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.     

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.     

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.     

A reaction center-light-harvesting 1 complex (RC-LH1) from a Rhodospirillum rubrum mutant with altered esterifying pigments: characterization by optical spectroscopy and cryo-electron microscopy

Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p4212 space group. A projection map calculated to 9 A shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.     

Isolation and purification of the reaction center (RC) and the core (RC-LH1) complex from Rhodobium marinum: the LH1 ring of the detergent-solubilized core complex contains 32 bacteriochlorophylls

The reaction center (RC) and the core (RC-LH1) complex were isolated and purified from Rhodobium marinum; together with the LH1 complex [Meckenstock et al. (1992a) FEBS Lett. 311: 128], a complete set of RC, LH1 and RC-LH1 from the same wild-type strain of a purple photosynthetic bacterium can therefore now be made. Comparison of the BChl a/BPhe a ratio (determined by HPLC) between the RC and the RC-LH1 complexes lead us to the determination of the number of BChls in the LH1 ring to be 32.06+/-2.90, indicating that the LH1 ring from Rh. marinum consists of 16 alphabeta subunits.     

Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center

Precise folding of photosynthetic proteins and organization of multicomponent assemblies to form functional entities are fundamental to efficient photosynthetic electron transfer. The bacteriochlorophyll b-producing purple bacterium Blastochloris viridis possesses a simplified photosynthetic apparatus. The light-harvesting (LH) antenna complex surrounds the photosynthetic reaction center (RC) to form the RC-LH1 complex. A non-membranous tetraheme cytochrome (4Hcyt) subunit is anchored at the periplasmic surface of the RC, functioning as the electron donor to transfer electrons from mobile electron carriers to the RC. Here, we use atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) to probe the long-range organization of the photosynthetic apparatus from Blc. viridis and the unfolding pathway of the 4Hcyt subunit in its native supramolecular assembly with its functional partners. AFM images reveal that the RC-LH1 complexes are densely organized in the photosynthetic membranes, with restricted lateral protein diffusion. Unfolding of the 4Hcyt subunit represents a multi-step process and the unfolding forces of the 4Hcyt α-helices are approximately 121 picoNewtons. Pulling of 4Hcyt could also result in the unfolding of the RC L subunit that binds with the N-terminus of 4Hcyt, suggesting strong interactions between RC subunits. This study provides new insights into the protein folding and interactions of photosynthetic multicomponent complexes, which are essential for their structural and functional integrity to conduct photosynthetic electron flow.     

Possible pathway for ubiquinone shuttling in Rhodospirillum rubrum revealed by molecular dynamics simulation

In the last decade, the structures of many components of the photosynthetic apparatus of purple bacteria, as well as the mutual organization of these components within the purple membrane, were resolved. One key question that emerged concerned the assembly of the core complex consisting of the reaction center (RC) and the light-harvesting 1 (LH1) complex. In some species, like Rhodobacter sphaeroides, the ring-shaped LH1 complex was found to be open, whereas other species, like Rhodospirillum rubrum, have a closed ring surrounding the reaction center. This poses the question of how the ubiquinone molecule that transports electrons and protons from the RC to the cytochrome bc(1) complex overcomes the apparent barrier of the LH1 ring. In this study, we investigated how, in the case of a closed LH1 ring, the ubiquinone molecule diffuses through the LH1 ring. For this purpose, the LH1 structure of R. rubrum was modeled and the potential of mean force along the diffusion pathway through the LH1 was determined by steered molecular-dynamics simulations. The potential was reconstructed using the fluctuation theorem in combination with the stiff spring approximation. An upper limit for the mean first-passage time for diffusion of ubiquinone through the LH1 ring, based on a worst-case scenario potential, was calculated as approximately 8 x 10(-3) s, which is still in agreement with known turnover rates of RC and RC-LH1 complexes in the range of approximately 1000 Hz.     

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.     

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.     

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.     

Three-dimensional reconstruction of a membrane-bending complex: the RC-LH1-PufX core dimer of Rhodobacter sphaeroides

A three-dimensional model of the dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) complex from Rhodobacter sphaeroides, calculated from electron microscope single particle analysis of negatively stained complexes, shows that the two halves of the dimer molecule incline toward each other on the periplasmic side, creating a remarkable V-shaped structure. The distribution of negative stain is consistent with loose packing of the LH1 ring near the 14th LH1 alpha/beta pair, which could facilitate the migration of quinone and quinol molecules across the LH1 boundary. The three-dimensional model encloses a space near the reaction center Q(B) site and the 14th LH1 alpha/beta pair, which is approximately 20 angstroms in diameter, sufficient to sequester a quinone pool. Helical arrays of dimers were used to construct a three-dimensional membrane model, which matches the packing lattice deduced from electron microscope analysis of the tubular dimer-only membranes found in mutants of Rba. sphaeroides lacking the LH2 complex. The intrinsic curvature of the dimer explains the shape and approximately 70-nm diameter of these membrane tubules, and at least partially accounts for the spherical membrane invaginations found in wild-type Rba. sphaeroides. A model of dimer aggregation and membrane curvature in these spherical membrane invaginations is presented.     

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.     

Conjugation-length dependence of the T1 lifetimes of carotenoids free in solution and incorporated into the LH2, LH1, RC, and RC-LH1 complexes: possible mechanisms of triplet-energy dissipation

In addition to the roles of antioxidant and spacer, carotenoids (Cars) in purple photosynthetic bacteria pursue two physiological functions, i.e., light harvesting and photoprotection. To reveal the mechanisms of the photoprotective function, i.e., quenching triplet bacteriochlorophyll to prevent the sensitized generation of singlet oxygen, the triplet absorption spectra were recorded for Cars, where the number of conjugated double bonds (n) is in the region of 9-13, to determine the dependence on n of the triplet lifetime. The Cars examined include those in (a) solution; (b) the reconstituted LH1 complexes; (c) the native LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, and Rps. acidophila 10050; (d) the RCs from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, and Rsp. rubrum S1; and (e) the RC-LH1 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, Rps. acidophila 10050, and Rsp. rubrum S1. The results lead us to propose the following mechanisms: (i) A substantial shift of the linear dependence to shorter lifetimes on going from solution to the LH2 complex was ascribed to the twisting of the Car conjugated chain. (ii) A substantial decrease in the slope of the linear dependence on going from the reconstituted LH1 to the LH1 component of the RC-LH1 complex was ascribed to the minor-component Car forming a leak channel of triplet energy. (iii) The loss of conjugation-length dependence on going from the isolated RC to the RC component of the RC-LH1 complex was ascribed to the presence of a triplet-energy reservoir consisting of bacteriochlorophylls in the RC component.     

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.     

Role of the H protein in assembly of the photochemical reaction center and intracytoplasmic membrane in Rhodospirillum rubrum

Rhodospirillum rubrum is a model for the study of membrane formation. Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus. This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC). The proteins of the photochemical components are encoded by the puf operon (LHalpha, LHbeta, RC-L, and RC-M) and by puhA (RC-H). R. rubrum puf interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent. The puh region was cloned, and DNA sequence determination identified open reading frames bchL and bchM and part of bchH; bchHLM encode enzymes of bacteriochlorophyll biosynthesis. A puhA/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation. Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly. This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane. In complemented strains, expression of puh occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector. The puhA gene alone was sufficient to restore phototrophic growth provided that recombination occurred.     

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.     

The accumulation of the light-harvesting 2 complex during remodeling of the Rhodobacter sphaeroides intracytoplasmic membrane results in a slowing of the electron transfer turnover rate of photochemical reaction centers

A functional proteomic analysis of the intracytoplasmic membrane (ICM) development process was performed in Rhodobacter sphaeroides during adaptation from high-intensity illumination to indirect diffuse light. This initiated an accelerated synthesis of the peripheral light-harvesting 2 (LH2) complex relative to that of LH1-reaction center (RC) core particles. After 11 days, ICM vesicles (chromatophores) and membrane invagination sites were isolated by rate-zone sedimentation and subjected to clear native gel electrophoresis. Proteomic analysis of gel bands containing the RC-LH1 and -LH2 complexes from digitonin-solubilized chromatophores revealed high levels of comigrating electron transfer enzymes, transport proteins, and membrane assembly factors relative to their equivalent gel bands from cells undergoing adaptation to direct low-level illumination. The GroEL chaperonin accounted for >65% of the spectral counts in the RC-LH1 band from membrane invagination sites, which together with the appearance of a universal stress protein suggested that the viability of these cells was challenged by light limitation. Functional aspects of the photosynthetic unit assembly process were monitored by near-IR fast repetition rate analysis of variable fluorescence arising from LH-bacteriochlorophyll a components. The quantum yield of the primary charge separation during the early stages of adaptation showed a gradual increase (variable/maximal fluorescence = 0.78-0.83 between 0 and 4 h), while the initial value of ~70 for the functional absorption cross section (σ) gradually increased to 130 over 4 days. These dramatic σ increases showed a direct relation to gradual slowing of the RC electron transport turnover rate (τ(QA)) from ~1.6 to 6.4 ms and an ~3-fold slowing of the rate of reoxidation of the ubiquinone pool. These slowed rates are not due to changes in UQ pool size, suggesting that the relation between increasing σ and τ(QA) reflects the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc(1) complex as the membrane bilayer becomes densely packed with LH2 rings.     

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.