title: Spectroscopy of individual chlorosomes from Chlorobaculum tepidum
Creators: name: Günther, Lisa M.
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: Spectroscopy
subject: Chlorosomes
subject: light-harvesting
subject: Chlorobaculum tepidum
subject: 500
subject: 530

description: Green sulfur bacteria survive under the lowest light conditions. They have perfectly adapted to this environment by evolving a unique light-harvesting apparatus, the chlorosome. This highly efficient antenna structure allows these bacteria to grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. The chlorosome is considered as a sack that accommodates hundreds of thousands bacteriochlorophyll (BChl) c, d or e molecules depending on the bacterial species and growth conditions. The BChls build supramolecular assemblies solely by self-assembly. This is in contrast to other photosynthetic light-harvesting antenna systems, for which a protein scaffold imposes the proper positioning of the chromophores. The electronically excited states of these molecular assemblies can be described as Frenkel excitons whose photophysical properties depend crucially on the mutual arrangement of the pigments. The high light-harvesting efficiency of the chlorosomes and the self-assembly of the BChls into secondary structures within the chlorosome has promoted enormous interest in the structure-function relations of these molecular assemblies, as they can serve as blueprints for artificial light-harvesting systems. However, details about their structural organization of the supramolecular BChl arrangement in the chlorosomes are the subject of an ongoing debate, and conclusive structural information at atomic resolution is not available yet. Several models for their secondary structures, including tubular and lamellar aggregates, have been proposed to date. This variety reflects the sample heterogeneity inherent to this natural system. The photophysical properties and their relation to the supramolecular organization of the chlorosome become accessible by optical spectroscopy. Therefore, in this thesis, spectroscopic techniques were used to study the structure-function relationship of the secondary structures in chlorosomes. However, the great heterogeneity of the samples leads to inhomogeneous broadening of the spectra, and subtle spectral features that might be characteristic for specific structural properties get obscured by ensemble averaging. In order to minimize the inherent sample heterogeneity polarization-resolved fluorescence-excitation spectroscopy was performed on individual chlorosomes from the photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum. This approach makes it possible to reveal the properties of the exciton states without ensemble averaging. Furthermore, this technique was combined with mutagenesis. Hence, chlorosomes from the wild type and two mutants of Cba. tepidum were studied, in particular the bchR single mutant and the bchQR double mutant. In these mutants, the various options for methylation of the bacteriochlorophyll molecules, which are a primary source of the structural and spectral heterogeneity of wild type chlorosome samples, are reduced via the deactivation of distinct genes. The spectra of all studied chlorosomes show a strong intensity modulation as a function of the polarization of the incident radiation, which clearly indicates that collective excitations dominate the spectrum. A global analysis of the spectra revealed that the broad ensemble absorption band around 740 nm consists of several spectral contributions. The observed photophysical properties of individual chlorosomes are consistent with cylindrical supramolecular structures. Here, the information obtained from polarization-resolved fluorescence-excitation spectroscopy, cryo-electron microscopy, and theoretical modeling is combined with results previously obtained from nuclear magnetic resonance spectroscopy and cryo-electron microscopy. It is demonstrated that only the combination of these techniques yields an unambiguous description of the molecular packing of bacteriochlorophylls in chlorosomes. The results of these techniques show that in contrast to some suggestions in the literature, for the chlorosomes from the wild type as well as for those from mutants, the dominant secondary structural element features tubular symmetry. Moreover, the secondary structures of all three species follow a very similar construction principle, which is rolling up the lattice structure reported in Ganapathy et al. 2009 under an angle δ onto a cylinder. The experimental results are compared with predictions from computer simulations of the cylindrical model structure. Studying variations of the structural parameters yields an explanation about the inhomogeneously broadened absorption bands. Moreover, the simulations provide a quantitative estimate of the curvature variation of these aggregates that might explain the ongoing debates concerning the chlorosome structure. From the correlation of spectral parameters, speculations about the degree of variation of the structural elements are made, and the composition of individual chlorosomes is deduced. Finally, a combination of all the results gives a conclusive picture of the interior of the chlorosomes.