Molecular stochastic simulations of chromatophore vesicles from Rhodobacter sphaeroides.

A kinetic model is presented for photosynthetic processes under varying illumination based on the recently introduced steady state model of the photosynthetic chromatophore vesicles of the purple bacterium Rhodobacter sphaeroides. A stochastic simulation system is built up from independent copies of the different transmembrane proteins, each encapsulating its own set of binding sites and internal states. The proteins are then connected through pools for each of the metabolites. A number of steady state and time-dependent scenarios are presented showing that even under steady state conditions the stochastic model exhibits a different behavior than a continuous description. We find that the electronic coupling between the light harvesting complexes increases the efficiency of the core complexes which eventually allows the bacteria to bridge short illumination outages at already lower light intensities. Some new experiments are proposed by which the DeltapH dependent characteristic of the bc(1) complex or the proton buffering capacity of the vesicle could be determined.

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition.

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.

Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence.

FOF1-ATP synthase converts two energetic "currencies" of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the single-enzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (<1 microM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of approximately 0.25 mol ADP per mole of enzyme. There was no detectable detachment of F1 from FO. (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (>1 microM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s(-1) at pH 8. Proton slip probably presents the free-wheeling of the central rotary shaft, subunit gamma, in an open structure of the (alphabeta)3 hexagon with no nucleotides in the catalytic sites.

Fusion of chromatophores from photosynthetic bacteria with a supported lipid layer: characterization of the electric units.

Direct electrometric measurements of membrane potential changes are a valuable tool for study of vectorial transfer of electrons, protons, and ions. Commonly model membrane systems are created by fusion of lipid/protein vesicles with lipid-coated thin films. We characterized the electric units resulting from this process using chromatophores from the purple bacterium Rhodobacter sphaeroides and either a Mylar film or a planar modified gold electrode as support. Investigation of the shunting activity of the ionophore gramicidin on the flash-induced potential change demonstrates fusion of individual chromatophores to form independent 'blisters', which preserve an interior aqueous compartment. Under current-clamp conditions the photovoltage follows the change of the membrane potential of the individual blisters.

Ultrafast exciton relaxation in the B850 antenna complex of Rhodobacter sphaeroides.

Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacterium Rhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.

Flash-induced electrogenic reactions in the SA(L223) reaction center mutant in Rhodobacter sphaeroides chromatophores.

The charge transfer events in the SA(L223) reaction center mutant Rhodobacter sphaeroides chromatophores were investigated by direct electrometry. Besides the primary charge separation, the small stigmatellin-sensitive electrogenic reaction due to the electron transfer from the primary to the secondary quinone acceptor in the reaction center complex was observed after the first flash. The second flash-induced electrogenic phase of the secondary quinone protonation and subsequent electrogenic reactions of the cytochrome bc1 complex were much slower than those in chromatophores of the wild type. It is suggested that replacement of Ser-L223 by Ala impairs both specific proton-conducting pathways leading to the secondary quinone QB.

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1.

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.

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.

On the dielectrically observable consequences of the diffusional motions of lipids and proteins in membranes. 2. Experiments with microbial cells, protoplasts and membrane vesicles.

The dielectric properties of suspensions of intact cells of Methylophilus methylotrophus, Paracoccus denitrificans and Bacillus subtilis have been measured in the frequency range 1 kHz to 13 MHz. All possess a pronounced dispersion corresponding in magnitude and relaxation time to the "beta-dispersion" in a terminology defined by Schwan [Adv. Biol. Med. Phys. 5:147-209 (1957)]. The latter two strains, but not M. methylotrophus, also possess a substantial alpha-dispersion. The relaxation time of the beta-dispersion of B. subtilis is significantly lower than that of the other two strains, due to the higher internal K+ content of this Gram-positive organism. Treatment of P. denitrificans or B. subtilis with lysozyme greatly reduces the magnitude of the alpha-dispersion; in the latter case it is virtually abolished. The magnitude of both the alpha- and beta-dispersions of protoplasts of these organisms is significantly decreased by treatment with the cross-linking reagent glutaraldehyde, indicating that diffusional motions of the lipids and/or proteins in the protoplast membranes contribute to the dielectric relaxations observed in this frequency range. Such motions cannot be unrestricted, as in the "fluid mosaic" model, since the relaxation times of the lipids and proteins, if restricted by hydrodynamic forces alone, should then correspond, in protoplasts of this radius (0.4-0.5 micron), to approximately 10 Hz. Even after treatment of the (spherical) protoplasts with glutaraldehyde, the breadth of the remaining beta-dispersion is still significantly greater than (a) that of a pure Debye dispersion and (b) that to be expected solely from a classical Maxwell-Wagner-type mechanism. It is recognised that the surfaces of the protein complexes in such membranes extend significantly beyond the membrane surface as delineated by the phospholipid head-groups; such molecular granularity can in principle account for the broadened dielectric relaxations in the frequency range above 1 kHz, in terms of the impediment to genuinely tangential counterion relaxation caused by the protruding proteins themselves. The relaxation time of a previously observed, novel, low-frequency, glutaraldehyde-sensitive (mu-) dispersion in bacterial chromatophore suspensions, as well as that of their alpha-dispersion, is significantly increased by increasing the aqueous viscosity with glycerol. This finding is consistent with the view that, from a dielectric standpoint, the motions of charged proteins (and lipids) in biological membranes are rather tightly coupled to those of the adjacent ions and dipoles in the electric double layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell-cycle-specific biosynthesis of the photosynthetic membrane of Rhodopseudomonas sphaeroides. Structural implications.

Structural changes association with the intracytoplasmic membrane during the cell cycle of the photosynthetic bacterium Rhodopseudomonas sphaeroides have been studied by freeze-fracture electron microscopy. The isolated intracytoplasmic membrane vesicles, chromatophores, were fused in order to obtain large fracture faces, allowing more precise measurements and statistical analysis of both intramembrane particle density and size determinations. The intramembrane particle density of the protoplasmic face (PF) of the intracytoplasmic membrane, (from 4970 to 8290/micrometers 2), was shown to be a linear function of the protein/phospholipid ratio (from 2.5 to 5.1, w/w) of the intracytoplasmic membrane. Under constant light intensity, both the average particle size and particle size distribution remained unchanged during the cell cycle. These results provide the structural basis for the earlier reported cell-cycle-specific variations in both protein/phospholipid ratio and alternation in phospholipid structure of the intracytoplasmic membrane of R. sphaeroides during photosynthetic growth. The average particle diameter in the PF face of the intracytoplasmic membrane was 8.25, 9.08 and 9.75 nm at incident light intensities of 4000, 500 and 30 ft X cd, respectively. When chromatophores were fused with small, unilamellar liposomes, the intramembrane particle density decreased as input liposome phospholipid increased, whereas the particle size remained constant and particle distribution became random.

[Role of cofactors in membrane potential generation by Rhodospirillum rubrum chromatophores incorporated in a teflon filter]. / Rol' kofaktorov v obrazovanii membrannogo potentsiala khromatoforami Rhodospirillum rubrum, vstroennymi v teflonovyi fil'tr.

The chromatophores of the bacterium Rhodospirillum rubrum were incorporated into a Teflon filter impregnated with a decane solution of phospholipids and the light-induced electric potential difference (delta psi) between the aqueous phases separated by the filter was measured. The generation of delta psi in such a system at continuous light requires the presence of cofactors, i. e. artificial electron donors and acceptors. These cofactors provide for a steady-state flow of e by regenerating the reduced form of the photo-oxidized reaction center bacteriochlorophyll and by reoxidizing the photo-reduced quinone acceptor of the reaction center. The most efficient donors are the reduced forms of nitrogen-containing redox mediators, e. g. TMPD, DAD, PMS, DCPIP and methylene blue, while p-benzoquinones with E07' approximately greater than 150 mV are practically inactive. The oxidized forms of nitrogen-containing mediators and a wide variety of p-quinones with E07' down to -220 mV can be used as electron acceptors. Using inhibitor analysis and compounds with different E0' it is shown that the cofactors donate electrons immediately to the reaction center bacteriochlorophyll and accept them from a low potential deprotonated form of the primary acceptor QI, substituting the secondary acceptor QII. The inability of hydrophilic sulfonate-substituted quinones to act as acceptors suggests that QI cannot be localized on the outer surface of the chromatophores.

[Study of microwave photolosses in chromatophores of photosynthesizing Rhodospirillum rubrum bacteria]. / Issledovanie mikrovolnovykh fotopoter' v khromatoforakh fotosinteziruiushchikh bakterii Rhodospirillum rubrum.

The study of photoinduced dielectric losses at frequency 10(10) c/s as function of samples humidity, heating and chemical treatment, intensity and wavelength of light is reported. The signals of microwave losses and differential spectrum signals are compared. The authors attribute the photolosses observed to the electrons localised in the realm between the primary and secondary acceptors during their reduction, probably being in the conductance quasiband of proteins in the photosynthetic reaction centre.

Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata.

The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesting II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynthesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes.

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum.

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.

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 salf 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, MgCl-2, 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 A2, and the surface potential of about -100 mV in the presence of about 0.1 mM divalent cation of 5 mM monovalent cation were calculated.

The orientations of reaction center transition moments in the chromatophore membrane of Rhodopseudomonas sphareroides, bases on new linear dichroism and photoselection measurements.

Chromatophore membranes from Rhodopseudomonas sphaeroides were oriented by drying suspensions on the surfaces of glass slides, Polarized spectra of light-induced absorption changes were obtained between 500 and 1000 nm. As observed earlier, these spectra showed negative bands, reflecting photooxidation of the bacteriochlorophyll 'special pair' in the reaction centers, centered near 870, 810, 630 and 600 nm. These bands have been designated BY1, BY2, BX1 and BX2, respectively, corresponding to two QY transitions and two QX transitions of the dimeric special pair. We found the BY1 and BX1 transition moments to be parallel (within 20 degrees) to the plane of the membrane, whereas the BX2 moment makes an angle of 55--63 degrees with the plane. Using the photoselection technique we found that the angle between the BY1 and BX1 transition moments is 30 degrees, while that between BY1 and BX2 is 75 degrees. The BX1 and BX2 moments were found to be orthogonal, consistent with the prediction of molecular exciton theory for a dimer. By combining these data, we have calculated the orientations of the transition moments of the bacteriochlorophyll dimer in spherical polar coordinates, with the pole of the coordinate system normal to the plane of the membrane. The orientations of the QY and QX transition moments of the two bacteriopheophytin molecules in the reaction center were also computed in this coordinate system by transforming the data reported by Clayton, C.N., Rafferty, R.K. and Vermeglio, A. ((1979) Biochim. Biophys. Acta 545, 58--68). We have derived the transformation equations for two polar coordinate systems: in one, the pole is an axis of symmetry as defined by the orientations of purified reaction centers in stretched gelatin films (Rafferty, C.N. and Clayton, R.K. (1979) Biochim. Biophys. Acta 545, 106--121). In the other, the pole is normal to the plane of the chromatophore membrane. These two polar axes are approximately orthogonal.

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.