Isolation, Size Estimates, and Spectral Heterogeneity of an Oligomeric Series of Light-Harvesting 1 Complexes from <i>Rhodobacter sphaeroides</i>

119

142

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-Å 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 ␣/␤-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of ϳ30-Å 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.In purple photosynthetic bacteria, highly organized transmembrane pigment-protein complexes perform absorption of light and its conversion into chemical energy. Two light-harvesting complexes (LH), 1 LH2 and LH1, ensure the collection of light. Then the excitation energy is funneled toward the special pair of bacteriochlorophylls in the reaction center (RC), followed by an electron transfer from the special pair of bacteriochlorophylls to the ubiquinone (Q) acceptors Q A and Q B . After two photoreactions and proton captures, ubiquinol (QH 2 ) is formed at the Q B site that dissociates from the RC into the membrane. The bc 1 complex utilizes QH 2 and oxidized cytochrome c 2 as reductant and oxidant, respectively. The net result is a cyclic electron transfer that promotes the formation of a proton gradient across the membrane, which is utilized for ATP synthesis by F 1 F 0 -ATP synthase (for a review, see Ref. 1).The mechanisms of excitonic energy migration and photoinduced electron transfer have been studied in detail using biophysical approaches and analyzed at the molecular level following the resolution of the atomic structures of the RC (2), LH2 (3, 4), and the bc 1 complex (5). However, despite the wealth of information available on the individual proteins, their supramolecular organization in the membrane remains undetermined. A crucial issue is the spatial organization between LH1 and RC, forming the so-called core complex, in which the transformation of light energy into charge separation occurs. So far, no atomic model of the core complex is available. Early studies by various electron microscopy (EM) techniques of native membranes of Rhodopseudomonas viridis and Ectothiorhodospira halochloris have shown a single RC surrounded by a closed circle of light-harvesting molecule...

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Structural Role of PufX in the Dimerization of the Photosynthetic Core Complex of Rhodobacter sphaeroides

Scheuring

Francia

Busselez

et al. 2004

119

142

“…6C. This difference is borne out by the facts that LH1 can be readily dissembled into individual ␣ 1 ␤ 1 Bchl 2 units, often termed B820 (33), and that it can also be fractionated into a series of LH1 oligomers that vary in size from (␣␤) 2-3 to (␣␤) 10 -11 (34,35). Neither of these types of subdivision of a nonameric LH2 complex has been reported, although the octomeric LH2 of Rhodospirillum molischianum has been successfully dissociated into B820 subunits (36).…”

Section: Structural Basis For Variations In Size and Shape Of The Lh1mentioning

confidence: 99%

Flexibility and Size Heterogeneity of the LH1 Light Harvesting Complex Revealed by Atomic Force Microscopy

Bahatyrova

Frese

Werf

et al. 2004

Self Cite

118

100

Previous electron microscopic studies of bacterial RC-LH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q B site of the RC to the quinone pool prior to reduction of the cytochrome bc 1 complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (␣ 1 ␤ 1 Bchl 2 ) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.Photosynthetic organisms harvest light energy and convert it to a chemically useful form, using light harvesting (LH) 1 and reaction center (RC) complexes. In the purple photosynthetic bacteria, the reaction center, which is the site of photochemistry, receives excitation energy from the light harvesting LH1 complex, which receives energy in turn from the LH2 complex (reviewed in Ref. 1). The atomic structure of the Rhodopseudomonas acidophila LH2 complex (2) and the cryo-electron microscopy (EM) structure of the Rhodobacter sphaeroides complex (3) both revealed a circular arrangement of nine protomers, each consisting of an ␣ and a ␤ polypeptide. The LH2␣ polypeptides formed an inner ring, with the ␤ ring outermost; in all, 27 bacteriochlorophyll (Bchl) molecules are bound to this structure (2). More recent work has established that LH1 surrounds the RC using an arrangement of 16 ␣␤ protomers and 32 Bchls (4) when there is no prulifloxacin PufX protein. In other bacteria, an LH1 ring of 15 ␣␤ protomers, together with either PufX or a putative PufX homologue (W), form a continuous ring of protein around the RC (5, 6). The demonstration of both circular and elliptical forms of this LH1 complex provided evidence for its flexibility (4). This property of the LH1 complex wa...

“…The observation that membranes composed nearly entirely of RC-LH1-PufX dimers form elongated tubular structures (28 -30), with a degree of order sufficient to enable calculation of a low resolution projection map of the complex in its native membrane (20,26,30), allows this dimer structure to be used as a exemplar for induced membrane curvature by an intrinsic membrane protein. It is not known how intrinsic membrane proteins impose curvature on the membrane in which they sit, although several fascinating examples have emerged (31)(32)(33).…”

mentioning

confidence: 99%

Three-dimensional Reconstruction of a Membrane-bending Complex

Qian

Bullough

Hunter

2008

Self Cite

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 ␣/␤ 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 ␣/␤ pair, which is ϳ20 Å 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 ϳ70-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.Photosynthetic bacteria provide an ideal system for investigating the conversion of light energy into chemical energy in nature. Only three different protein complexes are required for this conversion; the first is a so-called "core" complex composed of light-harvesting I (LH1) and reaction center (RC) complexes. Light energy absorbed by LH1 is transferred to the RC, where photochemical charge separation takes place (1, 2). This charge separation induces electron transfer coupled to production and migration of quinols to the cytochrome bc 1 complex (3, 4). Pumping of protons across the membrane forms an electrochemical potential, which drives various cellular processes including synthesis of ATP (5). Most of the purple bacteria contain a second light-harvesting complex, LH2; each subunit of LH2 consists of two integral transmembrane polypeptides called ␣ and ␤, which bind the bacteriochlorophyll (BChl) and carotenoid pigment molecules (6). The bacterial photosynthetic membrane is composed of closely packed arrays of LH2 and RC-LH1 complexes; atomic force microscopy (AFM) of membranes from several photosynthetic bacteria has revealed the organization of these arrays (7, 8). As a result of structural, functional, and AFM studies these are among the best characterized of any biological membrane. Recently, it has been possible to combine the data from structural and functional approaches to construct three-dimensional models of an entire membrane vesicle, comprising over a 100 complexes, at the atomic level (9).Despite these advances, several important aspects of structure and function remain: it is not known how quinol molecules, the product of photochemistry, pass through the apparently closed LH1 rings that encir...