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

The purpose of this study was to gain information on the functional consequences of the supramolecular organization of the photosynthetic apparatus in the bacterium Rhodobacter sphaeroides. Isolated complexes of the reaction center (RC) with its core antenna ring (light-harvesting complex 1 (LH1)) were studied in their dimeric (native) form or as monomers with respect to excitation transfer and distribution of the quinone pool. Similar issues were examined in chromatophore membranes. The relationship between the fluorescence yield and the amount of closed centers is indicative of a very efficient excitation transfer between the two monomers in isolated dimeric complexes. A similar dependence was observed in chromatophores, suggesting that excitation transfer in vivo from a closed RC⅐LH1 unit is also essentially directed to its partner in the dimer. The isolated complexes were found to retain 25-30% of the endogenous quinone acceptor pool, and the distribution of this pool among the complexes suggests a cooperative character for the association of quinones with the protein complexes. In chromatophores, the decrease in the amount of photoreducible quinones when inhibiting a fraction of the centers implies a confinement of the quinone pool over small domains, including one to six reaction centers. We suggest that the crowding of membrane proteins may not be the sole reason for quinone confinement and that a quinone-rich region is formed around the RC⅐LH1 complexes.

supporting

confidence: 73%

“…A small polypeptide, PufX, appears to play a key role in determining the structural organization. In its absence, the RC⅐LH1 complexes adopt a monomeric conformation with a closed LH1 ring of 16 ␣␤-heterodimers (23)(24)(25)27).…”

mentioning

confidence: 99%

Functional Consequences of the Organization of the Photosynthetic Apparatus in Rhodobacter sphaeroides

Comayras

Jungas

Lavergne

2005

“…Other modifications whose relation with the arrangement of RC⅐LH1 complexes is not straightforward are also observed. The structure of the intracytoplasmic membranes is affected (16,17), so membrane preparations do not yield sealed vesicles as in the WT. It may thus be hypothesized that the supramolecular arrangement of the RC⅐LH1 complexes affects the curvature of the membrane and controls in this manner its large-scale structure.…”

Section: Discussionmentioning

confidence: 99%

Functional Consequences of the Organization of the Photosynthetic Apparatus in Rhodobacter sphaeroides

Comayras

Jungas

Lavergne

2005

In the bacterium R. sphaeroides, the polypeptide PufX is indispensable for photosynthetic growth. Its deletion is known to have important consequences on the organization of the photosynthetic apparatus. In the wildtype strain, complexes between the reaction center (RC) and the antenna (light-harvesting complex 1 (LH1)) are associated in dimers, and LH1 does not fully encircle the RC. In the absence of PufX, the complexes become monomeric, and the LH1 ring closes around the RC. We analyzed the functional consequences of PufX deletion. Some effects can be ascribed to the monomerization of the RC⅐LH1 complexes: the number of RCs that share a common antenna for excitation transfer or a common quinone pool become smaller. We examined the kinetic effects of the closed LH1 ring on quinone turnover: diffusion across LH1 entails a delay of ϳ1 ms, and the barrier appears to be located directly against the quinone-binding (secondary quinone acceptor (Q B )) pocket. The diffusion of ubiquinol from the RC to the cytochrome bc 1 complex is ϳ2-fold slower in the mutant, suggesting an increased distance between the two complexes. The properties of the Q B pocket (binding of inhibitors, stabilization of Q B ؊ , and rate of Q B -H 2 formation) appear to be modified in the mutant. Another specificity of PufX ؊ is the accumulation of closed centers in the Q A ؊ (where Q A is the primary quinone acceptor) state as the secondary acceptor pool becomes reduced, which is probably the origin of photosynthetic incompetence. We suggest that this is related to the Q B pocket alterations. The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.

“…The availability of a wide array of genetic tools for R. sphaeroides (16) can be exploited for AFM studies of membrane organization, because it allows control to be exerted over the composition of the proteins present in the membrane. Mutants of R. sphaeroides, which form highly ordered tubular membranes containing only RC-LH1-PufX dimers, have been used to study the organization of complexes by electron microscopy (17,18), and similar techniques have been applied to photosynthetic proteins reconstituted into two-dimensional crystals (19 -21). However, because the purification process removes the native lipids, and, in the case of R. sphaeroides, reconstitutes the complexes in an alternating, and therefore nonbiological, "up-down-up" topology, such work is unsuitable for examining the native organization of the complexes.…”

mentioning

confidence: 99%

The Organization of LH2 Complexes in Membranes from Rhodobacter sphaeroides

Olsen

Tucker

Timney

et al. 2008

Self Cite

The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058 -1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of ϳ53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, <120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by ϳ1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.The biological membrane at its simplest is a lipid bilayer that divides cellular contents from the exterior medium. Yet the bilayer performs more than a simple barrier function as it plays host to a wide variety of integral membrane proteins that perform transport, sensing, motility, biosynthesis, energy generation (in the form of ATP), as well as energy harvesting in the case of phototrophic organisms. We have structural information about the membrane proteins involved in photosynthesis, e.g. the bacterial reaction center (1), the peripheral light-harvesting complex (2), and the photosystem I (3) and photosystem II (4) supercomplexes and in transport (5), sensing (6), and electron transport (7). However, there is a need now to gain information upon the organization of these proteins within the membrane in vivo. The use of AFM 2 to directly visualize membrane proteins in situ allows us to make precise measurements of the disposition of any protein within the membrane (8) under nearly native conditions. We have demonstrated that the comparatively disordered intracytoplasmic membrane from the model photosynthetic bacterium Rhodobacter sphaeroides can be successfully imaged by AFM (9) to reveal the hidden architecture ...