Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides

Article1 February 1999free access Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides Colette Jungas Colette Jungas CEA/Cadarache-DSV-DEVM Laboratoire de Bioenergetique Cellulaire, 13108 St Paul-lez-Durance, Cedex, France Search for more papers by this author Jean-Luc Ranck Jean-Luc Ranck Institut Curie, Section de Recherche, UMR 168 et LCR-CEA 8, 11 rue Pierre-et-Marie Curie, 75231 Paris, Cedex 05, France Search for more papers by this author Jean-Louis Rigaud Jean-Louis Rigaud Institut Curie, Section de Recherche, UMR 168 et LCR-CEA 8, 11 rue Pierre-et-Marie Curie, 75231 Paris, Cedex 05, France Search for more papers by this author Pierre Joliot Pierre Joliot Institut de Biologie Physico-Chimique, CNRS UPR 9072, 13 rue Pierre-et-Marie Curie, 75005 Paris, France Search for more papers by this author André Verméglio Corresponding Author André Verméglio CEA/Cadarache-DSV-DEVM Laboratoire de Bioenergetique Cellulaire, 13108 St Paul-lez-Durance, Cedex, France Search for more papers by this author Colette Jungas Colette Jungas CEA/Cadarache-DSV-DEVM Laboratoire de Bioenergetique Cellulaire, 13108 St Paul-lez-Durance, Cedex, France Search for more papers by this author Jean-Luc Ranck Jean-Luc Ranck Institut Curie, Section de Recherche, UMR 168 et LCR-CEA 8, 11 rue Pierre-et-Marie Curie, 75231 Paris, Cedex 05, France Search for more papers by this author Jean-Louis Rigaud Jean-Louis Rigaud Institut Curie, Section de Recherche, UMR 168 et LCR-CEA 8, 11 rue Pierre-et-Marie Curie, 75231 Paris, Cedex 05, France Search for more papers by this author Pierre Joliot Pierre Joliot Institut de Biologie Physico-Chimique, CNRS UPR 9072, 13 rue Pierre-et-Marie Curie, 75005 Paris, France Search for more papers by this author André Verméglio Corresponding Author André Verméglio CEA/Cadarache-DSV-DEVM Laboratoire de Bioenergetique Cellulaire, 13108 St Paul-lez-Durance, Cedex, France Search for more papers by this author Author Information Colette Jungas1, Jean-Luc Ranck2, Jean-Louis Rigaud2, Pierre Joliot3 and André Verméglio 1 1CEA/Cadarache-DSV-DEVM Laboratoire de Bioenergetique Cellulaire, 13108 St Paul-lez-Durance, Cedex, France 2Institut Curie, Section de Recherche, UMR 168 et LCR-CEA 8, 11 rue Pierre-et-Marie Curie, 75231 Paris, Cedex 05, France 3Institut de Biologie Physico-Chimique, CNRS UPR 9072, 13 rue Pierre-et-Marie Curie, 75005 Paris, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:534-542https://doi.org/10.1093/emboj/18.3.534 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and ∼24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 Å and allow the calculation of a projection map at 20 Å. The unit cell (a = 198 Å, b = 120 Å and γ = 103°) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of ∼112 Å in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex. Introduction In bacterial photosynthesis, absorption of light initiates a cyclic electron transfer coupled to proton translocation across the cytoplasmic membrane. Three membrane protein complexes are necessary to collect and convert light energy into chemical energy: the light-harvesting complexes (LHC), the reaction center (RC) and the cytochrome (cyt) bc1 complex. To ensure an efficient collection of light energy, the purple photosynthetic bacteria usually contain two types of LHC; LH1 and LH2. The amounts of LH2 are modulated by several factors, such as light intensity and oxygen tension, while LH1 are synthesized in stoichiometric amounts with the RC (Drews and Golecki, 1995). After light absorption by the LHC, the excitation energy is transferred to the RC where a charge separation occurs between donor and acceptor molecules. The photooxidized primary electron donor, a dimer of bacteriochlorophyll (Bchl) molecules, is reduced by the high potential carriers of the bc1 complex (Meyer and Donohue, 1995) via soluble carriers such as cyt c2. To complete the cyclic electron transfer, this complex is reduced in turn by the photoreduced acceptor. The mechanisms of excitonic energy migration and photoinduced electron transfer have been studied in detail using biophysical approaches, and analyzed at the molecular level following the resolution of the atomic structure of the RC, LH2 and bc1 complexes (Deisenhofer et al., 1985; Allen et al., 1986; McDermott et al., 1995; Xia et al., 1997; Zhang et al., 1998). However, despite the wealth of information available on the individual proteins, their supramolecular organization in the membrane remains unclear. A first issue is the spatial organization between LH1 complexes and RCs. Native membranes and LH1–RC complexes isolated from Rhodopseudomonas (Rp.) viridis (Miller, 1982; Engelhardt et al., 1986; Ikeda-Yamasaki et al., 1998), Ectothiorhodospira halochloris (Stark et al., 1984; Engelhardt et al., 1986), Rp.molischianum (Boonstra et al., 1994) and Rhodospirillum (Rs.) rubrum (Walz and Ghosh, 1997; Stahlberg et al., 1998) have been analyzed by various electron microscopic techniques. All these studies reveal a single RC inside a closed ring of LH1 complexes. Organization of LH1 complexes in a closed ring was also observed by a cryo-electron microscopy study of two-dimensional (2D) crystals of Rs.rubrum purified complexes (Karrasch et al., 1995). The 8.5 Å projection map, obtained in this latter study, showed 16 subunits arranged in a ring-like structure (116 Å overall diameter, 68 Å inner diameter) that could only accommodate one RC. The closed LH1 structure raised the question of how the quinone/quinol transfer took place between the RC acceptor site and the bc1 complex during cyclic electron transfer. Several hypotheses have been proposed, such as slight movements of the α-helices allowing diffusion of the quinone molecules through the ring (Karrasch et al., 1995). It has also been suggested that the presence in the ring of additional proteins such as PufX, an essential polypeptide for photosynthetic growth of Rhodobacter (Rb.) sphaeroides and Rb.capsulatus (Farchaus et al., 1990; Lilburn et al., 1992), or the Ω polypeptide in the case of Rs.rubrum (Ghosh et al., 1994; Stahlberg et al., 1998), may catalyze quinone transfer by forming specific channels through the ring of LH1 complexes (McGlynn et al., 1996). A second issue is the organization of the RC and bc1 complexes in the photosynthetic unit. A series of experiments performed with intact cells of Rb.sphaeroides prompted the proposal that the elements of the photosynthetic electron transfer chain might be organized in supercomplexes (Joliot et al., 1989), each one comprising two RCs, one cyt c2 and one bc1 complex. This proposal originated from the observation that the apparent equilibrium constant between the different reactants, measured during photooxidation of the donor chain under continuous illumination, was much lower than that deduced from their redox mid-point potentials measured at equilibrium (Joliot et al., 1989; Lavergne et al., 1989). The supercomplex hypothesis has been challenged by Fernàndez-Velasco and Crofts (1991), and an alternative model was recently proposed to explain the low value of the observed equilibrium constant (Crofts et al., 1998). The basic assumption in this alternative model is a heterogeneity in the ratio of electron transfer components in individual chromatophores acting as separate compartments. However, this model fails to explain the following important observations made on intact cells of Rb.sphaeroides: (i) the addition of subsaturating concentrations of myxothiazol, a specific inhibitor of the bc1 complex, decreases the number of active bc1 complexes but does not affect the rate of cyclic electron transfer for the uninhibited complexes (Verméglio et al., 1993; Joliot et al., 1996); (ii) the complete photoinduced cyclic electron flow functions even at −20°C in a frozen medium (Joliot et al., 1997); (iii) evidence, by freeze-fracture electron microscopy, of a dimeric arrangement of the photosynthetic membrane proteins (Golecki et al., 1991; Sabaty et al., 1994) in the tubular membranes of Rb.sphaeroides cells deleted in LH2 complexes (Hunter et al., 1988; Kiley et al., 1988) or grown in the presence of nitrate (Sabaty et al., 1994); and (iv) the very recent biochemical evidence that dimeric LH1–RC complexes can be isolated by gentle solubilization of photosynthetic membranes of Rb.sphaeroides (Francia et al., 1998). Here, we report on the biochemical and structural characterization of these tubular membranes. A projection map at 20 Å resolution was obtained by electron microscopy of negatively stained samples. The electron density distribution agrees with the supramolecular arrangement of the photosynthetic apparatus determined by functional approaches, in particular the dimeric association of the photosynthetic RCs (Joliot et al., 1989). An important additional finding is the organization of the LH1 complexes in C-shaped structures, which sheds new light on the coupling of electron transfer between RC and bc1 complex by the quinone molecules. Results Purification and biochemical characterization of the tubular membranes The cytoplasmic membrane of Rb.sphaeroides forma sp. denitrificans grown photosynthetically in the presence of 200 mM nitrate or under dark semi-aerobic conditions presents a large number of tubular membranes (Sabaty et al., 1994). The presence of these tubes is correlated with the decreased synthesis of LH2 complexes. Similar tubular membranes were found in various mutants of Rb.sphaeroides in which this complex is deleted (Hunter et al., 1988; Kiley et al., 1988). Figure 1A shows an electron micrograph of negatively stained spheroplasts of Rb.sphaeroides strain RCLH10. These tubes are between 0.5 and 2 μm long and ∼116 nm in diameter as shown by the freeze-drying electron micrograph depicted in Figure 1B (see also Golecki et al., 1991). Interestingly, these freeze-drying pictures present well-organized rows of dimeric particles, in agreement with freeze-fracture experiments (Golecki et al., 1991; Sabaty et al., 1994). We purified these tubes from cells of Rb.sphaeroides forma sp. denitrificans, and from mutant RCLH10 deleted in LH2 complexes, as described in the Materials and methods. The purity of the preparations was assayed by electron microscopy of negatively stained samples. The best samples contained <10% contamination in the form of cytoplasmic membrane fragments and chromatophores. The upper part of Figure 2 shows the absorption spectrum of purified tubular membranes recorded at 77 K. In the near-infrared spectral region, the main absorption band is centered at 880 nm and corresponds to the Qy transition of the LH1 complexes. In addition, typical bands of Bchl and bacteriopheophytin (Bpheo) molecules of the RC are centered at 804 and 760 nm, respectively. The blue region is dominated by the Qx transitions of the LH1 complexes at 590 nm and by the carotenoid bands centered at 445, 472 and 505 nm. The presence of bc1 complex is revealed by the characteristic α-bands of cyt c1, cyt bH and cyt bL centered at 548, 556 and 561 nm, respectively. From five different preparations of purified tubular membranes, a relative ratio of 0.9 ± 0.05 bc1 complex per 2 RCs was determined. This ratio did not significantly change during the purification procedure, and is similar to that measured for chromatophores of wild-type Rb.sphaeroides (van der Berg et al., 1979). The presence of RCs, bc1 complexes and LH1 complexes was further confirmed by gel electrophoresis (Figure 2B). This gel presents characteristic polypeptide bands of the RC at 28, 26 and 24 kDa. The bands of the cyt b, cyt c1, Rieske protein and subunit IV were observed at 43, 33, 20 and 14 kDa, respectively. These assignments were confirmed by Western blots (not shown). LH1 subunits were detected at 11 and 8 kDa. Part of the LH1 migrates as oligomers of high molecular weight as already observed by Hunter et al. (1988). We also measured the relative ratio of Bchl to RC, which we found to be 24–25 Bchl molecules per RC in agreement with previous work on membrane or isolated complexes (van Grondelle et al., 1994; McGlynn et al., 1996; Francia et al., 1998). Figure 1.(A) Electron micrograph of spheroplasts of Rb.sphaeroides strain RCLH10. Samples were negatively stained with 1% phosphotungstic acid. Bar = 500 nm. (B) Unidirectional shadowed tubular membrane of Rb.sphaeroides forma sp. denitrificans obtained by freeze-drying. Bar = 200 nm. Download figure Download PowerPoint Figure 2.(A) Absorption spectrum of purified tubular membranes of Rb.sphaeroides recorded at 77 K. The inset represents the dithionite-reduced minus ferricyanide-oxidized difference spectrum of the same preparation in the the α-band region. (B) Polypeptide composition of purified tubular membranes isolated from Rb.sphaeroides. RC indicates the three polypeptide subunits H, M and L of the reaction center migrating with apparent molecular weights of 28, 26 and 24 kDa, respectively. The four subunits of the cyt bc1 complex are indicated by FeS, cyt b, cyt c1 and IV. LH1 indicates the subunits of the LH1 complexes with apparent molecular weights of 11 and 8 kDa. Download figure Download PowerPoint Electron microscopy of the tubular membrane The high degree of organization observed by freeze-etching (Golecki et al., 1991; Sabaty et al., 1994) and freeze-drying (Figure 1B) suggests that the tubular membrane purified from Rb.sphaeroides is a natural 2D crystal of membrane proteins that can be analyzed by negative staining and electron microscopy. Figure 3A shows low-dose electron micrographs of negatively stained tubular membranes. Optical diffraction analysis of such images clearly shows two distinct patterns, both with sharp strong spots. The Fourier transform and the filtered image of one tubular crystal are depicted in Figure 3B and C, respectively. These two diffraction patterns correspond to two layered 2D crystals diffracting independently and originating from the top and bottom parts of the tube flattened on the electron microscope grid. Only the pattern with the strongest non-overlapping spots, which corresponds to the top layer, was subsequently used for image processing. The indexation clearly indicated a monoclinic unit cell with parameters: a = 198 Å, b = 120 Å and γ = 103°, and the resolution extended up to 25 Å. Eight images were selected, by optical diffraction, digitized and processed using the procedure of Crowther et al. (1996). Five images were kept for the final merging step. Reflections with quality index for image phases (IQ) values of 5 are visible out to 20 Å resolution (Figure 4A). A projection map was calculated (Figure 4B). The unit cell (198×120 Å) contains an elongated S-shaped supercomplex composed of two C-shaped structures of external diameter 112 Å, their open sides facing each other and enclosing a large protein mass. In addition to the density localized in the center of each ring, a positive density is detected between the two C-shaped structures. This S-shaped supercomplex corresponds closely to the dimeric appearance of the rows observed in freeze-dried (Figure 1B) and freeze-fractured samples (Golecki et al., 1991; Sabaty et al., 1994). However, at the higher resolution achieved in this study, the S-shaped arrangement displays a clear asymmetry for each individual picture (Figure 3C). Figure 3.(A) Low-dose electron micrographs of native tubular membranes from Rb.sphaeroides. Samples were negatively stained with 1% uranyl acetate. Bar = 100 nm for all the tubes. (B) Fourier transform of one tube from digitized scan. The arrow indicates the resolution at 25 Å. (C) Filtered image from masked Fourier transform of electron micrograph. Bar = 200 Å. Download figure Download PowerPoint Figure 4.(A) Diffraction properties of negatively stained native tubular membranes from Rb.sphaeroides. Plot showing the Fourier transform of five negatively stained images after processing and merging. The size of the circles indicates the IQ value. The two dotted rings are at radii corresponding to 1/40 and 1/20 Å−1. (B) Projection map at 20 Å resolution after processing and averaging of negatively stained native tubular flat membrane from Rb.sphaeroides. The unit cell (a = 198 Å, b = 120 Å and γ = 103°) is outlined in black. Positive density representing the protein is shown as solid lines and negative density as dotted lines. Download figure Download PowerPoint Discussion In this work, we purified and analyzed in detail the tubular membranes of Rb.sphaeroides induced by the absence of LH2 complexes. These membranes contain all the proteins necessary for photosynthetic activity: RC, LH1 and bc1 complexes. We found a bc1 complex to RC ratio of 0.5, and 24–25 Bchl molecules per RC. The organization of the photosynthetic unit was analyzed by electron microscopy, taking advantage of the 2D crystal organization of the native membranes. The excellent quality of these tubular membranes enabled us to record low dose images of negative stained specimens and to resolve, for the first time, the supramolecular organization of the complete photosynthetic apparatus in a native membrane. The 20 Å projection map revealed that the membrane proteins are arranged as two connected C-shaped structures enclosing a large protein mass. Taking into account the composition and stoichiometry of the membrane complexes in the tubular membranes, the following interpretation of the projection map shown in Figure 4 is proposed. Each C-shaped structure in the projection map corresponds to LH1 complexes. This attribution agrees with the 8.5 Å projection map of the LH1 complex of Rs.rubrum (Karrasch et al., 1995) which revealed a circular arrangement of 16 transmembrane αβ subunits in a 116 Å diameter ring with a 68 Å hole in the center, similar in size to that observed in our projection map. An important observation is the C-shape of the LH1 complex in the tubular membrane, as opposed to the closed ring observed for the reconstituted LH1 complexes of Rs.rubrum. Given that the 16 αβ subunits present in the closed ring of Rs.rubrum LH1 correspond to 32 molecules of Bchl, and that we measured ∼24 Bchl molecules per RC in the tubular membrane, we deduce that the C-shaped structure should contain ∼12 αβ subunits. The superimposition of 3/4 of the projected structure of the closed ring of LH1 complexes of Rs.rubrum with our projection map, shown in Figure 5, supports this stoichiometry. The protein density observed within each LH1 C-shaped structure can be attributed to one RC, in agreement with previous observations in other species (Miller, 1982; Stark et al., 1984; Engelhardt et al., 1986; Boonstra et al., 1994; Ikeda-Yamasaki et al., 1998). This protein density is well resolved, suggesting that the RC can occupy a single well-defined position with respect to the LH1. The remaining density, localized between the two C-shaped structures, can tentatively be attributed to the bc1 complex. This accounts for both the presence of the bc1 complex in the ratio of 1:2 RCs, and for the asymmetry of the unit cell. However, at the present stage it is difficult to localize the bc1 complex precisely in this negative-stain projection map. Because the stain does not penetrate the membrane, the density contribution in the projection map arises solely from the cytoplasmic surface of the flattened tube. The contribution of the bc1 complex in the projection map is thus expected to be very weak because most of the extramembranous parts of this complex (Rieske protein and cyt c1) are located in the internal membrane of the tube and do not contribute significantly to the projection map. Figure 5.Proposed model of the photosynthetic unit of Rb.sphaeroides viewed from above the membrane. The projection structure of the RC of Rb.sphaeroides (Allen et al., 1987; Ermler et al., 1994) and the projection map of 3/4 of LH1 of Rs.rubrum (Karrasch et al., 1995) are shown in red and green, respectively. Download figure Download PowerPoint The supramolecular organization of the photosynthetic components observed in this electron microscopic study agrees with the suggestion, based on diverse functional evidence, that the photosynthetic electron chain of Rb.sphaeroides is organized in supercomplexes that include two RCs and one bc1 complex as membrane partners (Joliot et al., 1989, 1996, 1997; Verméglio et al., 1993). The observation that only one cyt c2 can bind to two RCs even when an excess of cyt c2 is present in the periplasmic space was an important argument in favor of a dimeric association of the RCs in the supercomplex of Rb.sphaeroides (Verméglio et al., 1993). Similarly, analysis of flash-induced absorbance changes on intact cells of Rs.rubrum has shown that the anticooperative interactions between the two cyt c2-binding sites prevent the simultaneous binding of two cyt c2 molecules on the same dimer of RCs (Joliot et al., 1990). In the case of Rb.sphaeroides, this behavior can be tentatively explained by postulating that the RCs in the supercomplex are positioned so that their M-subunits, and hence the interaction site between the cyt c2 and the RC (Tiede et al., 1993; Adir et al., 1996) are close together. This positioning of the RCs would also allow an efficient exchange of quinone molecules between the RC and the bc1 complex (see below). A tentative match between our density map and a projection map derived from the coordinates of the Rb.sphaeroides RC determined by X-ray crystallography (Allen et al., 1986; Ermler et al., 1994) where the M-subunit and hence the secondary electron acceptor (QB) sites of both RCs are facing the open side of the two LH1 rings in the pseudo-dimeric association, is depicted in Figure 5. It must be emphasized that the functional evidence for an organization in supercomplexes of the photosynthetic electron carriers was not dependent on the LH2:LH1 ratio (Verméglio et al., 1993; Sabaty et al., 1994). This suggests that the supercomplex organization observed in the tubular membrane is also present in the invaginated membrane. The presence of LH2 complexes in non-stoichiometric amounts prevents a crystalline arrangement of the photosynthetic components and leads to the formation of the chromatophores. On the other hand, in the absence of LH2 the formation of the tubular membrane is induced by the crystallization of the membrane proteins present in a stoichiometric ratios. Finally, it is noteworthy that the dimeric organization of the RCs and LH1 complexes reported here is also consistent with the recent observation that dimeric cores composed of two RCs, two PufX polypeptides and ∼27 LH1 subunits can be isolated from wild-type Rb.sphaeroides membranes after gentle solubilization (Francia et al., 1998). From the functional results obtained with Rs.rubrum intact cells (Joliot et al., 1990), we postulate that a similar dimeric association of RCs is also present in this species, although further supporting structural and biochemical evidence is required. Finally, it is also interesting to note that PSII RCs, which are highly homologous to the purple bacteria RCs, have been suggested to present a dimeric association in vivo where the D1/D2 heterodimers are in close contact and surrounded by LHC (Rögner et al., 1996; Hankamer et al., 1997). Besides the structural evidence for a supercomplex organization of the photosynthetic chain of Rb.sphaeroides, other important information can be drawn from our electron microscopy study. One striking feature is the organization of the LH1 complexes in C-shaped structures. This finding contrasts with previous proposals based on image processing and analyses of electron microscopy pictures recorded for both highly organized native membranes and purified LH1–RC and LH1 complexes. The picture that emerges from these various studies is that the LH1 complexes form a closed ring surrounding the RC when present. In the work of Karrasch et al. (1995), it should be noted that the LH1 complexes of Rs.rubrum were dissociated into αβ dimers prior to crystallization, and therefore the closed ring structure occurring in the reconstituted system gives no direct information on the arrangement in the native membrane. Similarly, the formation of a closed ring in the isolated LH1–RC complexes of other species may be induced during solubilization, as discussed by Francia et al. (1998). When the dimeric arrangement is disrupted by solubilization, a closed ring of LH1 completely encircling the RC may well be more stable than a C-shaped structure. In the case of Rp.viridis, the circular appearance of the LH1 ring could be an artifact caused by averaging. Nevertheless, at the present stage, it is difficult to know how far the organization in C-shaped structures of LH1 observed in Rb.sphaeroides can be generalized to other species of purple bacteria, in particular to those containing a low bc1:RC ratio and (or) possessing no PufX-like polypeptide. Whatever the final answer to this question is, organization in C-shaped structures of the LH1 complexes of Rb.sphaeroides in native membrane has important functional and structural implications. This geometry would facilitate diffusion of quinone molecules between RCs and the bc1 complex, especially if the RCs are localized so that the QB sites, located on the M subunit, face the open side of the LH1 ring. Organization of LH1 complexes in C-shaped structures may also account for certain spectroscopic observations. Inhomogeneous absorption of LH1 complexes has been demonstrated by different approaches. Approximately 10% of the Bchl molecules of the LH1 complexes absorb at a long wavelength (Bchl 896) (Kramer et al., 1984) and present a high value (0.25) of time-independent anisotropy (van Grondelle et al., 1987, 1994; Hunter et al., 1989). This minor fraction of the Bchl molecules was suggested to act as a trap for the excitation energy (van Grondelle et al., 1994). One possible interpretation is that the two Bchl molecules localized at the extremities of the C-shaped structure correspond to this long wavelength form of LH1 and are a privileged site of energy transfer between the LH1 complexes and the RC. Several lines of evidence favor a model in which the formation of the C-shaped structures of LH1 we observed are related to the presence of the PufX polypeptide. The PufX protein is a small hydrophobic polypeptide found so far only in Rb.capsulatus and Rb.sphaeroides. The presence of this polypeptide is essential for photosynthetic growth (Farchaus et al., 1990; Lilburn et al., 1992) and facilitates the exchange of quinone molecules between the Qo site of the bc1 complex and the QB site of the RC (Farchaus et al., 1990; Lilburn et al., 1992; Barz et al., 1995a,b). The evidence for its involvement in the formation of C-shaped structures of LH1 can be summarized as follows: (i) deletion of the pufX gene induces a significant increase in the ratio of Bchl molecules to RC in both Rb.capsulatus and Rb.sphaeroides species (Farchaus et al., 1992; Westerhuis et al., 1993; McGlynn et al., 1996; Francia et al., 1998); (ii) low concentrations of the PufX protein inhibit the in vitro reconstitution of LH1 complexes for both Rb.sphaeroides and Rb.capsulatus (Recchia et al., 1998). This small polypeptide may also be an essential factor for the supramolecular organization of the photosynthetic apparatus because the presence of dimeric LH1–RC complexes depends strictly on the presence of PufX (Francia et al., 1998). Our results suggest that the bc1 complex occurs as a monomer in the tubular membrane of Rb.sphaeroides. Indeed, if the bc1 complex were present in a dimeric form, this dimer would have to be localized between two S-shaped structures to fulfill the 1:2 ratio of bc1 complex to RC. Thus, the unit cell would contain four RCs and be twice as large as observed. Our proposal of a monomeric bc1 complex is at odds with several lines of biochemical and structural evidence in favor of a dimeric form of this complex. First, the bc1 complex isolated from Rb.sphaeroides migrates as a dimer on sizing gels (Guergova-Kuras et al., 1998). Secondly, a symmetric dimer is also present in the crystal structure of the mitochondrial bc1 complex described recently at 2.9 Å resolution (Xia et al., 1997; Zhang et al., 1998). This dimeric association certainly stabilizes the isolated purified bc1 complex. In a native membrane, however, specific interactions between the bc1 complex and other membrane proteins involved in the formation of supercompl