Spectral dynamics of individual bacterial light-harvesting complexes: alternative disorder model

The bacterial (Rhodopseudomonas acidophila) photosynthetic peripheral light-harvesting complex of type 2 (LH2) exhibits rich fluorescence spectral dynamics at room temperature. The fluorescence spectrum of individual LH2 shifts either to the blue or to the red during the experimental observation time of a few minutes. These spectral changes are often reversible and occur between levels of a distinctly different peak wavelength. Furthermore, they are accompanied by a change of the spectral line shape. To interpret the dynamics of spectral changes, an energetic disorder model associated with easily explainable structural changes of the protein is proposed. This model assumes that each pigment in the tightly coupled ring of bacteriochlorophylls can be in two states of electronic transition energy due to the protein-pigment interaction. The transition between these structural, and hence spectroscopic, states occurs through the thermally induced conformational potential energy barrier crossing. Although simplified, the model allows us to reproduce the bulk fluorescence spectrum, the distribution of the single-molecule spectral peak wavelength and its changes, and the statistics of the duration of the spectral states. It also provides an intuitively clear picture of possible protein dynamics in LH2. At the same time, it requires additional sophistication since it essentially does not reproduce the red occurrences of single LH2 spectra.     

Chemical oxidation of the FMO antenna protein from Chlorobaculum tepidum

Effect of chemical oxidation by ferricyanide on bacteriochlorophyll a (BChl a) in the Fenna–Matthews–Olson protein (FMO) was studied using absorbance and fluorescence spectroscopy at ambient and cryogenic temperatures. Partially selective oxidation of pigments bound to the antenna complex was achieved and the probable absorption wavelength corresponding to the recently discovered bacteriochlorophyll No. 8 of 806 nm was obtained by comparative analysis of the effect of chemical oxidation and the effect of different isolation procedures. Formation of a stable product identified as a chlorophyll a derivative occurred upon chemical oxidation. This new pigment remained bound within the pigment–protein complex, and exhibited an efficient energy transfer to BChl a. Furthermore, complex effects of the pigment oxidation upon the fluorescence yield of the FMO protein were observed. Utility of this approach based on chemical modifications for the investigation of the native regulatory mechanisms involved in the energy transfer in the FMO protein is discussed.     

Optical and structural properties of chlorosomes of the photosynthetic green sulfur bacterium Chlorobium limicola

Isolated chlorosomes of the photosynthetic green sulfur bacterium Chorobium limicola upon cooling to 4 K showed, in addition to the near-infrared absorption band at 753 nm due to bacteriochlorophyll c, a weak band near 800 nm that could be attributed to bacteriochlorophyll a. The emission spectrum showed bands of bacteriochlorophyll c and a at 788 and 828 nm, respectively. The fluorescence excitation spectrum indicated a high efficiency of energy transfer from bacteriochlorophyll c to bacteriochlorophyll a. When all bacteriochlorophyll c absorption had been lost upon storage, no appreciable change in the optical properties of the bacteriochlorophyll a contained in these ‘depleted chlorosomes’ was observed. The fluorescence and absorption spectra of the chlorosomal bacteriochlorophyll a were clearly different from those of the soluble bacteriochlorophyll a protein present in these bacteria. The results provide strong evidence that bacteriochlorophyll a, although present in a small amount, is an integral constituent of the chlorosome. It presumably functions in the transfer of energy from the chlorosome to the photosynthetic membrane; its spectral properties and the orientation of its near-infrared optical transitions as determined by linear dichroism are such as to favor this energy transfer.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Chirality-based signatures of local protein environments in two-dimensional optical spectroscopy of two species photosynthetic complexes of green sulfur bacteria: simulation study

Two-dimensional electronic chirality-induced signals of excitons in the photosynthetic Fenna-Matthews-Olson complex from two species of green sulfur bacteria (Chlorobium tepidum and Prosthecochloris aestuarii) are compared. The spectra are predicted to provide sensitive probes of local protein environment of the constituent bacteriochlorophyll a chromophores and reflect electronic structure variations (site energies and couplings) of the two complexes. Pulse polarization configurations are designed that can separate the coherent and incoherent exciton dynamics contributions to the two-dimensional spectra.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q_y transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (α and β) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q_y transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 Å.     

Probing the binding sites of exchanged chlorophyll a in LH2 by Raman and site-selection fluorescence spectroscopies

In this work we have selectively released the 800 nm absorbing bacteriochlorophyll a molecules of the LH2 protein from the photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050, and replaced them with chlorophyll a (Chla). A combination of low-temperature electronic absorption, resonance Raman and site-selection fluorescence spectroscopies revealed that the Chla pigments are indeed bound in the B800 binding site; this is the first work that formally proves that such non-native chlorins can be inserted correctly into LH2.     

Isolation and properties of a pigment-protein complex associated with the reaction center of the green photosynthetic sulfur bacterium Prosthecochloris aestuarii

The membrane-bound pigment system of green sulfur bacteria consists of light-harvesting bacteriochlorophyll a-protein and a ‘core complex’ that is associated with the reaction center (Kramer, H.J.M., Kingma, H., Swarthoff, T. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 359–364). The isolation and properties of the core complex from Prosthecochloris aestuarii are described. The complex has a molecular mass of 200 ± 50 kDa and contains bacteriochlorophyll a, carotenoid and pigments absorbing near 670 nm (probably bacteriopheophytin c and an unidentified pigment). Fluorescence emission spectra and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the absence of light-harvesting bacteriochlorophyll a-protein. The preparation showed no reaction center activity. Circular and linear dichroism spectra indicated that the structure of the core complex was basically not altered by the isolation procedure. Comparison with the CD spectrum of the intrinsic membrane-bound pigment-protein complex indicates that the latter contains 14 bacteriochlorophyll a molecules (two subunits) belonging to the light-harvesting protein and about 20 bacteriochlorophyll a molecules belonging to the core complex.     

Bacteriochlorophyll a-protein interactions in a complex from Prosthecochloris aestuarii. A Resonance Raman study

Raman spectra of bacteriochlorophyll a (BChl) bound to the soluble protein complex from Prosthecochloris aestuarii have been obtained at low temperature, using the resonance effect on their Q_x for Soret electronic bands. These spectra show that the acetyl carbonyls of at least four of the seven molecules bound to the monomer subunit of the complex and the ketone carbonyls of at least five of them are oriented close to the mean plane of the conjugated part of the dihydrophorbin macrocycle. Up to three bacteriochlorophyll molecules may have their ketone carbonyls free from hydrogen-bonding and up to two may have their acetyl carbonyls similarly free. Several of the binding sites of the remaining conjugated carbonyls are probably the same as those binding the conjugated carbonyls of bacteriochlorophyll (and of bacteriopheophytin) in reaction centers and in antenna structures of purple bacteria and as those binding chlorophyll in the antenna of higher plants and algae. The present resonance Raman spectra confirm that the magnesium atoms of most of the seven bacteriochlorophylls are pentacoordinated. They also show that polarisation effects from their local environments induce changes in the groundstate structures of the dihydrophorbin skeletons of the complexed molecules with respect to those of isolated, monomeric bacteriochlorophyll. These changes are quasi-identical for the seven molecules. These environmental effects predominate over any structural change brought about by intermolecular bonding of the conjugated carbonyls or of the magnesium atoms. The dihydrophorbin rings of the seven molecules thus appear to be immersed in a nearly homogeneous medium of low permittivity, although specific van der Waals interactions may polarise the free carbonyls to quite different extents. The possible implications of these observations on the interpretation of the electronic spectrum of the set of complexed bacteriochlorophylls are discussed.     

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.     

Linear dichroism of electric field oriented bacteriochlorophyll a-protein from green photosynthetic bacteria

Bacteriochlorophyll a-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Q_y region of the bacteriochlorophyll a absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The ≈12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll a-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Q_y exciton transition observed in absorption at the same wavelength.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

Single tryptophan mutants of FtsZ: Nucleotide binding/exchange and conformational transitions

Cell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In this work, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottom protofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an intermediate that is stabilized by the nucleotide bound in the N-domain as well as by quaternary protein–protein interactions. In this context, we characterized the unfolding of the Trp mutants using time-resolved fluorescence and phasor plot analysis. A novel picture of the structural transition from the native state in the absence of denaturant, to the solvent-exposed unfolded state is presented. Taken together our results show that conformational changes between the GDP and GTP states of FtsZ, such as those observed in FtsZ unfolding, are restricted to the interaction surface between the N and C domains.     

Variable fluorescence in green sulfur bacteria

Green sulfur bacteria possess a complex photosynthetic machinery. The dominant light harvesting systems are chlorosomes, which consist of bacteriochlorophyll c, d or e oligomers with small amounts of protein. The chlorosomes are energetically coupled to the membrane-embedded iron sulfur-type reaction center via a bacteriochlorophyll a-containing baseplate protein and the Fenna-Matthews-Olson (FMO) antenna protein. The fluorescence yield and spectral properties of these photosynthetic complexes were investigated in intact cells of several species of green sulfur bacteria under physiological, anaerobic conditions. Surprisingly, green sulfur bacteria show a complex modulation of fluorescence yield upon illumination that is very similar to that observed in oxygenic phototrophs. Within a few seconds of illumination, the fluorescence reaches a maximum, which decreases within a minute of illumination to a lower steady state. Fluorescence spectroscopy reveals that the fluorescence yield during both processes is primarily modulated on the FMO-protein level, while the emission from chlorosomes remains mostly unchanged. The two most likely candidates that modulate bacteriochlorophyll fluorescence are (1) direct excitation quenching at the FMO-protein level and (2) indirect modulation of FMO-protein fluorescence by the reduction state of electron carriers that are part of the reaction center.     

Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer

Defferential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic trasitions with midpoints (Tm) at 59°C and 63°C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60°C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm=66°C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 begining at about 30°c (Tm=41°C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20°C, sensitive at 40°C and resistant when returned to 20°C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing termperature over the same range as the 41°C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 Which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.     

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl?d, Chl?f or bacteriochlorophyll (BChl) b to replace native BChl?a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg_?10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6?ps, energy transfer from Chl?a to B850 BChl?a remained highly efficient. We measured faster energy-transfer time constants for Chl?d (3.5?ps) and Chl?f (2.7?ps), which have red-shifted absorption maxima compared to Chl?a. BChl?b, red-shifted from the native BChl?a, gave extremely rapid (≤0.1?ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.     

Structure of a bacteriochlorophyll a-protein from the green photosynthetic bacterium Prosthecochloris aestuarii.

The three-dimensional structure of a water-soluble bacteriochlorophyll a-containing protein from the green photosynthetic bacterium Prosthecochloris aestuarii has been determined by X-ray crystallography from a 2.8 Å resolution electron density map based on four isomorphous derivatives. Details of the crystallographic procedures used to obtain the map are presented.The bacteriochlorophyll a-protein is shown to consist of three identical subunits, tightly packed around a 3-fold symmetry axis. Each subunit consists of a core of seven bacteriochlorophyll a molecules enclosed within a “bag” of protein. The polypeptide chain forms an extensive 15-strand β-sheet, which is almost planar in its central region, and twisted at its extremities, and wraps around the chlorophyll core to form an efficient amphipathic layer between the chlorophylls and the aqueous environment. There are extensive contacts between the phytyl chains of the seven bacteriochlorophylls within each subunit. These hydrocarbon chains constitute an inner hydrophobic core of the molecule which may be important in forming the complex. There are also extensive contacts between the protein and both the bacteriochlorophyll head groups and tails, but relatively few contacts between the respective head groups. The seven magnesiums all appear to be five co-ordinated. In five cases the presumed ligand is a histidine side-chain, in one case a polypeptide carbonyl oxygen, and in the other case a water molecule.At low temperature, both the absorption and circular dichroism spectra of the bacteriochlorophyll a-protein show splitting which can be interpreted in general terms as due to exciton interactions between the seven chromophores, but calculations of the expected splitting based on the bacteriochlorophyll co-ordinates determined crystallographically are in poor agreement with the observed spectra. Furthermore, the observed red shift of the Q_y absorption band of bacteriochlorophyll a, from about 770 nm in organic solvents to 809 nm in the bacteriochlorophyll a-protein, is not explained by the exciton calculations. It seems likely that the red shift is due to perturbations of the spectra of the individual bacteriochlorophylls by the protein environment, but, pending the determination of the amino acid sequence, it is not possible at this time to define in detail all the proteinchlorophyll interactions. It is suggested that the bacteriochlorophyll a-protein serves as a good model for the organization of chlorophyll in vivo, and that the types of interaction seen here between chlorophyll and protein are likely to be found in other chlorophyll proteins.     

Electrostatic charge controls the lowest LH1 Qy transition energy in the triply extremophilic purple phototrophic bacterium,Halorhodospira halochloris

Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile—the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Q_y absorption maximum at 1016 nm, which is the lowest transition energy among phototrophic organisms. Here we report that this extraordinarily red-shifted LH1 Q_y band of Hlr. halochloris exhibits interconvertible spectral shifts depending on the electrostatic charge distribution around the BChl b molecules. The 1016 nm band of the Hlr. halochloris LH1-RC complex was blue-shifted to 958 nm upon desalting or pH decrease but returned to its original position when supplemented with salts or pH increase. Resonance Raman analysis demonstrated that these interconvertible spectral shifts are not associated with the strength of hydrogen-bonding interactions between BChl b and LH1 polypeptides. Furthermore, circular dichroism signals for the LH1 Q_y transition of Hlr. halochloris appeared with a positive sign (as in BChl b-containing Blastochloris species) and opposite those of BChl a-containing purple bacteria, possibly due to a combined effect of slight differences in the transition dipole moments between BChl a and BChl b and in the interactions between adjacent BChls in their assembled state. Based on these findings and LH1 amino acid sequences, it is proposed that Hlr. halochloris evolved its unique and tunable light-harvesting system with electrostatic charges in order to carry out photosynthesis and thrive in its punishing hypersaline and alkaline habitat.     

Low Light Adaptation: Energy Transfer Processes in Different Types of Light Harvesting Complexes from Rhodopseudomonas palustris

Energy transfer processes in photosynthetic light harvesting 2 (LH2) complexes isolated from purple bacterium Rhodopseudomonas palustris grown at different light intensities were studied by ground state and transient absorption spectroscopy. The decomposition of ground state absorption spectra shows contributions from B800 and B850 bacteriochlorophyll (BChl) a rings, the latter component splitting into a low energy and a high energy band in samples grown under low light (LL) conditions. A spectral analysis reveals strong inhomogeneity of the B850 excitons in the LL samples that is well reproduced by an exponential-type distribution. Transient spectra show a bleach of both the low energy and high energy bands, together with the respective blue-shifted exciton-to-biexciton transitions. The different spectral evolutions were analyzed by a global fitting procedure. Energy transfer from B800 to B850 occurs in a mono-exponential process and the rate of this process is only slightly reduced in LL compared to high light samples. In LL samples, spectral relaxation of the B850 exciton follows strongly nonexponential kinetics that can be described by a reduction of the bleach of the high energy excitonic component and a red-shift of the low energetic one. We explain these spectral changes by picosecond exciton relaxation caused by a small coupling parameter of the excitonic splitting of the BChl a molecules to the surrounding bath. The splitting of exciton energy into two excitonic bands in LL complex is most probably caused by heterogenous composition of LH2 apoproteins that gives some of the BChls in the B850 ring B820-like site energies, and causes a disorder in LH2 structure.