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...

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 membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.

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