Limitations of Linear Dichroism Spectroscopy for Elucidating Structural Issues of Light-Harvesting Aggregates in Chlorosomes.

Linear dichroism (LD) spectroscopy is a widely used technique for studying the mutual orientation of the transition-dipole moments of the electronically excited states of molecular aggregates. Often the method is applied to aggregates where detailed information about the geometrical arrangement of the monomers is lacking. However, for complex molecular assemblies where the monomers are assembled hierarchically in tiers of supramolecular structural elements, the method cannot extract well-founded information about the monomer arrangement. Here we discuss this difficulty on the example of chlorosomes, which are the light-harvesting aggregates of photosynthetic green-(non) sulfur bacteria. Chlorosomes consist of hundreds of thousands of bacteriochlorophyll molecules that self-assemble into secondary structural elements of curved lamellar or cylindrical morphology. We exploit data from polarization-resolved fluorescence-excitation spectroscopy performed on single chlorosomes for reconstructing the corresponding LD spectra. This reveals that LD spectroscopy is not suited for benchmarking structural models in particular for complex hierarchically organized molecular supramolecular assemblies.

Structural Variations in Chlorosomes from Wild-Type and a bchQR Mutant of Chlorobaculum tepidum Revealed by Single-Molecule Spectroscopy.

Green sulfur bacteria can grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. They contain chlorosomes, perhaps the most efficient light-harvesting antenna system found in photosynthetic organisms. Chlorosomes contain supramolecular structures comprising hundreds of thousands of bacteriochlorophyll molecules, which are properly positioned with respect to one another solely by self-assembly and not by using a protein scaffold as a template for directing the mutual arrangement of the monomers. These two features-high efficiency and self-assembly-have attracted considerable attention for developing light-harvesting systems for artificial photosynthesis. However, reflecting the heterogeneity of the natural system, detailed structural information at atomic resolution of the molecular aggregates is not yet available. Here, we compare the results for chlorosomes from the wild type and two mutants of Chlorobaculum tepidum obtained by polarization-resolved, single-particle fluorescence-excitation spectroscopy and theoretical modeling with results previously obtained from nuclear-magnetic resonance spectroscopy and cryo-electron microscopy. Only the combination of information obtained from all of these techniques allows for an unambiguous description of the molecular packing of bacteriochlorophylls within chlorosomes. In contrast to some suggestions in the literature, we find that, for the chlorosomes from the wild type as well as for those from mutants, the dominant secondary structural element features tubular symmetry following a very similar construction principle. Moreover, the results suggest that 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 exploited by nature to achieve improved spectral coverage at the level of individual chlorosomes.

Structure of Light-Harvesting Aggregates in Individual Chlorosomes.

Among all photosynthetic organisms, green bacteria have evolved one of the most efficient light-harvesting antenna, the chlorosome, that contains hundreds of thousands of bacteriochlorophyll molecules, allowing these bacteria to grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. In contrast to other photosynthetic light-harvesting antenna systems, for which a protein scaffold imposes the proper positioning of the chromophores with respect to each other, in chlorosomes, this is accomplished solely by self-assembly. This has aroused enormous interest in the structure-function relations of these assemblies, as they can serve as blueprints for artificial light harvesting systems. In spite of these efforts, conclusive structural information is not available yet, reflecting the sample heterogeneity inherent to the natural system. Here we combine mutagenesis, polarization-resolved single-particle fluorescence-excitation spectroscopy, cryo-electron microscopy, and theoretical modeling to study the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum. We demonstrate that only the combination of these techniques yields unambiguous information on the structure of the bacteriochlorophyll aggregates within the chlorosomes. Moreover, we provide a quantitative estimate of the curvature variation of these aggregates that explains ongoing debates concerning the chlorosome structure.