Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

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.     

The membrane location of the B890-complex from Rhodospirillum rubrum and the effect of carotenoid on the conformation of its two apoproteins exposed at the cytoplasmic surface

The membrane location of the two B890-apoproteins from Rhodospirillum rubrum has been investigated by comparing the effects of mild proteolysis with proteinase K, and immunoprecipitation, with antibodies prepared against the B890-complex and its individual apoproteins, upon membrane vesicles. By using chromatophores (inside-out vesicles) and spheroplasts (right-side vesicles) both membrane surfaces have been interrogated. The N-terminal regions of both aproproteins are located at the cytoplasmic membrane surface, while the C-termini are most probably located at the periplasmic surface. The conformation of the N-terminal regions of the apoproteins is changed by the presence of carotenoid. In the carotenoid-containing strain S1, for example, the α-apoprotein is insensitive to digestion by proteinase K, while in the absence of the carotenoid in the mutant G9⁺ six amino acids are removed from the N-terminus of the α-aproprotein by proteinase K treatment.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

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

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

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

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.     

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, Δ$\text{G}$°, at 10 and 100 mM ionic strength (?7.85 and ?8.26 kcal/mol, respectively). Binding saturates at an area of 650 Ų per melittin molecule. A change in the surface dipole potential corresponding to ?1.1 debye/?_a ($\text{?}{}_{\mathrm{a}}\text{= dielectric constant of the adsorption region}$) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.     

OBSERVATIONS ON THE FINE STRUCTURE OF SPHEROPLASTS OF RHODOSPIRILLUM RUBRUM

Spheroplasts of the photosynthetic bacterium Rhodospirillum rubrum were prepared from cultures grown in either the presence or absence of light. Cells were converted into spheroplasts by using lysozyme and Versene and fixed in a sucrose-veronal-acetate buffer mixture containing osmium tetroxide. Some preparations were shadow-cast and examined whole; others were embedded in Epon 812 and sectioned. The action of lysozyme and Versene appears to result in removal of the cell wall in strips. The relationship of the chromatophores to the cytoplasmic membrane is readily visualized in sections of broken spheroplasts, and in areas the chromatophores are seen to be continuous with the membrane. In all preparations examined, no definite connections between individual chromatophores were observed. In some cells large spherical granules were evident which either possessed or lacked a clearly visible limiting membrane. On serial sectioning, all granules appeared bounded by a single membrane 40 A wide. The granule membrane was well defined only if the section came from the center of the granule. Sections at other levels showed either a diffuse membrane or no membrane at all. The reasons for this are discussed.     

The functional unit of electrical events and phosphorylation in chromatophores from Rhodopseudomonas sphaeroides

1. From electron micrographs of chromatophores from Rhodopseudomonas sphaeroides and from the estimated bacteriochlorophyll content of the sample a mean value of 4700 bacteriochlorophyll per chromatophore was estimated. The mean diameter of the chromatophore vesicles was 600 Å.2. The decay of the flash-induced electric potential across the chromatophore membrane measured by the carotenoid band shift was 20% accelerated by about one valinomycin molecule per 4700 bacteriochlorophyll, i.e. by one ionophore molecule per chromatophore.3. The inhibition of the flash-induced ATP formation by valinomycin followed a similar pattern to the accelerated decay of the electric potential.4. The single turnover flash yield of ATP synthesis gave a mean value of one ATP per 1470 bacteriochlorophyll or about 3 ATP per vesicle.5. With regard to the partitioning of the ionophore between the membrane (85%) and aqueous phase (15%) we conclude that one molecule of valinomycin per chromatophore is sufficient to begin to collapse the electrical potential and inhibit ATP synthesis. It is therefore suggested that the membrane potential is an essential component of the energized state which is used for phosphorylation.The results correspond to those obtained for the 100-fold larger vesicles in chloroplasts (thylakoids) where one molecule of ionophore is also sufficient to quench both events.     

Assessment of Rhodopseudomonas sphaeroides chromatophore membrane asymmetry through bilateral antiserum adsorption studies.

The asymmetric structure of the Rhodopseudomonas sphaeroides chromatophore membrane was examined in detail by crossed immunoelectrophoresis techniques. Because these methods are quantitative and allow increased resolution and sensitivity, it was possible to analyze simultaneously the relative transmembrane distribution of a number of previously identified antigenic components. This was demonstrated by analysis of immunoglobulin samples that were adsorbed by preincubation with either isolated chromatophores or osmotically protected spheroplasts. The photochemical reaction center, the light-harvesting bacteriochlorophyll a-protein complex, the L-lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) were found to be exposed on the chromatophore surface (cytoplasmic aspect of the membrane within the cell). Other antigenic components were found to be exposed on the surface of spheroplasts (periplasmic aspect of the in vivo chromatophore membrane). Antigens with determinants expressed on both sides of the chromatophore membrane were also identified. Charge shift crossed immunoelectrophoresis confirmed the suggested amphiphilic character of the pigment-protein complexes and identified several additional amphiphilic membrane components.     

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

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

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.     

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.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and P_i of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of the pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H⁺ consumption during the synthesis of ATP. The ratio of H⁺ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 $\text{H}{}^{\mathrm{+}}\text{ATP}$.2. The stimulation of the electrical potential decay by ADP and P_i was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No ‘critical’ or ‘threshold’ potential was observed.3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle.4. The stimulation of the electrical potential decay by ADP and P_i is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H⁺-release on the inside of the chromatophore vesicles following a flash.     

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v₁ and v₂) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v₁/intensity v₂ is a sensitive probe for absorption shifts. Changes in this ratio of ～20% were observed during a valinomycin induced diffusion potential. At 5°C changes in the average intensity ratio of +6, ?4 and ?14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5°C and for intact cells under growing conditions.     

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.