Light-induced red shift of the Qx absorption band of light-harvesting bacteriochlorophyll in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the Q_x band(s) of the light-harvesting bacteriochlorophyll (BChl) (λ_max ~590 nm) shifts to the red in response to a light-induced membrane potential, as indicated by the characteristics of the light-minus-dark difference spectrum. In green strains, containing light-harvesting complexes I and II, and one or more of neurosporene, methoxyneurosporene, and hydroxyneurosporene as carotenoids, the absorption changes due to the BChl and carotenoid responses to membrane potential in the spectral region 540–610 nm are comparable in magnitude and overlap with cytochrome and reaction center absorption changes in coupled chromatophores. In strains lacking carotenoid and light-harvesting complex II, the BChl shift absorption change is relatively smaller, due in part to the lower BChl/reaction center ratio.In the carotenoid-containing strains, the peak-to-trough absorption change in the BChl difference spectrum is 5–8% of the peak-to-trough change due to the shift of the longest-wavelength carotenoid band, although the absorption of the BChl band is 25–40% of that of the carotenoid band. The responding BChl band(s) does not appear to be significantly red-shifted in the dark in comparison to the total BChl Q_x band absorption.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

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.     

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.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides II. Analysis of the band shifts of carotenoid and bacteriochlorophyll

An analysis was made of the changes of pigment absorption upon illumination of chromatophores of Rhodopseudomonas sphaeroides at ?35 °C, described in the preceding paper (de Grooth, B. G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 237–246). Comparison of the light-induced difference spectra in the carotenoid region obtained without additions, and in the presence of N-methylphenazonium methosulphate and ascorbate as donor-acceptor system showed that the latter spectrum was not only about 10 times larger in amplitude, but also red-shifted with respect to the first one. Together with the shape of the difference spectrum, this indicated that the spectrum obtained in the presence of a donor-acceptor system is due to an electrochromic shift of the absorption spectrum of a carotenoid by a few nm towards longer wavelength, caused by a delocalized potential across the chromatophore membrane. The results of an analysis of the kinetics of the absorbance changes near the zero points of the spectrum were in quantitative agreement with the extent of the red shift and indicated a shift of 0.25 nm for a single electron transfer per reaction center, and shifts of up to 4 nm when the electron transport is stimulated by a donor-acceptor system. For bacteriochlorophyll B-850 the shift is three times smaller.Analysis of the overall absorption spectrum showed that there are at least two pools of carotenoid. The carotenoid that shows electrochromism has absorption bands at 452, 481 and 515 nm, and comprises about one-third of the total carotenoid present; the remaining pool absorbs at about 7 nm shorter wavelength and does not show an electrochromic response to illumination. Both pools presumably consist of spheroidene; the differences in band location may be explained by the assumption that only the first pool is subjected to a local electric field which induces an electric dipole even at zero membrane potential. Similar results were obtained at room temperature and with a mutant of Rps. sphaeroides (G1C)-containing neurosporene.     

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides

The pigment content of a B800–850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed.The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800–850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band.The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

Ferricyanide-induced absorbance change of carotenoid in chromatophores of Rhodopseudomonas spheroides correlated to the oxidation of bacteriochlorophyll 885

Addition of high concentrations (e.g., 1–100 mM) of ferricyanideto a chromatophorc suspension of Rhodopseudomonas spheroidescaused a change in the absorption spectrum of carotenoid (spheroidene),which was completely reversed by adding reducing reagents suchas ferrocyanide and ascorbate. The spectral change is representedby a shift in the absorption spectrum of carotenoid by 2 to2.5 nm towards the longer wavelength side. The presence of piericidinA, o-phenanthroline or Cl-CCP in the reaction mixture did notaffect the ferricyanide-induced absorbance change. Triton X-100markedly suppressed the magnitude of the change. The additionof ferricyanide also caused simultaneous absorbance changeswith maxima at 590 and 885 nm. These are ascribed to oxidationof the (bulk) bacteriochlorophyll, BChl 885. There was no absorptionchange at other peaks of bacteriochlorophyll in the infraredregion (i.e., 800 and 855 nm). Therefore, the ferricyanide-inducedabsorbance change of carotenoid did not represent an oxidation-reductionreaction of carotenoid but was intimately correlated with oxidationof BChl 885 in the chromatophores, as judged from similaritiesobserved with respect to the time course patterns, midpointpotential (545–555 mv) in the ferriferrocyanide reactionsystem, as well as behavior towards various reagents and inhibitorsadded. A similar change of carotenoid (i.e., 2–2.5 nmshift of absorption spectrum) was caused by addition of MgCl₂to the chromatophores, but this did not induce any change inthe absorption spectrum of bacteriochlorophyll. The nature ofthe spectral change of carotenoid in chromatophores is discussed. (Received April 16, 1970; )     

Singlet and triplet absorption spectra of carotenoids bound in the reaction centers of Rhodopseudomonas sphaeroides R26

The singlet and triplet state absorption spectra are reported for two carotenoids, methoxyneurosporene and spheroidene, incorporated into the reaction center protein of the photosynthetic bacterial carotenoidless mutant Rhodopseudomonas sphaeroides R26. The spectra for the two different carotenoid molecules are identical suggesting a strong interaction between the protein and the different chromophores. Combined effects of electrochromic band shifts and carotenoid structural changes are invoked to account for the spectral observations.     

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.     

Titles of related papers in other sections

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1 : 2 : 2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846 and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b. The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably, to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P⁺-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.     

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.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Ionophore-sensitive spectral shift of carotenoid induced by ferricyanide in chromatophores from Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides, ferricyanideinduced a change in the absorption spectrum of carotenoid. Theionophore-sensitive part of the ferri-cyanide-induced changewas similar to that induced by light or by diffusion potential.The ferricyanide-induced change is explained by the electrochromicshift of the carotenoid spectrum by the inside-positive electricalfield change which is probably caused by the electrogenic electronflow from a membrane redox component to ferricyanide in theouter aqueous phase. The ionophore-insensitive part is probablythe response of the carotenoid in another pool to the localfield change by oxidation of bacteriochlorophyll [Okada, M.and A. Takamiya (1970) Plant & Cell Physiol. 11: 713–721]. (Received January 21, 1980; )     

Effect of phenolic herbicides on the oxygen-evolving side of Photosystem II. Formation of the carotenoid cation

Phenolic herbicides were added to suspensions of spinach chloroplasts or to oxygen-evolving Photosystem II membranes. Flash absorption spectroscopy at 21°C around 1000 nm reveals that these chemicals lead to a flash-induced absorption increase attributed to the radical-cation of a carotenoid. The herbicides studied can be arranged in the following order of decreasing efficiency for the reported effect: i-dinoseb, bromonitrothymol, trinitrophenol, ioxynil, dinitroorthocresol, 2,4-dinitrophenol. A similar effect was not observed with atrazine, DCMU or o-phenanthroline. For a given herbicide concentration, the amount of flash-induced carotenoid cation increases sharply when the pH is lowered below 5.5. A similar effect does not take place with other molecules which induce the formation of a carotenoid cation: tetraphenylboron, FCCP, 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p). The previous effects are observed in both oxygen-evolving Photosystem II and in preparations in which oxygen evolution is inhibited with alkaline Tris. In untreated material, the carotenoid cation is formed with a half-time of 10–35 μs. After Tris treatment, this half-time is a little longer at low than at high pH. These results indicate the existence of a specific site where phenolic inhibitors interact in the oxygen-evolving site of Photosystem II