Spectroscopic Investigations of Light-Harvesting 2 Complexes from Rps. acidophila

A better understanding of light capturing and energy transfer processes in natural photosynthesis can contribute to the development of a highly efficient, artificial, molecular-based technology that utilizes the sun for mankind’s energy supply. Being a prominent example, the light-harvesting 2 (LH2) antenna complexes located in the photosynthetic apparatus of anoxygenic non-sulfur purple bacteria has already provided a deep insight into how nature has developed a pigment-protein complex with highly efficient light-harvesting and energy transfer characteristics on a confined nanoscale geometry. In order to gain more detailed information about the electronic structure in LH2 complexes from Rhodopseudomonas (Rps.) acidophila (strain 10050), the absorbing and emitting states of these antenna complexes have been studied in this thesis by means of ensemble as well as singlemolecule fluorescence-excitation and emission spectroscopy at low temperature. The outcome of this thesis helps to clarify some long-standing problems concerning the discrepancies between the absorption and emission characteristics of ensembles of isolated LH2 complexes from purple bacteria, as well as discrepancies between the spectroscopic results obtained from ensemble and single-molecule studies on these pigment-protein antenna complexes. It was shown that the optical spectra of the ensemble sample, either dissolved in bulk-buffer solution or, according to the single-molecule preparation method, embedded in a thin polymer film, strongly depend on the sample preparation conditions and temperature. For the first time, fluorescence-excitation and emission spectra of the same individual LH2 complexes could be recorded at 1.2 K. In these experiments a significant difference between the emission spectra of single complexes concerning the correlation between spectral positions and widths (full width at half maximum, FWHM) was found. This strongly implied the existence of a different electron-phonon coupling strength for each complex, and moreover, led to the conclusion that at least for some of the complexes an exciton self-trapping process is effective. In a subsequent experiment, series of emission spectra from individual LH2 complexes have been recorded at 1.2 K with a significantly improved spectral and temporal resolution. Drastic fluctuations of the emission profiles were found to occur within individual complexes. In addition, the correlation between the shape and the peak position of the emission spectra provides direct evidence for fluctuations of the electron-phonon coupling strength not only within single LH2 complexes, but also between different ones. Finally, a direct comparison of fluorescence-excitation and emission spectra that were recorded for the same individual LH2 complexes allowed, by reduction of spectral and temporal inhomogeneities, an unambiguous assignment of the lowest exciton state for some of the LH2 complexes. This experiment provides evidence that the emission of the lowest exciton state can result in a narrow zero-phonon line and that emission spectroscopy of isolated LH2 complexes is to great extent affected by fluctuations of the electron-phonon coupling strength in the individual complexes. The work has resulted in four publications which can be found in part II of the thesis. In a first step (publication P1), a comprehensive optical characterization of ensembles of isolated LH2 complexes has been performed as a function of sample preparation conditions and temperature by means of steady-state fluorescenceexcitation, emission and fluorescence-anisotropy excitation spectroscopy. Spectral parameters (peak position, FWHM of the absorption/emission band, Stokes shift, excitonic bandwidth, etc.) which have been obtained from the optical spectra of LH2 ensembles, either dissolved in a buffer-detergent solution (with/without glycerol) or embedded in a spin-coated thin polymer film, were compared with those from native membranes at ambient and cryogenic temperatures. While bufferdetergent solutions and the polymer film matrix are the environments commonly used in bulk and single-molecule spectroscopic studies, respectively, native membranes served as the reference in which the LH2 complexes are naturally embedded. The most remarkable finding was a significant blue-shift of the B850 excitonic absorption band at 5 K upon transfer of the LH2 complexes from bulk-buffer solution into the spin-coated polymer film. Within the molecular exciton model this shift could be disentangled into three parts, namely to an increase of the local site energies, a contraction of the exciton band, and a decrease of the displacement energy. These results help to facilitate the comparison of results from single-molecule studies with those obtained from ensemble studies. In the second step (publication P2), fluorescence-excitation and emission spectra of the same individual LH2 complexes have been recorded at 1.2 K for the first time. The recorded emission spectra could be related to two classes of complexes with distinctively different types of emission spectra. One class of antenna complexes showed spectra with a relatively narrow spectral profile and a clear signature of a zero-phonon line (ZPL), whereas the other class displayed spectra that consisted of a broad featureless band. Further analysis of the emission spectra revealed clear correlations between the peak position and width (FWHM) of the emission band. This observation clearly indicated that the electron-phonon coupling strength strongly varies from complex to complex, as well as being a function of the spectral peak position. Due to long integration times, however, the influence of fast unresolved spectral diffusion on the width of the broad spectra could not be fully ruled out. Next, emission spectra of single LH2 complexes were recorded at 1.2 K with enhanced spectral resolution and significantly reduced exposure times. This allowed recording of series of emission spectra (100 - 2000) with an unprecedented spectral and temporal resolution for 26 LH2 complexes. Narrow ZPLs as well as broad structureless spectra from the same individual complex could be observed, suggesting a strong spectral diffusion. A multivariate statistical algorithm (MSA) was applied to the emission spectra to surpass weak signal intensities and strong spectral diffusion, resulting in so-called class-averaged spectra (CAS). From analysis of the CAS in terms of spectral widths (FWHM) and peak positions it followed that for each complex the width of the red-most CAS is always larger than the blue-most CAS, whatever the actual profile of the spectrum looked like. This correlation of the spectral profiles with peak positions can be interpreted as different electronphonon coupling strengths within each individual LH2 complex. Moreover, this study revealed a much larger variety of the emission profiles than previously observed in P2 and testifies, for the first time, that the electron-phonon coupling of an individual pigment-protein complex cannot be regarded as static. The measured linewidths of the ZPLs (smaller/equal than 10 cm-1) in the individual spectra, are the narrowest ever observed in emission spectra of LH2 complexes. These results have been published in publication P3. Finally, the fluorescence-excitation and emission spectra that have been recorded from individual LH2 complexes at 1.2 K, both showing strong temporal and spectral fluctuations, were directly compared for each individual complex. This comparison revealed that for 2/3 of the complexes the fluorescence-excitation spectrum could not be fully recorded due to the cut-off of the detection filter characteristics. However, for those complexes with fully recorded fluorescence-excitation spectra, a correlation of the red-most spectral feature of the excitation spectrum with the blue-most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Thus, the combination of fluorescence-excitation and emission spectroscopy on a single-molecule level allowed to surpass the spectral and temporal inhomogeneities such that the presence of the lowest exciton state in the excited state manifold of individual LH2 complexes could be unmasked for the first time. The results of this approach, which are in good agreement with the data obtained from spectral hole-burning studies, have been published in publication P4.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2014

Author dc:creator

• Kunz, Ralf

Contributors dc:contributor

• Köhler, Jürgen

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/1682/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:1682