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.     

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.     

Photosynthetic electron transport and electrochromic effects at sub-zero temperatures.

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and "P-518" in the region from -35 to -50 degrees C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant. In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

Photosynthetic electron transport and electrochromic effects at sub-zero temperatures

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and “P-518” in the region from ?35 to ?50 °C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant.In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550–650 and 700–950 nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as Δa_v/Δa_h, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810 nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm, and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the “special pair” that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the “light-dark” difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

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.     

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.     

Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl₂, have been measured in the wavelength region 460–540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCl concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of P-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

Dichroism of transient absorbance changes in the red spectral region using oriented chloroplasts. I. Field indicating absorbance changes

The light-induced transient absorbance changes which are affected by valinomycin have been studied using magnetically oriented spinach chloroplasts and a polarized measuring beam. The ΔA spectra for the two polarizations parallel and perpendicular to the plane of the photosynthetic membranes have been recorded in the spectral range 630–750 nm. Large polarization effects are found in all the bands of the ΔA spectrum, shifts in the position of the extrema are observed and the two spectra cross each other at various wavelengths. A comparison of these spectral features with available data on the dichroism of the Stark effect on monomolecular films of chlorophyll a and b indicates similarities favoring the already well documented hypothesis of the electrochromic nature of these absorbance changes in vivo.The data on this electrochromic effect can be correlated with the linear dichroism of oriented chloroplasts and the ΔA_∥?ΔA_⊥ spectrum in the 645–655 nm region gives further evidence of the orientation out of the membrane plane of the red transition moment of chlorophyll b.     

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.     

Ascorbate-independent carotenoid de-epoxidation in intact spinach chloroplasts

Slow (> 1 s) light-induced absorbance changes in the 475–530 nm spectral region were examined in Type A chloroplasts from spinach. The most prominent absorption change occurred at 505 nm. The difference spectrum for this light-induced increase, its absence in osmotically shocked chloroplasts and restoration by ascorbate, and its sensitivity to dithiothreitol indicate that the absorption change is due to carotenoid de-epoxidation. The reaction in intact chloroplasts is characterized by its independence of exogenous ascorbate and a rate constant 3- to 8-fold higher than that reported previously for chloroplasts supplemented with ascorbate.The relevance of carotenoid de-epoxidation to other photosynthetic processes was examined by comparing their sensitivities to dithiothreitol. Levels of dithiothreitol that eliminate the 505 nm shift are without effect on saturated rates of CO₂ fixation and do not appreciably inhibit fluorescence quenching. We conclude that carotenoid de-epoxidation is not directly involved in the reactions of photosynthesis or in the regulation of excitation allocation between the photosystems.     

Light- and chemically-induced oxidation-reduction reactions in chromatophore fractions of Rhodospirillum rubrum

Chromatophore fractions of Rhodospirillum rubrum were prepared by centrifugation of a ‘classical’ chromatophore preparation in a sucrose density gradient. Most of the experiments were carried out with a fraction separating out at about 23% sucrose. Although no light-induced reactions of cytochromes could be detected, the presence of two bound cytochromes, C422 and C428, in this fraction was indicated by oxidation-reduction reactions induced by KIO₄ and dithionite. Cytochrome C422 appeared to be the ‘high-potential’ cytochrome detected earlier in preparations from this organism. Titration of chemically-induced absorbance changes due to a reduction of oxidized cytochrome C428 has led to an estimated redox midpoint potential of E_m7.5 = −0.01 V for this component in a four-electron transport reaction.

Light-induced absorbance changes in the near-infrared spectral region measured in the light chromatophore fraction at different environmental redox potentials suggested a bacteriochlorophyll component, P', with spectral characteristics which were different from those of the high-potential bacteriochlorophyll component P890. The spectral shifts observed upon oxidation, either by light or by air were a bleaching in the 860–880-nm spectral region and an appearance of an absorption band at about 900 nm. The component could be oxidized and reduced reversibly and had an estimated midpoint potential of +0.06 V in a one-electron reaction.     

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.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor.

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550-650 and 700-950nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as deltaav/deltaah, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm , and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the "special pair" that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the "light-dark" difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

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

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360–410 mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores.

The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C₃₄₀) which has an oxidation-reduction midpoint potential close to 340 mV (n = 1) at pH 6.0–7.6. The E_m of this component varies by 60 mV/pH unit between pH 7.6 and 9.2.

The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C₃₄₀.     

Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl2, have been measured in the wavelength region 460-540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCL concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of p-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

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).     

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.     

Electrochromic absorbance changes in the chlorophyll-c-containing alga Pleurochloris meiringensis (Xanthophyceae)

Flash-induced absorbance changes were measured in the Chl-c-containing alga Pleurochloris meiringensis (Xanthophyceae) between 430 and 570 nm. In addition to the bands originating from redox changes of cytochromes, three major positive and tow negative transient bands were observed both 0.7 and 20 ms after the exciting flash. These transient bands peaking at 520, 480 and 451 nm and 497 and 465 nm, respectively, could be assigned to an almost homogeneous shift of the absorbance bands with maxima at 506, 473 and 444 nm, respectively. The shape of the absorbance transients elicited from PS I or PS II was identical, and the two photosystems contributed nearly equally to the absorbance changes. Furthermore, the decay transients were sensitive to the preillumination of the cells. These data strongly suggest that the absorbance transients originate from an electrochromic response of carotenoid molecules. The pigment species responsible for the 506 nm absorption band, probably heteroxanthin or diatoxanthin, transferred excitation energy to both photosystems as shown by the aid of 77 K fluorescence excitation spectra.Abbreviation LHC light-harvesting complex     

Shifts of the bacteriochlorophyll absorption band at 880 nm in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum]

The redox potential dependency of the light-induced absorption changes of bacteriochlorophyll in the chromatophores and subchromatophore particles from Rhodospirillum rubrum has been studied. The highest values of the absorption changes due to the bleaching of P870 and the blue shift of P800 are observed within the redox potential range of 360--410. At the potential values below 300 mV the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in the subchromatophore particles and to longer wavelengths in the chromatophores. Redox titration revealed that the red and blue shifts of 880 nm bacteriochlorophyll band are caused by the functioning of a non-identified component (X) which has an oxidation -- reduction midpoint potential close to 340 mV (n = 1) within the pH range of 6,0--7,6. The Em for this component decreases by 60 mV/pH unit within the pH range of 7.6--9,2. The results obtained suggest that the red shift is due to the transmembrane, while the blue shift -- to the local intramembrane electric field. The generation of both the transmembrane and local intramembrane electric fields apparently depends on redox transitions of the component X.