Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (E_m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll alpha-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. The 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 alpha 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.     

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.     

Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.     

Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus.

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.     

Carbon monoxide-reacting haemoproteins in the mitochondrial fraction of Acanthamoeba castellanii.

1. Room-temperature (18 degrees C) CO difference spectra of mitochondrial fractions from the amoeba Acanthamoeba castellanii reveal the presence of at least four CO-reacting haemoproteins. As well as cytochrome a3, other components reacting with CO are: (i) a c-type cytochrome; (ii) a b-type cytochrome; and (iii) another a-type cytochrome. 2. The same components can be identified in low-temperature photodissociation experiments with intact cells or mitochondria. 3. The time of exposure to CO and the nature of the reductant are both important in identifying all the components present, in that the b-type cytochrome is more readily distinguished after longer exposure to CO and more of the c-type cytochrome is detectable when NADH is the reductant 4. Treatment of mitochondria with ultrasound releases two components, identifiable in low-temperature difference spectra as a c-type and a b-type cytochrome; only the latter appears to have any reaction with CO, and the CO-reacting c-type cytochrome is retained in submitochondrial particles. 5. The complexity of the CO-reacting haemoproteins in this organism is compared with the simpler systems found in other eukaryotic organisms.     

Development and growth of photosynthetic membranes of Rhodospirillum rubrum

In cell-free extracts from low-aeration suspensions of Rhodospirillum rubrum strain G-9, bacteriochlorophyll a was distributed in two bands after rate-zone sedimentation in sucrose density gradients. From the physicochemical properties of these fractions, it was concluded that the upper band consisted of small membrane fragments, whereas the major band was composed of fragmented vesicular intracytoplasmic membrane (chromatophores). After a pulse with L-[35S]methionine, apparent polypeptide subunits of the reaction center and light-harvesting complexes within the upper pigmented fraction were labeled more rapidly than those of chromatophores; after a chase with excess unlabeled L-methionine, radioactivity from these components within the upper band appeared to be chased into the corresponding polypeptides of chromatophores. These labeling patterns are interpreted to reflect growth initiation and maturation of the photosynthetic apparatus and may, in part, represent a general mechanism for the development of vesicular intracytoplasmic membranes.     

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides.

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll alpha-depleted cytoplasmic membrane in phototrophically grown cells.

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes.

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dislysis, the dislysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c2 photooxidation and cytochrome b photoreduction was demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane. The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides. Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c2 photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greated membrane cytochrome c2 photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0 degrees C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.     

The participation of cytochromes in the reduction of N20 to N2 by a denitryfying bacterium.

The oxidation of cytochromes during the reduction of N2O to N2 by a denitrifying bacterium was studied spectrophotometrically. The reduced b- and c-type cytochromes are partially oxidized when N2O is added to intact cells reduced with lactate under anaerobic conditions. The oxidation of cytochromes is inhibited non-competitively by azide, cyanide, 2,4-dinitrophenol and CuSO4, which inhibit the reduction of N2O to N2. In the presence of each inhibitor at a high concentration, at which the reduction of N2O to N2 is perfectly inhibited, cytochromes are not oxidized by N2O, while when an adequate, low concentration of inhibitor is added, b-type cytochrome is partially oxidized but c-type cytochrome is apparently not oxidized. In cell-free extracts, prepared by the sonic disruption of cells, that have entirely lost their activity in the reduction of N2O to N2, cytochromes are not oxidized by N2O. From the above results, it was concluded that b-type and c-type cytochromes should participate in the electron transport mechanism of the reduction of N2O to N2.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.     

Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide.

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda max at 549.5 +/- 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed. 3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b-90 was observed. 4. Difference spectra attributed to (BChl)2, (Formula: see text), c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata. 5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dialysis, the dialysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c₂ photooxidation and cytochrome b photoreduction were demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane.

The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides.

Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c₂ photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greater membrane cytochrome c₂ photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.