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).     

The energy-linked carotenoid band-shift in Chromatium vinosum.

Chromatium vinosum chromatophores contain an energy-linked pyrophosphatase that is insensitive to oligomycin and dicyclohexylcarbodiimide. Pyrophosphate hydrolysis produces a carotenoid band-shift similar to that resulting from illumination. The carotenoid band-shift can also be produced by a K⁺ diffusion potential (interior positive) and the magnitude of the band shift is proportional to the membrane potential over at least a 100-fold variation in K⁺ concentration. The light-induced carotenoid band-shift in whole cells is identical to that seen in chromatophores but K⁺ diffusion potentials (interior positive) produce a mirror image of the light-induced band-shift. These results are interpreted in terms of chromatophores being inside-out vesicles.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts, spheroplast membrane vesicles and chromatophores.

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Light-induced blue shift of the carotenoid spectrum in chromatophores of Chromatium vinosum strain D

In Chromatium chromatophores, the response of part of the carotenoid complement to a light-induced membrane potential is a shift to the blue of its absorption spectrum, as indicated by the characteristics of the light-minus-dark difference spectrum. The spectrum in the dark of the population of carotenoid which responds to a light-induced membrane potential is located at least 1–2 nm to the red in comparison to the total carotenoid absorption. The results indicate that the proposed permanent electric field affecting the responding population has a polarity with respect to the chromatophore membrane opposite to that in Rhodopseudomonas sphaeroides chromatophores. The carotenoid absorption change interferes seriously with measurements of cytochrome c-555 redox changes at its α band.     

The location and function of cytochrome c2 in Rhodopseudomonas capsulate membranes.

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate. Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9-aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift. It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophors and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.     

Identification of the carotenoid present in the B-800–850 antenna complex from Rhodopseudomonas capsulata as that which responds electrochromically to transmembrane electric fields

Mild proteolysis of Rhodopseudomonas capsulata chromatophores results in a parallel loss of the 800 nm bacteriochlorophyll absorption band and a blue shift in the carotenoid absorption bands associated with the B-800–850 light-harvesting complex. Both the light-induced and the salt-induced electrochromic carotenoid band shift disappear in parallel to the loss of the 800 nm bacteriochlorophyll absorption upon pronase treatment of chromatophores. During the time required for the loss of the 800 nm bacteriochlorophyll absorption and the loss of the electrochromic carotenoid band shift photochemistry is not inhibited and the ionic conductance of the membrane remains very low. We conclude that the carotenoid associated with the B-800–850 light-harvesting complex is the one that responds electrochromically to the transmembrane electric field. Analysis of the pigment content of Rps. capsulata chromatophores indicates that all of the carotenoid may be accounted for in the well defined pigment-protein complexes.     

Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides: an analysis and interpretation of the band shifts.

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20% of the total carotenoid content, is responsible for the light-induced absorbance changes. The 'remaining' pool absorbs at wavelengths which were about 5 nm lower than those at which the 'changing' pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields. The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 +/- 6 mV per nm shift.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides. An analysis and interpretation of the band shifts

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20 % of the total carotenoid content, is responsible for the light-induced absorbance changes. The ‘remaining’ pool absorbs at wavelengths which were about 5 nm lower than those at which the ‘changing’ pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields.

The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 ± 6 mV per nm shift.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.     

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 chromotophores; 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 (Em = +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.     

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.     

The kinetics of carotenoid absorption changes in intact cells of photosynthetic bacteria

The kinetics of carotenoid absorption changes have been measured in intact cells of Rhodopseudomonas capsulata after short flash excitation. The observed changes were consistent with the thesis that they indicate the development and dissipation of membrane potential. In the generation of the absorption changes in anaerobic cells, fast (complete in 0.5 ms) and slow (half-time 3 ms) components can be distinguished. The slow component corresponds kinetically to the rate of cytochrome c re-reduction and is similarly antimycin sensitive. These data are similar to those observed in isolated chromatophores which have been artifically poised with redox mediators. In aerobic intact cells the kinetic profile is altered, mainly because the decay of the carotenoid change is much faster. Inhibition of respiration with KCN leads to flash-induced changes similar to those in anaerobic cells. At least two components can be distinguished in the decay of the carotenoid absorption changes in anaerobic intact cells. Only the faster decay component was inhibited by venturicidin which suggests that it corresponds to H⁺ flux through the F₀F₁-ATPase during ATP synthesis. The contribution of the venturicidin-sensitive decay to the total decay was dependent upon the initial amplitude of the carotenoid absorption change produced by the flash group. This suggests that there is an apparent threshold of membrane potential for ATP synthesis. Supporting evidence was provided by the finding that venturicidin stimulated the steady-state light-induced carotenoid absorption change at high but not at low light intensities. The entire decay of the carotenoid absorption changes was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in a manner that can be interpreted as an ionophore catalysing the dissipation of membrane potential.     

Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga.

1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The responses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the ‘light-minus-dark’ difference spectrum of the chromatophores.The oxonols appear to respond to the light-induced ‘energization’ by shifting their absorption maxima. In the presence of K⁺, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift.The dye response has an apparent second-order rate constant of approx. 2 · 10⁶ M^?1 · s^?1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not posses intrinsic probes of potential.     

Evaluation of the electrical capacitance in biological membranes at different phospholipid to protein ratios

Photosynthetic chromatophores of Rhodobacter capsulatus were differently enriched in phospholipid content by freezing, thawing and sonicating in the presence of phospholipid vesicles. Closed vesicles, characterized by different phospholipid to protein molar ratios and increasing average radius at increasing phospholipid enrichment, were collected after sucrose density gradient sedimentation. The electrical capacitance of these systems was evaluated from the ratio of reaction center content, photooxidized by single turnover flash in the presence of antimycin, to the corresponding membrane potential difference, measured from the electrochromic red shift of the endogenous carotenoid band. The values obtained, normalized per protein content, increased at increasing phospholipid enrichment, and correlated linearly with the increasing phospholipid to protein molar ratios. The charging capacitance of chromatophores was evaluated to be 3–6×10^-17 F and was found to increase at increasing average radius of the phospholipid enriched vesicles, as predicted by the equation of the spherical shell dielectric. The carotenoid signal, elicited in the dark by imposing diffusion potentials of known extent with K⁺-valinomycin pulses, significantly decreased at high phospholipid enrichment, indicating that in the presence of large phospholipid excess, a partial displacement of the carotenoid molecules sensing the induced electric field is produced. Concomitantly, the energy transfer efficiency from carotenoids to core light harvesting complexes (B-875) was also partially affected, particularly at high phospholipid to protein molar ratio. All together, these results suggest that the reaction center complexes are dispersed within the lipid bilayer upon fusion and that carotenoids sense a delocalized light-induced transmembrane field.Abbreviations BChl bacteriochlorophyll - [BChl]₂ reaction center - PL phospholipid - cyt cytochrome - transmembrane electrical potential difference - TES 2-2-Hydroxy-1,1-bis-(hydroxymethyl)ethyl-amino-ethanosulfonic acid - mg_p mg protein     

Fusion of proteoliposomes containing the B800-850 light-harvesting complex with membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking B800-850

Intracytoplasmic membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking the lightharvesting complex B800-850 were fused with proteoliposomes containing the B800-850 complex. Fluorescence emission spectroscopy at 77K showed that after fusion the fluorescence of the B850 bacteriochlorophyll disappeared nearly completely and the B870 fluorescence became prominent. This result and control experiments with proteoliposome-chromatophore mixture and with chromatophore and solubilized B800-850 complexes, respectively, indicate that in fused membranes a reorientation of membrane particles took place and excitons migrated from B850 to B870 bacteriochlorophyll.In fused proteoliposome-chromatophore vesicles a light-induced carotenoid band shift was observed, reflecting the building of an electrical membrane potential due to chargeseparation. Carotenoid band shift was not observed in separated proteoliposomes and NK3 chromatophores.It is concluded that by membrane fusion and lateral diffusion of membrane particles reaction center-light-harvesting B870 complexes came in functional contact with B800-850 antenna complexes.Abbreviations Bchl bacteriochlorophyll - LDAO lauryl dimethylamine oxide - RC reaction center Dedicated to Professor R. Clinton Fuller, Amherst, MA, USA, on the occasion of his 60th birthday in recognition of his work on photosynthetic bacteria and the cooperation between our laboratories     

Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Correlation of membrane-potential-sensing carotenoid to pigment-protein complex II in Rhodopseudomonas sphaeroides

The changes in carotenoid absorbance induced by illumination or by a diffusion potential were larger in chromatophores from cells cultured under low light intensity than those in chromatophores from high-light culture in a photosynthetic bacterium, Rhodopseudomonas sphaeroides. The carotenoid molecules which are associated with the pigment-protein complex (with the infrared bacteriochlorophyll peaks at 800 and 850 nm) (complex II) probably respond to the electrical field changes in the chromatophore membrane.