Orientation of reaction center and antenna chromophores in the photosynthetic membrane of Rhodopseudomonas viridis.
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Biomedical subjects
Publications and source records attributed to J Breton.
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A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.
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The relative orientation of the pigments of reaction centers from Rhodopseudomonas sphaeroides has been studied by the photoselection technique. A high value (+0.45) of p=(delta AV--delta AH)/(delta AV + delta AH) is obtained when exciting and observing within the 870 nm band which is contradictory to the results of Mar and Gingras (Mar, T. and Gringras, G. (1976) Biochim. Biophys. Acta 440, 609-621) and Shuvalov et al. (Shuvalov, V.A., Asadov, A.A. and Krakhmaleva, I.N. (1977) FEBS Lett. 16, 240-245). It is shown that the low values of p obtained by both groups were erroneous due to excitation conditions. Analysis of the polarization of light-induced changes when exciting with polarized light in single transitions (spheroiden band and bacteriopheophytin Qx bands) enable us to propose a possible arrangement of the pigments within the reaction center. It is concluded that the 870 nm band corresponds to a single transition and is one of the two bands of the primary electron donor (P-870). The second band of the bacteriochlorophyll dimer is centered at 805 nm. The Qx transitions of the molecules constituting the bacteriochlorophyll dimer are nearly parallel (angle less than 25 degrees). The two bacteriopheophytin molecules present slightly different absorption spectra in the near infra-red. Both bacteriopheophytin absorption bands are subject to a small shift under illumination. The angle between the Qy bacteriopheophytin transitions is 55 degrees or 125 degrees. Both Qy transitions are nearly perpendicular to the 870 nm absorption band. Finally, the carotenoid molecules makes an angle greater than 70 degrees with the 870 nm band and the other bacteriochlorophyll molecules.
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Studies of the fluorescence quantum yield and decay times, determined at the emission maxima of 685 and 735 nm, using picosecond laser pulses for excitation, indicate that the pigments which are responsible for the 735 nm emission derive their energy by transfer of singlet excitons from the light-harvesting pigments and not by direct absorption of photons. Microsecond pulse laser studies of the fluorescence quantum yields at these two fluorescence wavelengths indicate that long lived quenchers (most probably triplet states), which quench singlet excitions, accumulate preferentially within the long wavelength pigment system which gives rise to the 735 nm emission band.
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 delta A 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 delta A spectrum, shifts in the position of the extrema are observed and the two features with available data on the dichroism of the Stark effect on monomolecular hypothesis of the electrochromic nature of these absorbance changes in vivo. The data on this electrochromic effect can be correlated with the linear dichorism of oriented chloroplasts and the (see article) 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.
The light induced transient absorbance changes associated with the trap of photosystem I have been studied using magnetically oriented spinach chloroplasts and a polarized measuring beam. The deltaA spectra for the two polarization parallel and perpendicular to the plane of the photosynthetic membranes have been recorded in the spectral range 630-850 nm. A dichroic ratio greater than two is observed both in the main band around 700 nm and in the radical cation band around 810 nm, leading to the conclusion that the far-red transition moment of the P-700 dimeric species is lying almost parallel to the membrane plane. Dichroic ratios smaller than one are reported in the 650-670 nm band of the delta A spectrum. The possible attribution of this band to excitonic interactions in the dimers favors the hypothesis of a tilting out of the membrane plane of this transition. This finding ruled out an orientation parallel to the membrane plane of the two chlorophyll molecules constituting the P-700 phototrap. A small residual transient absorbance change is observed in the absence artificial electron acceptor. Its spectrum shows significant differences as compared to the normal P-700 spectrum: the magnitude of the signal at 700 nm is only 15-25% of the normal signal, the half-band width of the band around 700 nm is nearly twice as large and the dichroic ratio in the band is only 1.5 +/- 0.1. In the presence of ferricyanide, this signal is still observed both for intact and osmotically broken chloroplasts, suggesting a heterogeneity in the population of traps in Photosystem I.
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The effects on the optical properties of photosynthetic membranes caused by several types of chlorophyll differing in resonance frequency and in spatial disposition are theoretically analyzed. Using a method of moments and the linear dichroism spectrum of the lamellae, we evaluated the mean angle (phi) between the transition moment of each chlorophyll and the normal to the lamellae. We have confirmed that at about 695 nm the transition moment is in the plane of the lamellae, and outside it for chlorophyll b (phi approximately 48.6 degrees). By integrating over frequency the absorption variations affected by ionophores, we show that they may be ascribed to a Stark effect, and we analyze the dependence of this effect on the orientation of the chlorophylls. From this dependence and the degree of polarization of the Stark effect, we calculate the spatial fluctuations of the angle phi. The calculation shows that a definite value of phi corresponds to each resonance frequency of chlorophyl a found in vivo. This proves that the chlorophylls a are not oriented partly random. For chlorophylls b, on the other hand, phi may fluctuate by some 10 degrees about its mean value. The structural consequences of these results are discussed.
The fluorescence yield (F) of spinach chloroplasts at 100 degrees K measured at 735 nm (photosystem I fluorescence-F 735) and at 685 nm (photosystem II fluorescence-F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-mus pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10(15) to 10(18) photons/cm(2) per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.
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The degree of fluorescence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400-680 nm) and emission wavelengths (675-750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, PIN and PAN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization PIN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Qy (approximately 660-680 nm) absorption band of chlorophyll in vivo. With 670-680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts PIN is shown to be +5+/-1%. The excitation wavelength dependence studies of PIN indicate that the carotenoid and chlorophyll Qy transition moments tends to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).
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A technique is described for the preparation of oriented samples from spinach chloroplasts whose linear dichroism is then studied by (flash) absorption spectroscopy. The chloroplasts are suspended in a glycerol-containing medium, oriented in a magnetic field, and slowly cooled in the magnet until the medium is rigid enough to avoid disorientation effects. The absorption spectra in polarized light have been measured at -50 degrees and -170 degrees C. They allow the orientation of chlorophyll b to be resolved, and the red transition moment is found to be tilted out of the membrane plane. A study of the flash-induced absorption changes linked to Photosystem-1 activity reveals a progressive evolution of the difference spectra and of the linear dichroism with decreasing temperatures. At -170 degrees C, the difference spectrum of P700 in the red is well resolved. All transition moments are found to be largely parallel to the membrane plane. The potential use of the technique for other experiments by differential absorption spectroscopy and by EPR techniques is discussed.
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