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 role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides

Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an "S"-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex.     

Identification of protein W,the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc₁ complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.     

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.     

Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides.

The core complex of Rhodobacter sphaeroides is formed by the association of the light-harvesting antenna 1 (LH1) and the reaction center (RC). The PufX protein is essential for photosynthetic growth; it is located within the core in a 1 : 1 stoichiometry with the RC. PufX is required for a fast ubiquinol exchange between the Q(B) site of the RC and the Qo site of the cytochrome bc1 complex. In vivo the LH1-PufX-RC complex is assembled in a dimeric form, where PufX is involved as a structural organizer. We have modified the PufX protein at the N and the C-terminus with progressive deletions. The nine mutants obtained have been characterized for their ability for photosynthetic growth, the insertion of PufX in the core LH1-RC complex, the stability of the dimers and the kinetics of flash-induced reduction of cytochrome b561 of the cytochrome bc1 complex. Deletion of 18 residues at the N-terminus destabilizes the dimer in vitro without preventing photosynthetic growth. The dimer (or a stable dimer) does not seem to be a necessary requisite for the photosynthetic phenotype. Partial C-terminal deletions impede the insertion of PufX, while the complete absence of the C-terminus leads to the insertion of a PufX protein composed of only its first 53 residues and does not affect the photosynthetic growth of the bacterium. Overall, the results point to a complex role of the N and C domains in the structural organization of the core complex; the N-terminus is suggested to be responsible mainly for dimerization, while the C-terminus is thought to be involved mainly in PufX assembly.     

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.     

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.     

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.     

The PuhB protein of Rhodobacter capsulatus functions in photosynthetic reaction center assembly with a secondary effect on light-harvesting complex 1

The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.     

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.     

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.     

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.     

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.     

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.     

Protein shape and crowding drive domain formation and curvature in biological membranes

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.     

Carotenoids are essential for normal levels of dimerisation of the RC-LH1-PufX core complex of Rhodobacter sphaeroides: characterisation of R-26 as a crtB (phytoene synthase) mutant

Carotenoids play important roles in photosynthesis where they are involved in light-harvesting, in photo-protection and in the assembly and structural stability of light-harvesting and reaction centre complexes. In order to examine the effects of carotenoids on the oligomeric state of the reaction centre-light-harvesting 1 -PufX (RC-LH1-PufX) core complex of Rhodobacter sphaeroides two carotenoid-less mutants, TC70 and R-26, were studied. Detergent fractionation showed that in the absence of carotenoids LH2 complexes do not assemble, as expected, but also that core complexes are predominantly found as monomers, although levels of the PufX polypeptide appeared to be unaffected. Analysis of R-26 membranes by electron microscopy and atomic force microscopy reveals arrays of hexagonally packed monomeric RC-LH1-PufX complexes. Transfer of the crtB gene encoding phytoene synthase to TC70 and R-26 restores the normal synthesis of carotenoids demonstrating that the R-26 mutant of Rba. sphaeroides harbours a mutation in crtB, among its other defects. The transconjugant TC70 and R-26 strains containing crtB had regained their ability to assemble wild-type levels of dimeric RC-LH1-PufX core complexes and normal energy transfer pathways were restored, demonstrating that carotenoids are essential for the normal assembly and function of both the LH2 and RC-LH1-PufX complexes in this bacterial photosystem.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH >11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH >9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q(A)Q(B)(-) state is stabilized by about 40 meV at 6.511. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and (31)P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

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.