Dimerisation of the Rhodobacter sphaeroides RC-LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide

In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC-LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC-LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC-LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC-LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC-LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC-LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.     

Experimental evidence that the membrane-spanning helix of PufX adopts a bent conformation that facilitates dimerisation of the Rhodobacter sphaeroides RC-LH1 complex through N-terminal interactions

The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC-LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC-LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC-LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and β polypeptides on the other half of the dimer.     

Solution structure of the Rhodobacter sphaeroides PufX membrane protein: implications for the quinone exchange and protein-protein interactions

PufX membrane protein is found in Rhodobacter species of purple photosynthetic bacteria and has been known to play an essential role in ubiquinone/ubiquinol exchange between the reaction center and cytochrome bc1 complex and also contribute to the dimerization of the reaction center-light-harvesting core complex. We have determined the solution structure of the Rhodobacter sphaeroides PufX using multidimensional NMR spectroscopy. The PufX, functionally expressed in Escherichia coli, forms a stable alpha helix consisting of 21 residues over the central transmembrane domain. The overall structure of the PufX is very similar to those of the LH1 alpha- and beta-polypeptides from Rhodospirillum rubrum and LH2 polypeptides. A short segment (Lys28-Gly35) rich in Gly and Ala residues revealed a relatively fast exchange between the backbone amide protons and deuteriums in the hydroxyl groups of the solvent, indicating that the backbone of this segment is more easily accessible to the surrounding solvent molecules compared to those of its neighboring portions. The Gly- and Ala-rich segment is located in the middle of the central helix and forms an extensive groove-like conformation on the surface with the neighboring residues, where the residues with large side chains are aligned on one side of the helix, and small residues are aligned on the other face. Such a structural motif may serve as a functional site responsible for ubiquinol transport from the core complex to the membrane phase and for sequence-specific helix-helix interactions with the neighboring polypeptides.     

Structure of the dimeric RC–LH1–PufX complex from Rhodobaca bogoriensis investigated by electron microscopy

Electron microscopy and single-particle averaging were performed on isolated reaction centre (RC)—antenna complexes (RC–LH1–PufX complexes) of Rhodobaca bogoriensis strain LBB1, with the aim of establishing the LH1 antenna conformation, and, in particular, the structural role of the PufX protein. Projection maps of dimeric complexes were obtained at 13 Å resolution and show the positions of the 2 × 14 LH1 α- and β-subunits. This new dimeric complex displays two open, C-shaped LH1 aggregates of 13 αβ polypeptides partially surrounding the RCs plus two LH1 units forming the dimer interface in the centre. Between the interface and the two half rings are two openings on each side. Next to the openings, there are four additional densities present per dimer, considered to be occupied by four copies of PufX. The position of the RC in our model was verified by comparison with RC–LH1–PufX complexes in membranes. Our model differs from previously proposed configurations for Rhodobacter species in which the LH1 ribbon is continuous in the shape of an S, and the stoichiometry is of one PufX per RC.     

Protein-Induced Membrane Curvature Investigated through Molecular Dynamics Flexible Fitting

In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)₂ protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of ∼250-550 Å, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even though the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.     

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.     

Characterization of a Highly Purified, Fully Active, Crystallizable RC–LH1–PufX Core Complex from Rhodobacter sphaeroides

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1–PufX complex (RC–LH1–PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQ_A → D⁺Q_A^-) in the RC as a function of light intensity. The RC–LH1–PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.     

Investigation of Rhodobacter capsulatus PufX interactions in the core complex of the photosynthetic apparatus

The photosynthetic apparatus of purple bacteria in the genus Rhodobacter includes a core complex consisting of the reaction centre (RC), light-harvesting complex 1 (LH1), and the PufX protein. PufX modulates LH1 structure and facilitates photosynthetic quinone/quinol exchange. We deleted RC/LH1 genes in pufX ⁺ and pufX ⁺⁺ (merodiploid) strains of Rhodobacter capsulatus, which reduced PufX levels regardless of pufX gene copy number and location. Photosynthetic growth of RC-only strains and independent assembly kinetics of the RC and LH1 were unaffected by pufX merodiploidy, but the absorption spectra of strains expressing the RC plus either LH1 α or β indicated that PufX may influence bacteriochlorophyll binding environments. Significant self-association of the PufX transmembrane segment was detected in a hybrid protein expression system, consistent with a role of PufX in core complex dimerization, as proposed for other Rhodobacter species. Our results indicate that in R. capsulatus PufX has the potential to be a central, homodimeric core complex component, and its cellular level is increased by interactions with the RC and LH1.     

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii

The photosynthetic membrane in purple bacteria contains several pigment–protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1–RC (light-harvesting complex 1 – reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1–RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC–LH1–PufX cores, as it has been proposed in Rb. sphaeroides.     

The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides wild-type cells. PufX is associated with the reaction center-light harvesting 1 (RC-LH1) core complex and plays a key role in lateral ubiquinone/ubiquinol transfer. We have determined the PufX/RC stoichiometry by quantitative Western blot analysis and RC photobleaching. Independent of copy number effects and growth conditions, one PufX molecule per RC was observed in native membranes as well as in detergent-solubilized RC-LH1 complexes which had been purified over sucrose gradients. Surprisingly, two gradient bands with significantly different sedimentation coefficients were found to have a similar subunit composition, as judged by absorption spectroscopy and protein gel electrophoresis. Gel filtration chromatography and electron microscopy revealed that these membrane complexes represent a monomeric and a dimeric form of the RC-LH1 complex. Since PufX is strictly required for the isolation of dimeric core complexes, we suggest that PufX has a central structural role in forming dimeric RC-LH1 complexes, thus allowing efficient ubiquinone/ubiquinol exchange through the LH1 ring surrounding the RC.     

The PufX protein of Rhodobacter capsulatus affects the properties of bacteriochlorophyll a and carotenoid pigments of light-harvesting complex 1

A pufX gene deletion in the purple bacterium Rhodobacter capsulatus causes a severe photosynthetic defect and increases core light-harvesting complex (LH1) protein and bacteriochlorophyll a (BChl) levels. It was suggested that PufX interrupts the LH1 alpha/beta ring around the reaction centre, allowing quinone/quinol exchange. However, naturally PufX(-) purple bacteria grow photosynthetically with an uninterrupted LH1. We discovered that substitutions of the Rhodobacter-specific LH1 alpha seryl-2 decrease carotenoid levels in PufX(-)R. capsulatus. An LH1 alphaS2F mutation improved the photosynthetic growth of a PufX(-) strain lacking the peripheral LH2 antenna, although LH1 BChl absorption remained above wild-type, suggesting that Rhodobacter-specific carotenoid binding is involved in the PufX(-) photosynthetic defect and LH1 expansion is not. Furthermore, PufX overexpression increased LH1-like BChl absorption without inhibiting photosynthetic growth. PufX(+) LH1 alphaS2-substituted mutant strains had wild-type carotenoid levels, indicating that PufX modulates LH1 carotenoid binding, inducing a conformational change that favours quinone/quinol exchange.     

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

The 8.5A projection structure of the core RC-LH1-PufX dimer of Rhodobacter sphaeroides

Two-dimensional crystals of dimeric photosynthetic reaction centre-LH1-PufX complexes have been analysed by cryoelectron microscopy. The 8.5A resolution projection map extends previous analyses of complexes within native membranes to reveal the alpha-helical structure of two reaction centres and 28 LH1 alphabeta subunits within the dimer. For the first time, we have achieved sufficient resolution to suggest a possible location for the PufX transmembrane helix, the orientation of the RC and the arrangement of helices within the surrounding LH1 complex. Whereas low-resolution projections have shown an apparent break in the LH1, our current map reveals a diffuse density within this region, possibly reflecting high mobility. Within this region the separation between beta14 of one monomer and beta2 of the other monomer is approximately 6A larger than the average beta-beta spacing within LH1; we propose that this is sufficient for exchange of quinol at the RC Q(B) site. We have determined the position and orientation of the RC within the dimer, which places its Q(B) site adjacent to the putative PufX, with access to the point in LH1 that appears most easily breached. PufX appears to occupy a strategic position between the mobile alphabeta14 subunit and the Q(B) site, suggesting how the structure, possibly coupled with a flexible ring, plays a role in optimizing quinone exchange during photosynthesis.     

Overexpression of Rhodobacter sphaeroides PufX-bearing maltose-binding protein and its effect on the stability of reconstituted light-harvesting core antenna complex

The PufX protein, encoded by the pufX gene of Rhodobacter sphaeroides, plays a key role in the organization and function of the core antenna (LH1)-reaction centre (RC) complex, which collects photons and triggers primary photochemical reactions. We synthesized a PufX/maltose-binding protein (MBP) fusion protein to study the effect of the PufX protein on the reconstitution of B820 subunit-type and LH1-type complexes. The fusion protein was synthesized using an Escherichia coli expression system and purified by affinity chromatography. Reconstitution experiments demonstrated that the MBP-PufX protein destabilizes the subunit-type complex (20°C), consistent with previous reports. Interestingly, however, the preformed LH1-type complex was stable in the presence of MBP-PufX. The MBP-PufX protein did not influence the preformed LH1-type complexes (4°C). The LH1-type complex containing MBP-PufX showed a unique temperature-dependent structural transformation that was irreversible. The predominant form of the complex at 4°C was the LH1-type. When shifted to 20°C, subunit-type complexes became predominant. Upon subsequent cooling back to 4°C, instead of re-forming the LH1-type complexes, the predominant form remained the subunit-type complexes. In contrast, reversible transformation of LH1 (4°C) and subunit-type complexes (20°C) occurs in the absence of PufX. These results are consistent with the suggestion that MBP-PufX interacts with the LH1α- polypeptide in the subunit (α/β)-type complex (at 20°C), preventing oligomerization of the subunit to form LH1-type complexes.     

Carotenoid to bacteriochlorophyll energy transfer in the RC–LH1–PufX complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> containing the extended conjugation keto-carotenoid diketospirilloxanthin

RC–LH1–PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC–LH1–PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S₂–Q_x pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC–LH1–PufX we observe an additional, minor energy transfer pathway associated with the S₁ state of diketospirilloxanthin. By comparing the spectral properties of the S₁ state of diketospirilloxanthin in solution, in LH2, and in RC–LH1–PufX, we propose that the carotenoid-binding site in RC–LH1–PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S₁/ICT-mediated energy transfer channel.     

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.     

Structure, function and interactions of the PufX protein

The PufX protein is an important component of the reaction centre-light-harvesting 1 (RC-LH1) complex of Rhodobacter species of purple photosynthetic bacteria. Early studies showed that removal of the PufX protein causes changes in the structure of the RC-LH1 complex that result in a loss of the capacity for photosynthetic growth, and that this loss can be overcome though further mutations that change the structure of the LH1 antenna. More recent studies have examined interactions of the PufX protein with other components of the RC-LH1 complex. This review considers our current understanding of the structure and function of the PufX protein, how this protein interacts with other components of the photosynthetic membrane, and its influence on the oligomeric state of the RC-LH1 complex and the larger-scale architecture of the photosynthetic membrane.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes

In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 A by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of approximately 300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved alpha/beta heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the Q(B) site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G(31)XXXG(35) dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.