Metrohm potentiometer (Metrohm Ltd., Herisau, Switzerland) provided with a combined PtAg/AgCl microelectrode (Microelectrodes Inc, Bedford, NH USA) previously calibrated against a saturated solution of quinhydrone (E’m7 = +280 mV at 20 ºC). Differential spectra of Cyt b559 were obtained by subtracting the absolute spectra recorded at each Eh during titrations from the spectra of the fully oxidized cytochrome. The absorbance difference at 559 nm minus 570 nm 16 already have the Phe codon at that position). This change due to RNA-editing that occurs in certain higher plants is not possible in bacteria (Maier et al. 1996). Thus β subunit from sugar beet expressed in bacteria maintained a Ser at position 26 instead of a Phe. To determine if the presence of Ser26 in the recombinant β subunit sequence impaired its reconstitution, we expressed the β subunit from maize (MBP-Msubβ) in E. coli (note that psbF gene from maize already encodes Phe residue at position 26). When the redox differential absorption spectrum of membrane fragments containing the MBP-Msubβ from maize was compared with that of the β subunit from sugar beet, no differences were observed between both samples (data not shown). Thus the Phe26 residue was not essential for in vivo formation with the β subunit. Since β subunit from Synechocystis was the only polypeptide able to form spontaneously a Cyt b559-like structure in the host E. coli inner membrane, we further characterized such a protein structure by EPR spectroscopy and potentiometry. To distinguish between the properties of the formed (ββ) Cyt b559-like structure from other b-type endogenous cytochromes and other potential interferences induced by the expressed protein carrier, MBP, we also studied the bacterial membrane fragments expressing the empty pMAL-c2X vector (negative control). CW-EPR spectra (Fig. 7a) showed features at gef = 6.0, gef = 4.3 and gef = 2.0 values that were present both in the negative control and in the membrane fragments containing (ββ) homodimer. They corresponded to HS heme, non-heminic iron or endogenous radicals located in the E. coli inner membrane. Besides, LS heme contributions were also detected both in the negative control and in the membrane fragments containing (ββ) homodiner. A part of these signal intensities come from endogenous heme centers located in the E. coli inner membrane but the LS heme contribution in the CW-EPR spectrum of the negative control (not shown) was clearly much less intense than the one detected for membrane fragments containing (ββ) homodimers. When the CW-spectrum of the negative control was subtracted from that of the 17 (ββ) homodimer sample (Fig. 7a), the remaining features at gef = 2.96 and gef = 2.28 should correspond to the (ββ) homodimeric Cyt b559–like structure (Fig. 7a). The ei-EPR spectrum showed again intense features at gef ≈ 2.08 (Cu(II) signal) and gef = 2.0 (signal from endogenous free radicals). These features are truncated in Fig. 7b for a shake of clarity. Besides, the ei-EPR spectrum displayed characteristic features (gef = 2.96, gef = 2.28, and gef = 1.5), typical of a LS heme analogous to that previously reported for native Cyt b559 (Babcock et al. 1985; Stewart and Brudvig 1998; Yruela et al. 2003). Thus this sample showed only a LS form due to (ββ) homodimeric Cyt b559-like structure within E. coli membrane that corresponds to a heme with a planar axial bis-histidine coordination as in the case of native Cyt b559 (Babcock et al. 1985; Stewart and Brudvig 1998; Yruela et al. 2003). Potentiometric reductive titrations were carried out at pH 6.5 (Fig. 8) in the same redox conditions than those for in vitro reconstitution. E. coli inner membrane fragments containing β subunit exhibited two redox species with different Em (i.e., +58 mV and +126 mV) (Fig. 8). Note that the measurements were achieved in E. coli membrane fragments, where endogenous cytochromes were also present as mentioned above. As in the case of absorption and EPR measurements, these endogenous hemoproteins should also be detected in the potentiometric measurements. As a control, potentiometric reductive titrations of E. coli inner membrane fragments containing MBP only were carried out, and only one Em of +60 mV was calculated (Fig. 8). These results suggested that Em = +58 mV form, found in membrane fragments containing β subunit was due to bacterial endogenous cytochromes, and the Em = +126 mV form corresponded to the formation of (ββ) homodimeric Cyt b559-like structure. The higher Em (+126 mV) obtained in the bacterial membranes should not be due to the fact that the β polypeptide is forming a fusion protein with the MBP, a soluble moiety. Preliminary experiments indicated that the fusion proteins are anchored within the bacterial cytoplasmic membrane by the alpha-helix of the Cyt b559 subunits since protease treatment actually liberated the MBP 18 moiety, remaining the alpha-helix in the membrane pellet after centrifugation (data not shown). So the soluble moiety of the fusion proteins is very well separated from the cytochrome subunits making it difficult to have any significant influence on the redox properties of in vivo formed Cyt b559-like structure. Discussion In recent years, in vitro reconstitutions of various b-type cytochromes have been reported. For instance, recombinant membrane proteins as Cyt b5 (Mulrooney and Waskell 2000), Cyt b6 (Kroliczewski and Szczepaniak 2002) and (ββ) homodimer Cyt b559–like structure (Prodohl et al. 2005), have been described. In the present study, we report for the first time in vitro reconstitution of the photosynthetic Cyt b559, a cytochrome comprised of two different protein subunits that coordinate a heme group. It has to be noted that the spectral α-band around 559 nm of the heterodimer appeared somewhat less distorted and with higher amplitude than those from the homodimers, indicating thar (αβ) heterodimerization dominates over (αα) or (ββ) homodimerization. This may explain why nature has selected the heterodimer as the functional form in oxygenic photosynthesis. However, we still cannot discard the formation of some (αα) and (ββ) homodimers during the in vitro (αβ) heterodimer reconstitution. We have tried to separate the different potential dimeric forms (αβ, αα, ββ) of the reconstitution mixture by using native PAGE gel with mild conditions and probe with the corresponding antibodies, but all dimeric forms were destroyed during the electrophoresis, the subunits α and β appearing as separated bands. Such instability of the in vitro reconstituted Cyt b559 is not totally surprising, considering the 3-D structure of this cytochrome, where the three moieties (α, β, heme) are highly exposed to the electrophoresis reactants. Besides the native EPR form LS1, two additional forms, HS and LS2 were found in the reconstituted heterodimeric sample from plants (Fig. 4). Both HS and LS2 forms have also 19 been found in native Cyt b559 (Loew 1983; Scheidt and Gouterman 1983; Shuvalov et al. 1995; Kropacheva et al. 2003) and, according to the features observed in other metalloproteins such as catalase, haemoglobin and myoglobin (Blumberg and Peisach 1971; Fiege et al. 1995), assigned to heme distorted environment (HS) and to heme centers with one of the coordinated histidines displaced by hydroxyl group (LS2). The intensity of the LS2 signal in the native Cyt b559 EPR spectra depended on sample integrity and treatments (Fiege et al. 1995; Bianchetti et al. 1998). A model has been suggested in which the two subunits remained linked, but one histidine ligand was replaced by an –OH group as second axial ligand (Shuvalov et al. 1994). It is well possible that some heterodimers are not perfectly assembled in vitro, because the protein subunits are free in solution, in contrast to more restricted conditions within the biological membrane. The detergent present in the solution could also add some structural distortion, although small amounts of SDS detergent was proved even to facilitate the in vitro reconstitution (Weber et al. 2011). The Em (+36 mV) of our in vitro reconstituted samples was very similar to that found in isolated native Cyt b559 (Matsuda and Butler 1983; Ortega and Hervás 1989), though this redox potential is influenced by the pH and the hydrophobicity surrounding the Cyt b559 heme center (Ortega et al. 1988; Ahmad et al. 1993; Kaminskaya et al. 1999; Roncel et al. 2001; Weber et al. 2011). Expressed foreign integral membrane proteins in E. coli have a natural tendency to aggregate into inclusion bodies or insert into the bacterial inner membrane (Drew et al. 2003). Examples of spontaneous insertion into the bacterial membrane are the recombinant LHCP (Kohorn and Auchincloss 1991), Cyt f (Rothstein et al. 1985), Cyt b5 (Smith et al. 1994) and Cyt b6 (Kroliczewski et al. 2005). But these three cytochromes were made of a single polypeptide, and were obtained as holoprotein using bacterial endogenous heme. Recent works have also described transformant bacterial membranes with the Cyt b559 β subunit from Synechocystis 20 (Prodohl et al. 2005) and Synechococcus (Yu et al. 2003), with spectral properties analogous to those of native Cyt b559. According to our SDS-PAGE analysis, both Cyt b559 subunits from the different organisms used in the present study were inserted spontaneously into the bacterial inner membrane (Fig. 1). However, among all Cyt b559 subunits analyzed in the present study, β subunit from Synechocystis was the only polypeptide able to form a Cyt b559-like structure spontaneously in E. coli inner membrane. The negative results obtained with α subunit from all organisms tested, suggest that there are some constrains for the correct heme assembly independently of protein insertion within the bacterial membrane. This negative result was not due to less stability of the recombinant α polypeptide within the bacterial membrane due to, for instance, specific membrane protease degradation since its size remained the same as the native one (Fig. 1). Note that we also detected a poorer in vitro reconstitution capacity for this subunit (see Fig. 3). At present we do not have any clue to explain why overexpressed β subunit from plant sugar beet unlike Synechocystis was unable to form a Cyt b559-like structure in E. coli inner membrane, though its sequence is highly conserved and in both cases the subunits were integrated in the bacterial membrane as full polypeptides. The chloroplastic origin of our plant protein subunits does not seem to account for it since recombinant Cyt b6 from spinach was successfully reconstituted in bacteria (Kroliczewski et al. 2005). EPR analysis of bacterial inner membrane fragments containing the β subunit from Synechocystis showed only a LS1 form, with similar spectroscopic features to those of native Cyt b559 (Babcock et al. 1985; Stewart and Brudvig 1998; Yruela et al. 2003). This proves the heme coordination to in vivo formed (ββ) homodimer. It is worth of noting that LS2 was not detected in these samples, indicating that the formation of a distorted Cyt b559 was restricted within the biological membrane most probably due to less hydroxylation activity within the hydrophobic membrane matrix. In addition, the EPR features of this LS1 form shows a small 21 shifting when compared to those of the LS1 form found in in vitro reconstituted Cyt b559. These discrepancies may be connected with some small differences in the environment surrounding the LS heme centers in both cases (Yruela et al. 2003). Furthermore, previous Em measured for the (ββ) homodimer (+50 mV) were smaller than that obtained in the present work (+126 mV) (Francke et al. 1999; Yu et al. 2003). It has to be noted that these potentiometric measurements were carried out in samples where the (ββ) homodimer was outside of a biological membrane and, therefore, close to that of our in vitro reconstitution. The observed differences indicate the important role of the environment surrounding the heme on its Em value (Ortega et al. 1988; Ahmad et al. 1993; Kaminskaya et al. 1999; Roncel et al. 2001). In fact, the heme environment of the in vivo formation and the natural thylakoid membrane conditions is more hydrophobic than the aqueous buffer solutions used for in vitro reconstitution. Interestingly, the native Cyt b559 LP form in PSII membranes exhibits a redox potential value of +110 mV (Roncel et al. 2001), similar to that measured in this work in bacterial inner membrane fragments. Concluding, we have developed a methodology to reconstitute the αβ heterodimeric form of a plant Cyt b559. This can be useful tool to future studies to elucidate the mechanism by which Cyt b559 matures, and to explore the molecular mechanisms that regulate the intriguing redox potential variability of this elusive metalloprotein. This methodology can even be more powerful if used together with site-directed mutagenesis techniques, and the application of advanced spectroscopic techniques with no interferences from other tetrapyrrol molecules such as chlorophylls as normally occurred when using photosynthetic materials. Future work will also aim to determine the different behaviour of α subunit compared to β subunit for in vivo formation despite their structural similarity. Acknowledgements 22 This work was supported by Grants AGL2008-00377, MAT2008-03461, and BFU2007-68107C02-01 from the Spanish Ministry of Science and Innovation (MICINN), PADI CVI-261 from the Andalusia Regional Government, and DGA-GC E33 and DGA-GE B18 from Aragon Regional Government. All these Grants were partially financed by the EU FEDER Program. M. A. Luján would like to thank to the FPI Fellowship Program of the MICINN for financial support. References Ahmad I, Giorgi LB, Barber J, Porter G, Klug DR (1993) Redox potentials of cytochrome b-559 in the D1/D2/cytochrome b-559 reaction centre of Photosystem II. Biochim Biophys Acta 1143 (2):239-242 Babcock GT, Widger WR, Cramer WA, Oertling WA, Metz JG (1985) Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure. Biochemistry 24 (14):3638-3645 Betton JM, Hofnung M (1996) Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. 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