Jakub Pšenčík

Two-Dimensional Electronic Spectroscopy Reveals Ultrafast Energy Diffusion in Chlorosomes.

Chlorosomes are light-harvesting antennae that enable exceptionally efficient light energy capture and excitation transfer. They are found in certain photosynthetic bacteria, some of which live in extremely low-light environments. In this work, chlorosomes from the green sulfur bacterium Chlorobaculum tepidum were studied by coherent electronic two-dimensional (2D) spectroscopy. Previously uncharacterized ultrafast energy transfer dynamics were followed, appearing as evolution of the 2D spectral line-shape during the first 200 fs after excitation. Observed initial energy flow through the chlorosome is well explained by effective exciton diffusion on a sub-100 fs time scale, which assures efficiency and robustness of the process. The ultrafast incoherent diffusion-like behavior of the excitons points to a disordered energy landscape in the chlorosome, which leads to a rapid loss of excitonic coherences between its structural subunits. This disorder prevents observation of excitonic coherences in the experimental data and implies that the chlorosome as a whole does not function as a coherent light-harvester.

Excitation Energy Transfer Dynamics and Excited-State Structure in Chlorosomes of Chlorobium phaeobacteroides

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 10(11) photons x pulse(-1) x cm(-2) were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (approximately 200-500 fs), manifesting itself as a rise in the red part of the Q(y) absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl e-->BChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10-20 ps) whereas it decays more rapidly in the BChl a region ( approximately 1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.

Structure of Chlorosomes from the Green Filamentous Bacterium Chloroflexus aurantiacus

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

In situ mapping of the energy flow through the entire photosynthetic apparatus.

Absorption of sunlight is the first step in photosynthesis, which provides energy for the vast majority of organisms on Earth. The primary processes of photosynthesis have been studied extensively in isolated light-harvesting complexes and reaction centres, however, to understand fully the way in which organisms capture light it is crucial to also reveal the functional relationships between the individual complexes. Here we report the use of two-dimensional electronic spectroscopy to track directly the excitation-energy flow through the entire photosynthetic system of green sulfur bacteria. We unravel the functional organization of individual complexes in the photosynthetic unit and show that, whereas energy is transferred within subunits on a timescale of subpicoseconds to a few picoseconds, across the complexes the energy flows at a timescale of tens of picoseconds. Thus, we demonstrate that the bottleneck of energy transfer is between the constituents.

Effect of Carotenoid Biosynthesis Inhibition on the Chlorosome Organization in Chlorobium phaeobacteroides Strain CL1401

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid‐depleted chlorosomes were obtained by means of 2‐hydroxybiphenyl–supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and β‐Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30–40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid‐depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid‐depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady‐state fluorescence emission anisotropy in the BChl e region dropped from ∼0.24 for native chlorosomes to ∼0.14 for carotenoid‐depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid‐depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid‐depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

Phosphorescence of singlet oxygen and meso-tetra(4-sulfonatophenyl)porphin: time and spectral resolved study

Highly sensitive spectroscopic experimental setup was built to detect infrared luminescence with both time and spectral resolution. In this contribution, phosphorescence of meso-tetra(4-sulphonatophenyl)porphin, which is used as a photosensitizer for photodynamic therapy of cancer, and emission of singlet oxygen were studied. Two-dimensional matrices of data (counts as a function of time and wavelength) were obtained. From these matrices, 1.7 μs lifetime of the photosensitizer triplet state and the same rise-time of the singlet oxygen emission were resolved. Further, 3.7 μs lifetime of singlet oxygen phosphorescence was determined.

Effect of Carotenoids and Monogalactosyl Diglyceride on Bacteriochlorophyll c Aggregates in Aqueous Buffer: Implications for the Self-assembly of Chlorosomes¶

Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light‐harvesting complex of green bacteria, has been studied in aqueous buffer. Unlike other chlorophyll‐like molecules, BChl c is rather soluble in aqueous buffer, forming dimers. When BChl c is mixed with carotenoids (Car), the BChl c Qy transition is further redshifted, in respect to that of monomers and dimers. The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c. The redshift of the BChl c Qy band is proportional to the Car concentration. In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the nonpolar esterifying alcohol, does not form aggregates with Car in aqueous buffer or nonpolar solvents. Instead, the position of the BChlide c Qy transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer. Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car. The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the nonpolar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes.

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S1 state was determined to be ∼10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S2 state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Qy band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm−1 and |E|=0.0039 cm−1 (chlorobactene), and |D|=0.0355 cm−1 and |E|=0.0039 cm−1 (isorenieratene). In the intermediate frequency range 300–1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm−1 and |E|=0.0050 cm−1.

The lamellar spacing in self-assembling bacteriochlorophyll aggregates is proportional to the length of the esterifying alcohol

Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In order to gain insight into their structure we examined hexane-induced aggregates of purified bacteriochlorophyll c by X-ray scattering. The bacteriochlorophyll c aggregates exhibit scattering features that are virtually identical to those of native chlorosomes demonstrating that the self-assembly of these pigments is fully encoded in their chemical structure. Thus, the hexane-induced aggregates constitute an excellent model to study the effects of chemical structure on assembly. Using bacteriochlorophyllides transesterified with different alcohols we have established a linear relationship between the esterifying alcohol length and the lamellar spacing. The results provide a structural basis for lamellar spacing variability observed for native chlorosomes from different species. A plausible physiological role of this variability is discussed. The X-ray scattering also confirmed the assignments of peaks, which arise from the crystalline baseplate in the native chlorosomes.