Estimation of the bacteriochlorophyll c oligomerisation extent in Chloroflexus aurantiacus chlorosomes by very low-frequency vibrations of the pigment molecules: A new approach.

In green photosynthetic bacteria, the chlorosomal bacteriochlorophyll molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. For the first time, we demonstrated by coherent femtosecond spectroscopy at cryogenic temperature that the very low-frequency (~101 cm-1) vibrations of bacteriochlorophyll c pigments in isolated Chloroflexus aurantiacus chlorosomes are sensitive to their oligomerisation extent which depends on the light intensity during the growth of the cell cultures. We explained this sensitivity in terms of the coupling of delocalised vibration modes of BChl c molecules aggregated into chains within their antenna unit building blocks. These findings, together with previously obtained spectroscopy and microscopy data, confirmed that the unit building blocks functioning within Chloroflexus aurantiacus chlorosomal antenna are built up from the rather short (2-5 BChl c pigments) quasi-linear chains. The approach presented here seems to be perspective since it directly reveals structural and dynamical properties of the oligomeric systems.

Size variability of the unit building block of peripheral light-harvesting antennas as a strategy for effective functioning of antennas of variable size that is controlled in vivo by light intensity.

This work continuous a series of studies devoted to discovering principles of organization of natural antennas in photosynthetic microorganisms that generate in vivo large and highly effective light-harvesting structures. The largest antenna is observed in green photosynthesizing bacteria, which are able to grow over a wide range of light intensities and adapt to low intensities by increasing of size of peripheral BChl c/d/e antenna. However, increasing antenna size must inevitably cause structural changes needed to maintain high efficiency of its functioning. Our model calculations have demonstrated that aggregation of the light-harvesting antenna pigments represents one of the universal structural factors that optimize functioning of any antenna and manage antenna efficiency. If the degree of aggregation of antenna pigments is a variable parameter, then efficiency of the antenna increases with increasing size of a single aggregate of the antenna. This means that change in degree of pigment aggregation controlled by light-harvesting antenna size is biologically expedient. We showed in our previous work on the oligomeric chlorosomal BChl c superantenna of green bacteria of the Chloroflexaceae family that this principle of optimization of variable antenna structure, whose size is controlled by light intensity during growth of bacteria, is actually realized in vivo. Studies of this phenomenon are continued in the present work, expanding the number of studied biological materials and investigating optical linear and nonlinear spectra of chlorosomes having different structures. We show for oligomeric chlorosomal superantennas of green bacteria (from two different families, Chloroflexaceae and Oscillochloridaceae) that a single BChl c aggregate is of small size, and the degree of BChl c aggregation is a variable parameter, which is controlled by the size of the entire BChl c superantenna, and the latter, in turn, is controlled by light intensity in the course of cell culture growth.

[Search for an optimal orientational ordering of Qy transition dipoles of subantennae molecules in superantenna of photosynthetic green bacteria. Model calculations].

This work continues a series of our investigations on efficient strategies of functioning of natural light-harvesting antennae, initiated by our concept of rigorous optimization of photosynthetic apparatus structure by functional criterion. Using computer modeling for the functioning of the natural antennae, we suggested some basic principles for designing optimal model systems. Targeted searches for these principles in in vivo systems allowed us to recognize some of them in natural antennae. This work deals with the problem of the structure optimization of nonuniform superantennae of photosynthetic green bacteria. These superantennae consist of several uniform subantennae which produces a problem of their optimal coordination. In this work, we used mathematical modeling for the functioning of these natural superantennae to consider a possible way to optimize the superantenna structure using optimization of mutual spatial orientation of Qy transition dipoles of subantennae pigments. This allowed us to determine some modes of optimal orientational ordering of Qy transition dipoles of subantennae pigments in the model of the green bacterium Chloroflexus aurantiacus superantenna. It was shown that the optimal mutual orientation of Qy transition dipoles of subantennae pigments (resulting in stable minimizing of the energy transfer time within the superantenna and, as a consequence, in decrease in energy losses) ensures the high efficiency and stability of the superatenna functioning.

[Analysis of spectral conjugation of nonuniform subantennae in the light-harvesting superantenna of Oscillochloridaceae photosynthetic green bacteria].

The study is concerned with the problem of optimal spectral coupling of subantennal as a strategy of effective functioning of light-harvesting antennal of photosynthesizing organisms. A theoretical analysis of the optimality of spectral coupling of currently known spectrally inhomogeneous subantennal (B750, B805-860) in the superantenna of green bacteria Oscillochloris trichoides (from the new family Oscillochloridaceal discovered by Russian researchers in 2000), performed in the study, showed that the spectral composition of subantennal is functionally nonoptimal. This made it possible to predict the occurrence of an additional subantenna (B(x)) with an intermediate energy position (750 nm < X < 805 nm) for the optimization of energy transfer along this superantenna by the functional criterion.

Optimal spectral coordination of subantennae in natural antennae as an efficient strategy for light harvesting in photosynthesis.

This work continues a series of our investigations on efficient strategies of functioning of natural light-harvesting antennae, initiated by a concept of rigorous optimization of photosynthetic apparatus by functional criterion, and deals with the problem of an optimal spectral coordination of subantennae in photosynthetic superantenna of the green bacterium Oscillochloris trichoides from a new family of green bacteria Oscillochloridaceae based in 2000. At present, two subantennae were identified surely: chlorosomal BChl c subantenna B750 and membrane BChl a subantennae B805-860. Some indirect experiments indicated on the presence of minor amounts of BChl a in isolated chlorosomes which allowed us to propose on the existence of an intermediate-energy subantenna which can connect the chlorosomal BChl c and the membrane BChl a ones. However, in the absorption spectra of isolated chlorosomes, this BChl a subantenna was not visually identified. This promoted us to perform a theoretical analysis of the optimality of spectral coordination of Oscillochloris trichoides subantennae. Using mathematical modeling for the functioning of the natural superantenna, we showed that an intermediate-energy subantenna, connecting B750 and B805-860 ones, allows one to control superantenna efficiency, i.e. to optimize the excitation energy transfer from B750 to B805 by functional criterion, and hence, the existence of such intermediate-energy subantenna is biologically expedient.

[A comparative study of the fluorescence properties of the chlorosomal antenna of the green bacterium from the family Oscillochloridaceae and the members from two other families of green bacteria].

The fluorescence properties of bacteriochlorophylls (BChl) of the chlorosomal light-harvesting antenna of Oscillochloris trichoides (strain DG-6) from a new family of green filamentous bacteria Oscillochloridaceae were investigated in comparison with green bacteria from two other families. A strong dependence of the fluorescence intensity of chlorosomal bacteriochlorophyll c of Osc. trichoides on the redox potential of medium was found, which previously was observed only in green sulfur bacteria. The presence of BChl a in chlorosomes did not appear in their absorption spectra but was visualized by fluorescence spectroscopy at 77 K. From the comparative analysis of fluorescence spectral data for the chlorosomal light-harvesting antenna of Osc. trichoides and similar spectral data for green bacteria from two other families, it was concluded that, in some fluorescence spectral features (spectral position of bacteriochlorophyll c/a fluorescence bands; shape and full width at half maximum fluorescence band of chlorosomal bacteriochlorophyll c; the Stokes shift value of bacteriochlorophyll c band; a high molar ratio of bacteriochlorophyll c : bacteriochlorophyll a in chlorosomes that makes the bacteriochlorophyll a fluorescence band unresolved at room temperature; and highly redox-dependent fluorescence intensity of chlorosomal bacteriochlorophyll c), Osc. trichoides chlorosomes are close to the chlorosomal antenna of Chlorobiaceae species.

[A study of the content of pigments in the light-harvesting antenna of the green bacterium from the new family Oscillochloridaceae]. / Izuchenie sostava pigmentov svetosobiraiushchei antenny zelenykh bakterii novogo semeistva Oscillochloridaceae.

The properties of the light-harvesting superantenna of the photosynthesizing bacteria from the new family of green filamentous bacteria Oscillochloridaceae were investigated by optical spectroscopy. The antenna of Oscillochloris trichoides consists of peripheral chlorosomal and membrane subantennas. A method of isolation of Osc. trichoides chlorosomal antenna was developed using the chaothropic agent sodium thiocyanate, which simultaneously acts to stabilize chlorosomal activity. An analysis of the second derivatives of the absorption spectra of isolated chlorosomes and their acetone-methanol extracts suggested that BChl c was a predominant light-harvesting pigment in Osc. trichoides chlorosomes. Besides, it was found that, in addition to the BChl c-antenna, chlorosomes contain a minor BChl a-antenna. It was shown that the membrane BChl a-subantenna is a light-harvesting complex with absorption maxima in the near infrared region at 805 and 860 nm. Analysis of the spectral data obtained suggested that the Osc. trichoides chlorosomal antenna resembles those from Chlorobiaceae species, whereas the membrane B805-860 BChl a antenna of Osc. trichoides is close to the membrane B808-866 BChl a antenna of Chloroflexaceae species.

[Survival strategy of photosynthetic organisms. 1. Variability of the extent of light-harvesting pigment aggregation as a structural factor optimizing the function of oligomeric photosynthetic antenna. Model calculations]. / Strategiia vyzhivaniia fotosinteziruiushchikh organizmov. 1. Variabel'nost' stepeni agregatsii svetosobiraiushchikh pigmentov kak strukturnyi faktor, optimiziruiushchii funktsiiu oligomernoi fotosinteticheskoi antenny. Model'nye raschety.

In accordance with our concept of rigorous optimization of photosynthetic machinery by a functional criterion, this series of papers continues purposeful search in natural photosynthetic units (PSU) for the basic principles of their organization that we predicted theoretically for optimal model light-harvesting systems. This approach allowed us to determine the basic principles for the organization of a PSU of any fixed size. This series of papers deals with the problem of structural optimization of light-harvesting antenna of variable size controlled in vivo by the light intensity during the growth of organisms, which accentuates the problem of antenna structure optimization because optimization requirements become more stringent as the PSU increases in size. In this work, using mathematical modeling for the functioning of natural PSUs, we have shown that the aggregation of pigments of model light-harvesting antenna, being one of universal optimizing factors, furthermore allows controlling the antenna efficiency if the extent of pigment aggregation is a variable parameter. In this case, the efficiency of antenna increases with the size of the elementary antenna aggregate, thus ensuring the high efficiency of the PSU irrespective of its size; i.e., variation in the extent of pigment aggregation controlled by the size of light-harvesting antenna is biologically expedient.

[Survival strategy of photosynthetic organisms. 2. Experimental proof of the size variability of the unit building block of light-harvesting oligomeric antenna]. / Strategiia vyzhivaniia fotosinteziruiushchikh organizmov. 2. Eksperimetnal'noe dokazatel'stvo variabel'nosti razmera edinichnogo stroitel'nogo bloka oligomernoi antenny.

The present series of papers is part of an integrated research program to understand the effective functional strategy of native light-harvesting molecular antennae in photosynthetic organisms. This work tackles the problem of the structural optimization of light-harvesting antennae of variable size. In vivo, the size responds to the illumination intensity, thus implying more sophisticated optimization strategies, since larger antenna size demands finer structural tuning. Earlier modeling experiments showed that the aggregation of the antenna pigments, apart from being itself a universal structural factor of functional antenna optimization with any (!) spatial lattice of light-harvesting molecules, determines the antenna performance provided that the degree of aggregation varies: the larger the unit building block, the higher the efficacy of the whole structure. It means that altering the degree of pigment aggregation in response to the antenna size is biologically expedient. In the case of the oligomeric chlorosomal antenna of green bacteria, the strategy of variable antenna structural optimization in response to the illumination intensity was demonstrated to take place in vivo and facilitate high antenna performance regardless of its size, thus allowing bacteria to survive in diverse illumination conditions.

[Model of aggregation of pigments in the chlorosomal antenna of the green bacteria Chloroflexus aurantiacus]. / Model' agregatsii pigmentov v khlorosomnoi antenne zelenykh bakterii Chloroflexus aurantiacus.

Independent experimental and theoretical evaluation was performed for the adequacy of our previously proposed general molecular model of structural organization of light-harvesting pigments in chlorosomal bacteriochlorophyll (BChl) c/d/e-containing superantenna of different green bacteria. Simultaneous measurement of hole burning in the optical spectra of chlorosomal BChl c and temperature dependence of steady-state fluorescence spectra of BChl c was accomplished in intact cells of photosynthetic green bacterium Chloroflexus aurantiacus; this allows unambiguous determination of the structure of exciton levels of BChl c oligomers in this natural antenna, which is a fundamental criterion for adequacy of any molecular model for in vivo aggregation of antenna pigments. Experimental data were shown to confirm our model of organization of oligometric pigments in chlorosomal BChl c antenna of green bacterium Chloroflexus aurantiacus. This model, which is based on experimental data and our theory of spectroscopy of oligomeric pigments, implies that the unit building block of BChl c antenna is a cylindrical assembly containing six excitonically coupled linear pigment chains whose exciton structure with intense upper levels provides for the optimal spectral properties of the light-harvesting antenna.

Light control over the size of an antenna unit building block as an efficient strategy for light harvesting in photosynthesis.

It was shown that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon lowering growth light intensities led to enhancement of the hyperchromism of the BChl c Q(y) absorption band of the green photosynthetic bacterium Chloroflexus aurantiacus. With femtosecond difference absorption spectroscopy, it was shown that the amplitude of bleaching of the oligomeric BChl c Q(y) band (as compared to that for monomeric BChl a) increased with increasing BChl c content in chlorosomes. This BChl c bleaching amplitude was about doubled as the chlorosomal antenna size was about trebled. Both sets of findings clearly show that a unit BChl c aggregate in the chlorosomal antenna is variable in size and governed by the grow light intensity, thus ensuring the high efficiency of energy transfer within the BChl c antenna regardless of its size.

Exciton levels structure of antenna bacteriochlorophyll c aggregates in the green bacterium Chloroflexus aurantiacus as probed by 1.8-293 K fluorescence spectroscopy.

We have demonstrated temperature-dependence of the steady-state fluorescence lineshape of the bacteriochlorophyll (BChl) c band measured for intact cells of the green bacterium Chloroflexus aurantiacus over the 1.8-293 K range. The measured temperature-dependence has been shown to be in good agreement with the theoretical one, calculated for our original model of pigment organization in the chlorosomal oligomeric antenna of green photosynthetic bacteria based on spectral hole-burning studies (Fetisova, Z.G. et al. (1996) Biophys. J. 71, 995-1010). This model implies that the BChl c antenna unit is a tubular aggregate of six exciton-coupled linear pigment chains having the exciton level structure with strongly allowed higher levels.

Exciton delocalization in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy.

A model of pigment organization in the B808-866 bacteriochlorophyll a antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed. The building block of the antenna was assumed to be structurally similar to that of the B800-850 light-harvesting 2 (LH2) antenna of purple bacteria and to have the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers, where N = 24 or 32. We have shown that the Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43 degrees +/- 3 degrees and 8 degrees +/- 4 degrees, respectively, with the plane of the corresponding rings. Using the exciton model, we have obtained a quantitative fit of the pump-probe spectra of the B866 and B808 bands. The anomalously high bleaching value of the B866 band with respect to the B808 monomeric band provided the direct evidence for a high degree of exciton delocalization in the BChl866 ring antenna. The coherence length of the steady-state exciton wave packet corresponds to five or six BChl866 molecules at room temperature.

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy.

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Qy transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7-8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.

Pigment organization and exciton dynamics in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus.

The model for the B808-866 antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed. The three-dimensional structure of the B808-866 antenna is assumed to be similar to the structure of the B800-850 antenna of purple bacteria, i.e. it has the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers. The Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43 degrees +/- 3 degrees and 8 degrees +/- 4 degrees, respectively, with the plane of the corresponding rings. The lowest limit of the BChl866 aggregate size is N = 18. The anomalously high bleaching value of the BChl866 band with respect to the monomeric BChl808 band provides evidence for a high degree of exciton delocalization. To account for this phenomenon, the exciton model describing exciton dynamics in the spectrally disordered circular BChl866 aggregate is developed. According to this model, the effective exciton size in this antenna (Neff) is 6-8.

Energy transfers in the B808-866 antenna from the green bacterium Chloroflexus aurantiacus.

Energy transfers within the B808-866 BChl a antenna in chlorosome-membrane complexes from the green photosynthetic bacterium Chloroflexus aurantiacus were studied in two-color pump-probe experiments at room temperature. The steady-state spectroscopy and protein sequence of the B808-866 complex are reminiscent of well-studied LH2 antennas from purple bacteria. B808-->B866 energy transfers occur with approximately 2 ps kinetics; this is slower by a factor of approximately 2 than B800-->B850 energy transfers in LH2 complexes from Rhodopseudomonas acidophila or Rhodobacter sphaeroides. Anisotropy studies show no evidence for intra-B808 energy transfers before the B808-->B866 step; intra-B866 processes are reflected in 350-550 fs anisotropy decays. Two-color anisotropies under 808 nm excitation suggest the presence of a B808-->B866 channel arising either from direct laser excitation of upper B866 exciton components that overlap the B808 absorption band or from excitation of B866 vibronic bands in nontotally symmetric modes.

Functioning of oligomeric-type light-harvesting antenna.

In our previous work, we developed, for the first time, a theory of excitation energy transfer within an oligomeric-type light-harvesting antenna and, in particular, within the chlorosome of green bacteria (Biophys.J., 1996, vol.71, pp.995-1010). The theory was recently developed for a new original exciton model of aggregation of chlorosomal pigments, bacteriochlorophylls (BCh1) c/d/e (Biochem, Mol.Biol.Int., 1996, vol.40, No.2, pp. 243-252). In this paper, it was demonstrated with picosecond fluorescence spectroscopy that this theory explains the antenna-size-dependent kinetics of fluorescence decay in chlorosomal antenna, measured for intact cells of different cultures of the green bacterium Chlorobium limicola with different chlorosomal antenna size determined by electron microscopic examination of the ultrathin sections of the cells. According to our model, the energy transfer dynamics within the chlorosome imply the formation of a cylindrical exciton, delocalized over a tubular aggregate of BCh1 c chains, and inductive-resonance-type transfer of such a cylindrical exciton between the nearest tubular BCh1 c aggregates and to BCh1 a of the chlorosome.