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.     

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.     

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.     

Electron transport in chromatophores from Rhodopseudomonas sphaeroides GA fused with liposomes

Chromatophores from Rhodopseudomonas sphaeroides GA were fused with liposomes in order to dilute the components of the cyclic photosynthetic electron-transport chain within the membrane. This dilution led to a decrease in the rate of cytochrome b-561 reduction. The original rates could be restored at potentials around 100 mV (where a large part of the quinone pool is chemically reduced), if ubiquinone was incorporated into the liposomes prior to fusion. Similar dilution effects could be observed in synchronized cultures. The membrane obtained after division contained about twice the amount of phospholipids per reaction center when compared to chromatophores prepared from cells harvested just before division. Chromatophores from synchronized cultures are more uniform with respect to the concentration of the different electron-transport components in the membrane than the membranes from normally grown cells. The kinetic behaviour both of fused chromatophores and of membranes from synchronized cultures are in agreement with a modified Q-cycle model for photosynthetic electron transport in Rps. sphaeroides. The results presented in this paper cannot be explained by postulating the presence of a firmly bound quinone, Qz, in the ubiquinol: cytochrome c2 oxidoreductase, as previously proposed.     

Lipid analysis of cells and chromatophores of Rhodopseudomonas sphaeroides

The phospholipids and fatty acid analysis of four strains of Rhodopseudomonas sphaeroides and of chromatophores from two strains show some differences and also show the presence of an unusual polar neutral lipid which is ninhydrin positive and which on acid hydrolysis yields ornithine and an unidentified amino compound. This lipid is called aminolipid-X and has a fatty acid composition very different from the phospholipids. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contain a very small amount of plasmalogen forms as determined by combined mild alkaline hydrolysis, acetic acid hydrolysis and phospholipase A₂ hydrolysis.The reaction of intact cells and chromatophores with trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and isethionylacetimidate (IA) show that 78% of the total PE in chromatophores is localized on the outer membrane surface. In intact cells about 15–35% of the total PE is localized on the outer surface of the plasma membrane.     

Free-energy change accompanying the reduction of the reaction center secondary quinone in Rhodopseudomonas sphaeroides chromatophores

The standard free-energy change accompanying the electron transfer from Q_A to Q_B was estimated from the intensity of the delayed fluorescence in chromatophores of Rhodopseudomonas sphaeroides. The value of 120 meV (at pH 8) suggests that Q_B⁻ is more stable in the chromatophore membrane than in the isolated reaction center.     

Light-induced proton gradients and internal volumes in chromatophores of Rhodopseudomonas sphaeroides

To test the predictions of the chemiosmotic hypothesis, it is essential to have sensitive and accurate measures of the aqueous volume and pH within membrane compartments. One unique feature of the present investigation is the application of electron spin resonance probes to determine internal aqueous volume and pH changes in bacterial chromatophores under virtually identical conditions. Volumes of the chromatophores ranged from 6 to 16 microliter/mg bacteriochlorophyll among different preparations, and were sensitive to the osmolarity of the suspending buffer. pH gradients reached two units in illuminated chromatophores as determined with ESR methods, and increased when KCl and valinomycin were added to the assay. Measurements with the fluorescent dye 9-amino-acridine yielded similar pH gradients, provided that an operational vesicle volume, which corrected for the binding of the dye to the membrane, was used in the calculation. The sensitivity of the ESR method allowed the measurement of pH gradients resulting from only a few light flashes. A plot of pH gradients versus number of flashes was linear up to about 30 flashes, and intercepted the origin. This result is consistent with proton release into the bulk aqueous phase after only a single light flash. This ability to measure small pH gradients offers new opportunities for the study of energy-transducing mechanisms.     

Ionophore-sensitive spectral shift of carotenoid induced by ferricyanide in chromatophores from Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides, ferricyanideinduced a change in the absorption spectrum of carotenoid. Theionophore-sensitive part of the ferri-cyanide-induced changewas similar to that induced by light or by diffusion potential.The ferricyanide-induced change is explained by the electrochromicshift of the carotenoid spectrum by the inside-positive electricalfield change which is probably caused by the electrogenic electronflow from a membrane redox component to ferricyanide in theouter aqueous phase. The ionophore-insensitive part is probablythe response of the carotenoid in another pool to the localfield change by oxidation of bacteriochlorophyll [Okada, M.and A. Takamiya (1970) Plant & Cell Physiol. 11: 713–721]. (Received January 21, 1980; )     

Membranes of Rhodopseudomonas sphaeroides. IV. Assembly of chromatophores in low-aeration cell suspensions

Chromatophore membrane formation was induced in low-aeration suspensions of Rhodopseudomonas sphaeroides and highly purified chromatophore preparations were isolated at various intervals between 4 and 18 h. The levels of several functional components associated with the isolated structures were investigated. B-875, the light-harvesting bacteriochlorophyll complex associated with the reaction center, was preferentially inserted into the chromatophore membrane during the early stages of induction, and thereafter its levels reached a steady state; b- and c-type cytochromes were also maintained at essentially constant levels. In contrast, the levels of B-850, the accessory light-harvesting bacteriochlorophyll, together with its associated protein, continued to increase throughout the induction process. Increases in the levels of the major carotenoid component followed a similar course. These findings are consistent with a stepwise assembly mechanism for associated bacteriochlorophyll and protein components and suggest that separate regulatory mechanisms control the levels of functionally essential and accessory components within the membrane.     

The interrelation of the two c-type cytochromes in Rhodopseudomonas sphaeroides photosynthesis.

Photosynthetic electron flow in the bacterium Rhodopseudomonas sphaeroides involves two c-type cytochromes, one membrane-bound and the other a soluble protein, cytochrome c2. Membranes deficient in cytochrome c2 were used for photo-oxidation studies, with and without the addition of purified cytochrome c2. The results favour a series interrelation, membrane cytochrome c-cytochrome c2-reaction centre.     

Membranes of Rhodopseudomonas sphaeroides. IV. Assembly of chromatophores in low-aeration cell suspensions.

Chromatophore membrane formation was induced in low-aeration suspensions of Rhodopseudomonas sphaeroides and highly purified chromatophore preparations were isolated at various intervals between 4 and 18 h. The levels of several functional components associated with the isolated strucures were investigated. B-875, the light-harvesting bacteriochlorophyll complex associated with the reaction center, was preferentially inserted into the chromatophore membrane during the early stages of induction, and thereafter its levels reached a steady state; b- and c-type cytochromes were also maintained at essentially constant levels. In contrast, the levels of B-850, the accessory light-harvesting bacteriochlorophyll, together with its associated protein, continued to increase throughout the induction process. Increases in the levels of the major carotenoid component followed a similar course. These findings are consistent with a stepwise assembly mechanism for associated bacteriochlorophyll and protein components and suggest that separate regulatory mechanisms control the levels of functionally essential and accessory components within the membrane.     

Ubiquinone reduction and proton uptake by chromatophores of Rhodopseudomonas sphaeroides R-26: periodicity of two in consecutive light flashes.

Chromatophores of Rhodopseudomonas sphaeroides strain R-26 were subjected to a series of brief flashes of light in the presence of diaminodurene as an electron donor. Odd-numbered flashes induced the reduction of ubiquinone to the anionic semiquinone, as indicated by absorbance changes near 450 nm. This reaction was not attended by proton binding. Even-numbered flashes caused disappearance of the semiquinone, presumably by conversion to the fully reduced form. This reaction was attended by proton uptake.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.     

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.     

Porin from Rhodopseudomonas sphaeroides

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.