Reconstituted energy transfer from antenna pigment-protein to reaction centres isolated from Rhodopseudomonas sphaeroides

Efficient energy transfer has been reconstituted between an antenna pigment-protein and reaction centres isolated from the photosynthetic membrane of Rhodopseudomonas sphaeroides. The reconstituted system has fluorescence induction kinetics and fluorescence yields similar to those obtained from antenna bacteriochlorophyll in chromatophores. The results indicated that closed reaction centres quench fluorescence from the antenna pigment-protein, although not as strongly as photochemically active reaction centres. The measurement of fluorescence yields from chromatophores of the reaction centreless mutant PM-8 and of the parent strain Ga confirmed these observations.The fluorescence yield from the reconstituted system was approximately the same whether the reaction centres had been closed by photo-oxidation of the bacteriochlorophyll electron donor or chemical reduction of the primary acceptor, indicating a similar lifetime for the excited singlet state in both states of the reaction centres.     

Reconstitution of carotenoids into the light-harvesting pigment-protein complex from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26

Two carotenoids, neurosporene and spheroidene, have been successfully added to chromatophores from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26. Carotenoids reconstituted in this way into the B-850 light-harvesting pigment-protein complex both sensitise bacteriochlorophyll fluorescence and protect the complex from the photodynamic reaction.     

Investigation of spatial relationships and energy transfer between complexes B800-850 and B890-RC from Chromatium minutissimum reconstituted into liposomes.

Spatial relationships between different pigment-protein complexes in the membranes of the purple photosynthetic bacterium, Chromatium minutissimum, have been studied. The possibility of restoring the function of efficient excitation energy transfer from bacteriochlorophyll molecules to the reaction centers in the system of soybean liposomes, reconstituted with pigment-protein complexes B800-850 and B890-RC from C. minutissimum, has been explored. The chemical cross-linking method, together with stationary and picosecond spectrally resolved fluorescence measurements were employed. It has been shown that after the incorporation of the complexes into the liposome membranes conditions for directed excitation energy transfer from the light-harvesting pigments to the reaction centers are created, which are less optimal, however, than those in the native state. Possible reasons are considered.     

The incorporation of reaction centres into membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides.

Reaction centres purified from a blue-green mutant R-26 of Rhodopseudomonas sphaeroides can be incorporated into bacteriochlorophyll-less membranes purified from an aerobically-grown bacteriochlorophyll-less mutant 01 of R. sphaeroides. This can be accomplished by raising the temperature of the mixture or by addition of the detergent sodium cholate and its subsequent removal by dilution or dialysis. Optimum conditions for the reconstitution are at 4 degrees C in the presence of 1% cholate and soybean phospholipid (2 : 1, w/w, with membrane protein). Isopycnic sucrose density gradient centrifugation of such preparations shows that reaction centres and light-harvesting pigment-protein complex bind to the membranes. Reconstituted membranes exhibit light-induced steady-state cytochrome absorbance changes resembling those observed in chromatophores prepared from the photosynthetically-grown mutant R-26. The effect on these absorbance changes of varying reaction centre content in the membrane has been studied, and the time course of the interaction between 01 membrane cytochrome c2 and added reaction centre examined. Cytochrome b photoreduction and cytochrome c2 photo-oxidation were observed in the reconstituted preparation; each increased following the addition of antimycin A, suggesting that a cyclic light-driven system had been reconstituted.     

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.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes.

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associated with planar azolectin membrane, proteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgCl electrodes and voltmeter. The direction of the electric field in proteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

The incorporation of reaction centres into membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides

Reaction centres purified from a blue-green mutant R-26 of Rhodopseudomonas sphaeroides can be incorporated into bacteriochlorophyll-less membranes purified from an aerobically-grown bacteriochlorophyll-less mutant 01 of R. sphaeroides. This can be accomplished by raising the temperature of the mixture or by addition of the detergent sodium cholate and its subsequent removal by dilution or dialysis. Optimum conditions for the reconstitution are at 4°C in the presence of 1% cholate and soybean phospholipid (2 : 1, w/w, with membrane protein). Isopycnic sucrose density gradient centrifugation of such preparations shows that reaction centres and light-harvesting pigment-protein complex bind to the membranes. Reconstituted membranes exhibit light-induced steady-state cytochrome absorbance changes resembling those observed in chromatophores prepared from the photosynthetically-grown mutant R-26. The effect on these absorbance changes of varying reaction centre content in the membrane has been studied, and the time course of the interaction between 01 membrane cytochrome c₂ and added reaction centre examined.Cytochrome b photoreduction and cytochrome c₂ photo-oxidation were observed in the reconstituted preparation; each increased following the addition of antimycin A, suggesting that a cyclic light-driven system had been reconstituted.     

Light- and diffusion-potential-induced shift of carotenoid spectrum in reconstituted vesicles of Rhodopseudomonas sphaeroides.

Proteoliposomes were reconstituted from detergent-solubilized pigment.protein complexes of chromatophores of Rhodopseudomonas sphaeroides and soybean phospholipids. The reconstituted vesicles showed a photooxidation of reaction center bacteriochlorophyll and a light-induced spectral shift of carotenoid to longer wave-lengths. The red shift similar to that in intact cells or chromatophores, indicates the generation of local fields in the membrane of proteoliposomes. When inside-positive membrane potential was induced by adding valinomycin and potassium salt, a shift of carotenoid spectrum to shorter wavelengths was observed. Therefore, the reconstituted vesicles, at least in the major part of population, produced the light-induced local field in the same direction as in intact cells, which is inside negative. Sidedness of the membrane structure and the direction of electric field formation in reconstituted vesicles were opposite to those in chromatophores (inside-out vesicles.     

Light- and diffusion-potential-induced shift of carotenoid spectrum in reconstituted vesicles of Rhodopseudomonas sphaeroides

Proteoliposomes were reconstituted from detergent-solubilized pigment · protein complexes of chromatophores of Rhodopseudomonas sphaeroides and soybean phospholipids. The reconstituted vesicles showed a photooxidation of reaction center bacteriochlorophyll and a light-induced spectral shift of carotenoid to longer wavelengths. The red shift similar to that in intact cells or chromatophores, indicates the generation of local fields in the membrane of proteoliposomes. When inside-positive membrane potential was induced by adding valinomycin and potassium salt, a shift of carotenoid spectrum to shorter wavelengths was observed. Therefore, the reconstituted vesicles, at least in the major part of population, produced the light-induced local field in the same direction as in intact cells, which is inside negative. Sidedness of the membrane structure and the direction of electric field formation in reconstituted vesicles were opposite to those in chromatophores (inside-out vesicles).     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Energy transfer to the reaction centres in bacterial photosynthesis

From a combined study of (1) bacteriochlorophyll fluorescence lifetimes, (2) relative yields and (3) differential absorption changes corresponding to the reaction centres photooxidation, the absolute values of fluorescence lifetimes and quantum yields for two bacteriochlorophyll fractions have been calculated. The main bacteriochlorophyll fraction (80–90%) serving as a light-gathering antenna for reaction centresP ₈₉₀ is characterized by dark values of fluorescence lifetimes of the order of 10^–11 sec and fluorescence yields of 10^–3. The remaining part of the bulk pigment, not associated withP ₈₉₀ as far as excitation energy transfer is concerned, has an approximately constant fluorescence yield of about 5–8% and lifetime of about 10^–9 sec. Basing on these results, excitation jump times and intermolecular coupling energies were estimated to be 10^–13 sec and 10^–2 ev respectively. The conclusion is made that excitation energy transfer in the main part of bacteriochlorophyll occurs by the exciton mechanism at moderate intermolecular energies.     

Probing the fluorescence emission kinetics of the photosynthetic apparatus of Rhodopseudomonas sphaeroides, strain 1760-1, on a picosecond pulse fluorometer

Using the pulse picosecond fluorometric technique the fluorescence properties of intact cells, isolated chromatophores and photosynthetic reaction centres were studied in bacteria Rhodopseudomonas sphaeroides, strain 1760-1.The fluorescent emission from reduced reaction centres excited by 694.3 nm light has a biphasic character, the lifetimes of the components being τ₁ = 15±8 ps and τ₂ = 250 ps. The faster component, τ₁, contributes to the integral fluorescence in the long wavelength region. It disappears with oxidation of the reaction centres and is attributed to photoactive bacteriochlorophyll P870. The slow component, τ, is apparently due to both bacteriochlorophyll P800 and bacteriopheophytin. The fluorescence from intact cells exhibits a monophasic pattern and decays with τ = 200 ps.The fluorescence emitted by chromatophores comprises two components with τ₃ = 200 ps and τ₄ = 4200 ps. The duration of fluorescence τ₃ increases to its maximum of 500–550 ps, as P870 is oxidized chemically or photochemically, while τ₄ remains unchanged. The fluorescence with a lifetime of 200 ps was ascribed to the photosystem and the 4200-ps fluorescence to bacteriochlorophyll which had lost its functional links with the photosystem.The rise time of the fluorescence emitted by chromatophores varies from 60 or 70 ps to 350 ps depending on the wavelength of the exciting light and the recorded spectral region. On the basis of our findings the rate for energy migration was estimated to be 10⁹ s^?1.     

Energy transfer and bacteriochlorophyll fluorescence in purple bacteria at low temperature

Emission spectra of bacteriochlorophyll a fluorescence and absorption spectra of various purple bacteria were measured at temperatures between 295 and 4 K. For Rhodospirillum rubrum the relative yield of photochemistry was measured in the same temperature region. In agreement with earlier results, sharpening and shifts of absorption bands were observed upon cooling to 77 K. Below 77 K further sharpening occurred. In all species an absorption band was observed at 751-757 nm. The position of this band and its amplitude relative to the concentration of reaction centers indicate that this band is due to reaction center bacteriopheophytin. The main infrared absorption band of Rhodopseudomonas sphaeroides strain R26 is resolved in two bands at low temperature, which may suggest that there are two pigment-protein complexes in this species. Emission bands, like the absorption bands, shifted and sharpened upon cooling. The fluorescence yield remained constant or even decreased in some species between room temperature and 120 K, but showed an increased below 120 K. This increase was most pronounced in species, such as R. rubrum, which showed single banded emission spectra. In Chromatium vinosum three (835, 893 and 934 nm) and in Rps. sphaeroides two (888 and 909 nm) emission bands were observed at low temperature. The temperature dependence of the amplitudes of the short wavelength bands indicated the absence of a thermal equilibrium for the excitation energy distribution in C. vinosum and Rps. sphaeroides. In all species the increased in the yield was larger when all reaction centers were photochemically active than when the reaction centers were closed. In R. rubrum the increase in the fluorescence yield was accompanied by a decrease of the quantum yield of charge separation upon excitation of the antenna but not of the reaction center chlorophyll. Calculation of the F?rster resonance integral at various temperatures indicated that the increase in fluorescence yield and the decrease in the yield of photochemistry may be due to a decrease in the rate of energy transfer between antenna bacteriochlorophyll molecules. The energy transfer from carotenoids to bacteriochlorophyll was independent of the temperature in all species examined. The results are discussed in terms of existing models for energy transfer in the antenna pigment system.     

Effect of dehydration on the primary picosecond stages of charge separation in Rhodospirillum rubrum

Effects of dehydration on the quantum yield of charge separation in the reaction centres, fluorescence and nanosecond recombination luminescence in R. rubrum chromatophores have been investigated. It has been shown that dehydration results in more than a 10 times decrease in the quantum efficiency of photosynthesis. Besides, photoinduced fluorescence changes practically disappear in dehydrated samples and the parameters of nanosecond luminescence substantially change. These observations indicate that strong dehydration causes a deterioration of the primary charge separation process at the early picosecond stages of excitation energy transduction into energy of separated charges. This is, probably, due to either changes in the dynamic characteristics of the reaction centre pigment-protein complex or alteration in the structure state (spacings and mutual orientations) of the primary reactants involved in the primary charge separation.     

Singlet-triplet fusion in Rhodopseudomonas sphaeroides chromatophores. A probe of the organization of the photosynthetic apparatus

Chromatophores from various strains of Rhodopseudomonas sphaeroides were excited with laser flashes lasting about 20 ns. Fluorescence from the antenna bacteriochlorophyll of the photosynthetic apparatus was measured both during the laser flash, and during a weak Xe flash following the laser flash. Strong laser flashes caused severe quenching of the fluorescence, which could be correlated with the formation of triplet states of the antenna pigments. Triplet states of both BChl and carotenoids acted as quenchers, but bacteriochlorophyll triplets were the more effective of the two. In the double-flash experiments, the reciprocal of the fluorescence yield was proportional to the concentration of triplet quenchers remaining at the time of the second flash. This relationship indicates that singlet excitations can migrate over large domains in the antenna, rather than being restricted by boundaries separating individual reaction centers. Comparisons of chromatophores from different strains and from cells grown under different conditions showed that excitations are concentrated rapidly in the antenna complexes with the longest wavelength absorption bands (B870), and that the migration of excitations to trapping sites is relatively insensitive to the amount of antenna bacteriochlorophyll absorbing at shorter wavelengths (B800–B850). This suggests that the B870 complexes are organized in the membrane so as to interconnect many reaction centers, and that the B800–B850 complexes are arranged peripherally.     

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.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 +/- 20 mM--1 . CM--1. The molecular weight based on protein and chromophore assays was found to be 1.5 . 10(5); the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum.

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360-410mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores. The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C340) which has an oxidation-reduction midpoint potential close to 340 mV (n=1) at pH 6.0--7.6. The Em of this component varies by 60 mV/pH unit between pH 7.6 and 9.2. The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C340.     

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.