The Structure of the RC-LH1 Complex from Rps. acidophila: Optical Single-Molecule Spectroscopy and Numerical Simulations

The topic of this thesis was the investigation of the specific arrangement of the bacteriochlorophyll (BChl) a molecules in the light-harvesting 1 (LH1) complex from Rhodopseudomonas (Rps.) acidophila. In purple bacteria the LH1 complex forms an interconnected unit with a reaction-center (RC) complex, where the LH1 complex directly surrounds the RC to form the so called RC-LH1 core complex. As part of a cyclic electron transport pathway in bacterial photosynthesis, reduced ubiquinone molecules, Q_B H_2, have to leave the RC and pass through the LH1 complex into the membrane space. By now, it is well established that for some purple bacterial species the LH1 complex has a gap, presumably to facilitate the shuttling of Q_B H_2, whereas for other species, with a completely closed LH1 ring, channels in the LH1 aggregate might enable the transfer of Q_B H_2. Before this work, it was not known to which of these two types of RC-LH1 complexes (interrupted vs. closed LH1 ring), the RC-LH1 complex from Rps. acidophila belonged, let alone an idea of the exact arrangement of the BChl a molecules in the LH1 aggregate. In the following a short summary of the results achieved in this thesis will be presented. In a first series of experiments the RC-LH1 complex from Rps. acidophila was revisited for single-molecule spectroscopy. Thereby, the pigment-protein complexes were stabilized in the relatively mild, non-denaturating detergent dodecyl-β-D-maltoside (DDM), instead of the more deactivating detergent lauryldimethylamine N-oxide (LDAO) which was used in a previous study. In total, low-temperature (1.2 K) fluorescence-excitation spectra have been recorded from 61 individual RC-LH1 complexes stabilized in DDM, in comparison to only 24 complexes measured in the precursor study. According to their spectral characteristics, the complexes were either considered as intact or broken. The fraction of broken (dissociated) RC-LH1 complexes could be reduced significantly by using DDM instead of LDAO as detergent, namely from 37.5% in the precursor work to 11.5% in this thesis. Due to the enhanced quality of the stock solution, it was possible to perform polarization-resolved fluorescence-excitation spectroscopy, and to determine the distributions of the spectral properties for the intact RC-LH1 complexes with an improved statistics. Both these distributions and characteristic spectral features, such as a narrow line occurring on the red end of the spectra for most of the complexes, show a remarkable resemblance with the data obtained on RC-LH1 complexes from Rps. palustris in an earlier study. For the Rps. palustris RC-LH1 complex a low-resolution crystal structure is available. Hence, taking also the aforementioned similarity of the spectral characteristics into account, it is concluded that the LH1 complexes from Rps. acidophila and Rps. palustris can be described by the same structural model, namely an elliptical arrangement of BChl a molecules interrupted by a gap. Thus, by successfully applying low-temperature single-molecule spectroscopy as a tool for structural investigation, it was revealed for the first time that the RC-LH1 complex from Rps. acidophila rather belongs to the RC-LH1 complexes with an interrupted LH1 aggregate, than to those with a completely closed LH1 ring. In a second experimental series the influence of the environment on the spectra of the RC-LH1 complexes from Rps. acidophila was investigated. Thereby, the complexes were stabilized in detergent (DDM) buffer solution and reconstituted into a phospholipid bilayer, and the results were compared with the outcome of the first experimental series (vide supra), conducted on DDM stabilized RC-LH1 immobilized in a polyvinyl alcohol (PVA) matrix (DDM/PVA). The aim of these experiments was to test whether the immobilization of the RC-LH1 complexes in PVA might lead to a deformation of the LH1 structure, thereby limiting the significance of the results obtained from optical spectroscopy. In ensemble absorption and fluorescence-excitation spectra, spectral shifts were observed both as a function of the matrix as well as, as a function of temperature. Regarding the latter, varying spectral shifts were observed in the three environments upon cooling the sample. This could be explained consistently with an increase of the local pressure exerted on the complexes in the lipid bilayer and the DDM buffer solution, whereas for DDM/PVA a decrease of the local pressure is conceivable, since, due to a negative thermal expansion coefficient for thin polymer films below the glass transition temperature, an expansion rather than a contraction of the PVA-film is expected upon temperature reduction. In low-temperature single-molecule measurements it was found that the complexes dissolved in DDM buffer solution, without stabilization in a PVA matrix, are subjected to fast spectral dynamics preventing the extraction of meaningful data from single-molecule spectroscopy. Furthermore, for the complexes reconstituted into a lipid bilayer it was revealed that the reconstitution process results in a significantly larger fraction of broken complexes with respect to the preparation of the complexes in a PVA film. However, it was also found that for the intact complexes the statistics of the key spectral features, such as the spectral separation of the bands and the mutual orientation of their transition-dipole moments, show no difference as a function of using either a lipid bilayer or PVA as a matrix. Given the additional effort involved in the reconstitution process, the lower amount of intact RC-LH1 complexes, and, concerning the decisive spectral details, the identical results with respect to embedding the complexes in a PVA matrix, led to the conclusion that the immobilization of these pigment-protein complexes in a PVA matrix is a good choice for conducting low-temperature experiments on individual light-harvesting complexes. Next, the statistical distributions of the spectral features from the intact RC-LH1 complexes were compared with the corresponding data from numerical simulations of various LH1 model structures. Thereby, it was sought to learn more about the specific arrangement of the BChl a molecules in the LH1 complex from Rps. acidophila. All proposed LH1 model structures were within the resolution limit of the Rps. palustris RC-LH1 crystal structure and consequently all models had a gap in their BChl a arrangement. In comparison to an earlier simulation study on the RC-LH1 complex from Rps. palustris, more realistic simulation parameters were applied in this thesis, both with respect to the structural buildup of the LH1 models and regarding the coupling strength between the pigments. Initially three different LH1 model structures were tested. In two of these models fixed BChl a dimer units were placed equidistantly on an ellipse or a rectangle with rounded corners, whereas in the third model the placement of the BChl a molecules was equivalent to the irregular arrangement of the pigments in the Rps. palustris crystal structure. As a first result of the significantly increased interaction strengths in the simulations of this work, the site energy of the pigments in all proposed LH1 models adopts plausible values. However, it was also found that none of the three initial structures is able to reproduce the experimental distributions of the spectral features of the RC-LH1 complexes from Rps. acidophila. A careful analysis of the localization patterns of the LH1 exciton states revealed that the two lowest exciton states, k=1 and k=2, each localize in characteristic areas of the LH1 aggregates, where the values for the nearest-neighbor interactions between the pigments are significantly increased. With this knowledge it was possible, by slightly rearranging only four dimer units in one of the regular LH1 models (rectangle with rounded corners), to create a new LH1 model whose spectral properties are in satisfying agreement with the experimental single-molecule data. The structural modifications were inspired by the RC-LH1 palustris crystal structure and it could be argued that they are due to specific interactions between the RC and the LH1 complex. The final LH1 model clearly outperforms the refined Rps. palustris LH1 model introduced in an earlier study, since for the present model both more realistic simulation parameters were applied and it shows a much better structural overlap with the Rps. palustris RC-LH1 crystal structure. However, it has to be noted that in the only high-resolution crystal structure available for a RC-LH1 complex so far, no evidence can be found for the specific modifications performed on the regular LH1 structure. Additionally, it should be clear that similar structural modifications, as the one described above, can also lead to a LH1 model within the resolution of the palustris x-ray structure, being able to reproduce the experimental spectra. Therefore, it will not be claimed that in the final LH1 model the `one and only' structural solution for the LH1 complex from Rps. acidophila has been found, but it was shown how, in the limits of a rather simple simulation approach (Heitler-London approximation and dipole-dipole interaction between the pigments), a LH1 model with a regular placement of the pigments can be modified, to achieve a satisfying reproduction of the experimental distributions of the spectral features. Consequently, the more important result of this part is, that in excitonic systems which are dominated by the modulation of the nearest-neighbor interaction rather than by the diagonal disorder, the two lowest exciton states localize in specific areas of the LH1 aggregates, coinciding with the maxima of the nearest-neighbor interaction. This finding might be of general interest for any strongly coupled pigment array. Finally, triggered by a recent work on LH2 complexes from Rps. acidophila, a careful reinspection of the spectra from the individual RC-LH1 complexes considered as intact, revealed that, instead of the narrow line on the low-energy end of the spectra, a broad low-intensity band, occurring red-shifted with respect to the narrow line, might represent the lowest exciton state, k=1, of the LH1 complexes. In future work this new interpretation could be tested, by acquiring fluorescence-excitation and emission spectra from the same individual RC-LH1 complexes. According to the work on LH2, in these spectra the criterion for the identification of the lowest exciton state should be, that the emission spectrum has to emerge directly from the spectral region of the lowest exciton state in the excitation spectrum. Referring to the numerical simulations from the previous part, it is argued that not the long excited state lifetime of the k=1 exciton state in comparison to the higher exciton states might be responsible for the characteristic narrow line in the RC-LH1 fluorescence-excitation spectra, but the localization of the k=1 and k=2 exciton states on opposite sides of the LH1 aggregate. This would affect the relaxation of the k=2 state, now attributed to the narrow line, to k=1, as the rate for this process is amongst others determined by the spatial overlap between the corresponding exciton wavefunctions. Accordingly, due to the negligible overlap between the k=2 and k=1 exciton wavefunctions, it might be, that the narrow line reflects the comparatively slow relaxation of the k=2 state to k=1. In contrast, much more extended spatial overlaps with the k=1 exciton wavefunction were found for the higher exciton states (k≥3), making a fast relaxation to k=1 plausible and explaining the broad bands observed for these states in the excitation spectra. If this new interpretation of the spectral characteristics turns out to be true, on the one hand this would prove the necessity of acquiring, at the same time, fluorescence-excitation and emission spectra from individual LH complexes to ensure the completeness of the excitation spectra for these complexes. On the other hand, this would call for a reinterpretation of the spectra not only from the preceding parts of this work, but also from previous single-molecule studies on RC-LH1 complexes.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2015

Author dc:creator

• Böhm, Paul Sebastian

Contributors dc:contributor

• Köhler, Jürgen

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/1952/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:1952