Binding of carotenoids on reaction centers from Rhodopseudomonas sphaeroides R 26.

The carotenoid-less reaction centers isolated from Rhodopseudomonas sphaeroides (strain R 26) bind pure all-trans spheroidene as well as spheroidenone in a nearly 1 : 1 molar ratio with respect to P-870. Neither beta-carotene nor spirilloxanthin, both absent from wild-type Rps. sphaeroides, could be bound in appreciable amounts. Resonance Raman spectra of the carotenoid-reaction center complex indicate that the carotenoid is bound as a cis isomer, its conformation being very close, although probably not identical, to that assumed by the carotenoid in the wild-type reaction centers. The electronic absorption spectra of the carotenoid-reaction center complexes are in good agreement with such a interpretation. When bound to the R 26 reaction centers, spheroidene displays light-induced absorbance changes identical in peak wavelengths and comparable in amplitudes to those observed in the wild-type reaction centers. Thus the binding of the carotenoid to the R 26 reaction centers most likely occurs at the same proteic site as in the wild-type reaction centers. This site shows selectivity towards the nature of carotenoids, and has the same sterical requirement as in the wild type, leading to the observed all-trans to cis isomerisation.

Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form.

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from alpha-beta heterodimers containing three bacteriochlorophylls and one carotenoid each. The alpha subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51-71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 A resolution were calculated, demonstrating the nonameric ring-like organization of alpha-beta subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.

On the state of carotenoids bound to reaction centers of photosynthetic bacteria: a resonance Raman study.

The carotenoids bound to reaction centers of wild, Ga and GIC strains of Rhodopseudomonas spheroides, of Rhodospirrillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15'-cis-beta-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of P-870. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.

Preliminary characterization by X-ray diffraction of crystals of photochemical reaction centres from wild-type Rhodopseudomonas spheroides.

Reaction centres from wild-type Rhodopseudomonas spheroides (strain Y) in a solution of octylglucoside have been crystallized with polyethylene glycol as precipitant, either by vapour diffusion or dialysis. Orthorhombic crystals (space group P2(1)2(1)2(1)) diffract to 3.5 A resolution. The unit cell parameters are a = 142.5 A, b = 141.5 A, c = 80 A; they are compatible with the presence of one reaction centre per asymmetric unit.

A specific carotenoid is required for reconstitution of the Rubrivivax gelatinosus B875 light harvesting complex from its subunit form B820.

The core light-harvesting complex B875 from Rubrivivax gelatinosus has been reconstituted from its subunit form B820 with hydroxyspheroidene, the carotenoid which is bound to native B875 antenna. Other carotenoids which are chemically similar to hydroxyspheroidene (spheroidene, spheroidenone, neurosporene and spirilloxanthin) gave only low levels of partial reconstitution. Absorption and circular dichroism spectra of the hydroxyspheroidene-containing, reconstituted B875 were identical with those of original B875 antenna isolated directly from chromatophores, indicating that the two hydroxyspheroidene molecules bind to their native sites within the (alpha beta)Bchl2 subunit during the reconstitution process. These observations point to a structural role for this carotenoid in determining the architecture of Rv. gelatinosus B875 antenna.

Solubility diagram of the Rhodobacter sphaeroides reaction center as a function of PEG concentration.

In order to quantify the effect of polyethylene glycol 4000 (PEG) on the solubility of an integral membrane protein, we have crystallized the photochemical reaction center from Rhodobacter sphaeroides Y by batch method on a large range of PEG. The measurement of the solubility diagram display a semi-logarithmic dependence of solubility versus PEG concentration. Comparison of our results with previously published ones [Odahara, T., Ataka, M. and Katsura, M. (1994) Acta Cryst. D50, 639-642] suggests a notable effect of additional 1,2,3-heptane-triol and/or temperature on photochemical reaction center solubility.

Structure of spheroidene in the photosynthetic reaction center from Y Rhodobacter sphaeroides.

The structure of the reaction center of Y Rhodobacter sphaeroides has been solved at 3 A resolution, using the atomic coordinates of the reaction center from the carotenoidless mutant R26 Rhodobacter sphaeroides. The structure has been refined by a stimulated annealing with the computer program X-PLOR, leading to a crystallographic R factor of 0.22 using reflections between 8 and 3 A. The spheroidene molecule which is bound to the Y reaction center has been fitted in the electron density map as a 15-cis isomer with a highly asymmetric structure. The cis-bond is located at proximity from ring 1 of the accessory bacteriochlorophyll on the inactive M side. The nature of the cis-bond was confirmed by resonance Raman spectra obtained from Y reaction center crystals. The structure of spheroidene in Y reaction center is compared to that proposed for 1,2-dihydroneurosporene in Rhodopseudomonas viridis reaction center crystals.

Towards the understanding of the function of Rb sphaeroides Y wild type reaction center: gene cloning, protein and detergent structures in the three-dimensional crystals.

We report various experiments aimed at the resolution of the 3-dimensional structure of the photosynthetic reaction center from wild type Y Rhodobacter sphaeroides. The genes encoding the L and M polypeptides have been cloned and sequenced. They bear 2 mutations each when compared to those already sequenced in another Rb sphaeroides strain (2.4.1). In the L gene, these codon changes are silent. In the M gene, one is silent and the other one leads to a Leu-Met substitution at position 140. At the present stage of the refinement of the X-ray data (0.3 nm resolution) the structure of the Y reaction center is shown to be highly similar to that of the Rhodopseudomonas viridis reaction center. The binding of spheroidene on the M side of the Y reaction center is shown to be determined by hydrophobic interactions with neighboring amino acids and by steric factors. Preliminary results concerning the localization of the detergent (beta-octylglucoside) in the unit cell are presented. This method combines low angle neutron scattering at different contrasts in H2O/D2O with X-ray crystallographic data.

Thickness measurements of single walled dimyristoyl phosphatidylcholine vesicles by neutron scattering.

A method of deriving by neutron scattering thicknesses of lamellae in suspensions has been applied to single-walled vesicles of dimyristoyl phosphatidylcholine. The contrast variation method, based on data obtained for a range of isotope mixtures, has been used to extract a dimension Dw related to the lipid bilayer thickness and a measure alpha of the difference of density within the lamellae. Isotope mixtures for the lipid were used to optimize the information available. Dw is compared with results from multilayer stacks of lipid layers. The thickness for the low temperature L beta, structure has been observed to be higher than for the high temperature L alpha structure. Preliminary experiments on the kinetics of the mixing of the lipid isotope species are reported, and evidence is shown that the species are not segregated for lipids either above or below the transition temperature.

Structure of the body-centered cubic phase of lipid systems.

A new model is proposed for the structure of the body-centered cubic phase of lipid systems. Infinite rods of polar groups (and water) are arranged with axes parallel to the four cubic [unk]1 1 1[unk] directions. The hydrocarbon chains fill the space between the rods to form a continuous matrix. With this unified topology, the model explains satisfactorily the x-ray diffraction patterns of strontium soaps, lecithin, galactolipids, potassium soaps, and hexadecyltrimethylammonium bromide and explains the transition between cubic/H(II) phases. The paradoxical thermal effects on the lipid cubic phase, in particular the decrease of unit cell dimensions with increasing temperature, can be explained with the proposed model by mechanisms similar to those used for the monodimensional and bidimensional (mesomorphic) phases.

Resolution of Rhodocyclus gelatinosus photoreceptor unit components by temperature-induced phase separation in the presence of decyltetraoxyethylene.

A simple method for dissociating photoreceptor units from Rhodocyclus gelatinosus is described. Incubation of a chromatophore extract (Agalidis, I., Rivas, E. and Reiss-Husson, F. (1990) Photosynth. Res. 23, 249-255) at 4 degrees C with decyltetraoxyethylene and octyl-beta-D-thioglucopyranoside, followed by temperature-induced phase separation at 20 degrees C, led to the formation of three phases: a pellet composed of pure B875 antenna; an oily layer containing cytochrome c and other proteins; a detergent-poor supernatant containing crude reaction centers. This method provided a first step towards further purification of reaction centers and B875 antenna, respectively.

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides.

A cell envelope fraction had been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm3) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm3) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O2 pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm3). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.