Rotational mobility of the photoreaction center in chromatophore membranes of Rhodospirillum rubrum

We investigated the rotational mobility of the photoreaction center in chromatophores of Rhodospirillum rubrum by studying the photoinduced linear dichroism of absorption changes at 865 nm. The study was carried out in suspensions of chromatophores treated with ferricyanide in order to bleach their antenna bacteriochlorophyll and thus minimize depolarization by energy transfer. Very little depolarization of the photoinduced absorbance change at 865 nm was observed at room temperature for chromatophores immersed in a highly viscous medium over the time range 0–10 ms following an exciting light flash. In the light of independent evidence for transmembrane arrangement of the photoreaction center, we conclude that the photoreaction center protein is immobilized in the chromatophore membrane for at least 10 ms.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM RUBRUM

Sections of Rhodospirillum rubrum cells from cultures of different ages have been examined to obtain information on the development of chromatophores in this organism. Cells from the 12-hour cultures studied contain neither distinct invaginations of the cytoplasmic membrane nor distinct chromatophores. The first structures that can be related to chromatophore development occur peripherally in the cells, are relatively few in number, relatively high in density, and have an indistinct membrane. In cells from 26-hour cultures numerous distinct invaginations of the cytoplasmic membrane are present, and all layers of the cytoplasmic membrane are involved in the formation of each invagination. As the invaginations become more numerous, the ends of the invaginations become constricted to form one or more structures similar to the chromatophores previously described in this organism. Cells of R. rubrum, therefore, develop a structural continuum which initially consists of invaginations of the cytoplasmic membrane, and later of the chromatophores produced by and attached to these invaginations. The presence of this continuum, however, does not necessarily exclude the existence of discrete chromatophores within these cells. Several other structures previously reported in this organism are described in greater detail.     

Exogenous cytochrome c-dependent light-induced membrane potential generation in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores associated with a planar phospholipid macromembrane by bivalent cations in the presence of quinone, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate generate a transmembrane electrical potential difference in the light. Photoelectrical activity is also observed if chromatophores are preincubated with cytochrome c; the maximum values of responses are reached upon subsequent addition of ascorbate and menadion in the absence of bivalent cations and TMPD. The cytochrome c-dependent responses of the illuminated chromatophores are inhibited by Ca²⁺ and prevented by quinones. The possibility of cytochrome c (c₂) translocation across the chromatophore membrane and the mechanism of charge transfer across the planar phospholipid membrane are discussed.     

Distribution of phosphatidylethanolamine in the lipid bilayer of chromatophores of the photosynthetic bacterium rhodospirillum rubrum

The distribution of phosphatidylethanolamine in the two lipid layers of chromatophores ofRhodospirillum rubrum has been analysed by chemical modification of phosphatidylethanolamine (PE) with trinitrobenzenesulfonic acid (TNBA) at low temperatures. Around 45±1% of the total phosphatidylethanolamine is labelled by this procedure independent on chromatophore purity, vesicle size, action of proteases and growth state of the cells. This demonstrates a complete modification of the accessible phosphatidylethanolamine and an asymmetric distribution of phosphatidylethanolamine, with 45% of the phosphatidylethanolamine in the outer part of the bilayer.Abbreviations TNBA 2,4,6 trinitrobenzenesulfonic acid - PE phosphatidylethanolamine - PMS phenazinmethosulfate     

Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum

Acetylene reduction catalyzed by nitrogenase from Rhodospirillum rubrum has low activity and exhibits a lag phase. The activity can be increased by the addition of a chromatophore membrane component and the lag eliminated by preincubation with this component, which can be solubilized from chromatophores by treatment with NaCl. It is both trypsin- and oxygen-sensitive. Titration of the membrane component with nitrogenase and vice versa shows a saturation point. The membrane component interacts specifically with the Fe protein of nitrogenase, the interaction being ATP- and Mg²⁺-dependent.     

Assembly of plasmid DNA and chromatophore inRhodospirillum rubrum

Summary Rhodospirillum rubrum, a photosynthetic bacterium, contains many photosynthetic vesicular membranous structures called chromatophores. The organism contains a 55 kb specific plasmid which is essential for photosynthesis, but the exact relationship between the chromatophore and the plasmid is uncertain. In this study we examined the precise localization of the plasmids, especially in relation to the chromatophores. Fluorescence in situ hybridization indicated that there are several copies of the plasmid per cell and that some plasmids are localized close to the cellular envelope. In situ hybridization at the electron-microscopic level further revealed that the plasmid localized to the periphery of the chromatophore close to the envelope. Moreover, when the chromatophore fraction was purified from cells, the plasmid DNA was observed as a cluster around the chromatophore vesicles. The assembly of the plasmid and chromatophore may be related to chromatophore formation by invagination of cell membrane.     

Strong orientational ordering of the near-infrared transition moment vectors of light-harvesting antenna bacterioviridin in chromatophores of the green photosynthetic bacterium Chlorobium limicola

The direction of the transition moments of chlorosome pigments in chromatophores of the green photosynthetic bacterium Chlorobium limicola was studied by linear dichroism. Orientation of chromatophores was achieved by stretching a polyacrylamide gel in which they were packed. It was shown that in each individual chromatophore the Q_y transition moment vectors of the whole chlorosome bacterioviridin are parallel to each other and are practically ideally oriented along the chlorosome long axis. The exact value of the angle α between the bacterioviridin transition moments and the long axis of the chlorosome is calculated to be α = 0°, the mean square deviation being 7°.     

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores.

The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P_i exchange reactions ofR. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.     

Membrane proteins of Rhodopseudomonas spheroides IV. Characterization of chromatophore proteins

Less than 5% of the protein isolated from Rhodopseudomonas spheroides chromatophores (designated Fraction P₁) is insoluble in 2-chloroethanol. Electrophoresis of these proteins on dodecyl sulphate-polyacrylamide gels reveals a gel pattern similar to those obtained from anaerobic and aerobic cell envelope proteins. Chromatophore P₁ is shown to be part of the chromatophore structure and its presence in the chromatophore is not due to contamination from the cytoplasmic membrane.Preparative dodecyl sulphate-polyacrylamide gel electrophoresis was performed to purify chromatophore P_ll proteins, which comprise 95% of the total chromatophore protein. These proteins contain approximately 60–65 mole% non-polar amino acids. Comparison studies of the amino acid compositions, tryptic and chymotryptic maps, molecular weights, and antigenic reactivity of chromatophore proteins demonstrate the existence of protein heterogeneity in chromatophores. These investigations lead us to suggest that chromatophore-specific proteins do not appear in other particulate or soluble fractions derived from either aerobic or anaerobic-grown cells.     

Two regimens of electrogenic cyclic redox chain operation in chromatophores of non-sulfur purple bacteria A study using antimycin A

Antimycin A causes a biphasic suppression of the light-induced membrane potential generation in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores incubated anaerobically. The first phase is observed at low antibiotic concentrations and is apparently due to its action as a cyclic electron transfer inhibitor. The second phase is manifested at concentrations which are greater than 1–2 μM and is due to uncoupling that may be connected with an antibiotic-induced dissipation of the electrochemical H⁺ gradient across the chromatophore membrane. The inhibitory effect of anti-mycin added at low concentrations under aerobic conditions is removed by succinate to a large extent. It is expected that the electrogenic cyclic redox chain in the bacterial chromatophores incubated under conditions of continuous illumination may function at two regimes: (1) as a complete chain involving all the redox components, and (2) as a shortened chain involving only the P-870 photoreaction center, ubiquinone and cytochrome c₂.     

Effects of electron donors and acceptors on the kinetics of the photoelectric responses in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores

Chromatophores of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides were adhered to one side of a collodion film impregnated with a phospholipid solution in decane and 20 ns laser flashes were delivered to produce an electrical potential difference generated across the collodion film in less than 0.2 μs (resolution time of the apparatus). The kinetics of Δψ decay in the dark was studied. In the absence of additions there occurs a ‘rapid’ decay of photoelectric potential (τ ≈ 70 ms) corresponding to charge recombination within the primary dipole P-870⁺-Q^-_A. The rapid decay of Δψ is prevented by ascorbate in the presence of permeable redox dyes which can reduce the photooxidized P-870⁺ rapidly. Under these conditions, Δψ dissipates with τ > 0.5 s typical of a passive discharge of the chromatophore membrane. Prevention of the rapid decay of Δψ by 70–75% can be observed upon addition of excess ubiquinone-10 to the solution of phospholipids used to impregnate the collodion film, and to a lesser extent by addition of some other quinones. The effect of quinones is inhibited by o-phenanthroline. The data obtained show that upon association of chromatophores with the collodion film, the secondary quinone acceptor is extracted from its binding site into a hydrophobic volume of the macroscopic membrane, and this effect can be reversed by exogenous ubiquinone. About 4-times less Q-10 is required to reconstitute Q_B function in chromatophores from Rps. sphaeroides than in those from R. rubrum, which points to a tighter binding of the secondary acceptor in the former. No evidence for electrogenic nature of Q⁻_A → Q_B electron transfer could be obtained in experiments with Q_B-replenished chromatophores.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. This material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll a and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

Laser raman studies of lipid disordering by the B-protein of fd phage

Complexes of the B-protein of fd phage with the model lipid dipalmitoyl phosphatidylcholine (DPPC) were made by sonication of the fd phage in the presence of dipalmitoyl phosphatidylcholine. Both laser Raman spectra and circular dichroism show the protein in the membrane to be almost entirely in the β-sheet conformation. This β-sheet conformation is found to be independent of the temperature between 10° C and 50° C. On the other hand, the protein has a very dramatic effect on the organization of the lipid bilayer. An aqueous dispersion of 1 : 1 lipid/protein mixture gives a broad conformational transition of DPPC which occurs between 10° C and 30° C. This contrasts markedly with simple aqueous DPPC dispersions which show a sharp transition at 41°C. This appears to be the first reported example of the lowering of the conformational transition of a membrane bilayer by an intrinsic membrane protein.     

The effect of bovine myelin basic protein on the phase transition properties of sphingomyelin

The basic protein of myelin can spontaneously associate with the synthetic phospholipid N-palmitoylsphingosinephosphatidylcholine. The protein alters the phase transition properties of the lipid from a single transition at 41.5°C to two overlapping transitions, one being slightly above and the other slightly below the transition temperature of the pure lipid. The effect was not seen upon the addition of poly(l-lysine) to this lipid nor does the myelin basic protein alter the phase transition properties of dimyristoylphosphatidylcholine. The results thus demonstrate that the myelin basic protein can interact with a major zwitterionic lipid component of myelin in addition to acidic phospholipids.     

Localization of chromatophore proteins of Rhodobacter sphaeroides. I. Rapid Ca(2+)-induced fusion of chromatophores with phosphatidylglycerol liposomes for proteinase delivery to the luminal membrane surface.

A protease delivery system was developed for the exclusive and controlled digestion of proteins exposed at the morphological inside (periplasmic surface) of Rhodobacter sphaeroides chromatophores. In this procedure, proteinase K is encapsulated within large unilamellar liposomes which are fused to the chromatophores in the presence of Ca2+ ions. The liposomes were prepared by a detergent dialysis procedure from native phosphatidylglycerol and found to undergo rapid bilayer fusion with purified chromatophore preparations above a threshold concentration of 12.5 mM CaCl2. The fusion process was complete within 10 min at 35 mM Ca2+ with about 80% of the pigment located in the fusion products. Electron micrographs of freeze-fracture replicas confirmed the intermixing of the lipid bilayers and the unilamellar structure of the fused membrane vesicles. The procedure did not affect the labile B800 chromophore of the B800-850 antenna complex, but reduced slightly the absorption due to the B875 core antenna. Emission from both light-harvesting complexes was increased in the fused membranes, suggesting a partial dissociation of photosynthetic units in the expanded bilayer. The results, together with those presented in the following paper (Theiler, R., and Niederman, R. A. (1991) J. Biol. Chem. 266, 23163-23168), demonstrate that this new method fulfills the stringent requirements for a successful delivery of macromolecules to the chromatophore interior.     

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP)

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.