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Time-resolved spectroscopic analysis of fucoxanthin-chlorophyll proteins and isolated carotenoids
(2011)
The aim of this thesis was to elucidate the excitation energy transfer in the fucoxanthin-chlorophyll proteins (FCPs) isolated from the diatom Cyclotella meneghiniana in detail and to clarify the role of the different pigments contained. In a first step the excited state dynamics of the free pigments were studied by means of time-resolved absorption spectroscopy. The FCPs contain three different carotenoid species. Besides the main light-harvesting carotenoid fucoxanthin (fx) the xanthophyll cycle pigments diadinoxanthin (ddx) and diatoxanthin (dtx) are found in substoichiometric amounts. Fx is contained in an unusual carotenoid-to-chlorophyll ratio of about one. In case of ddx and dtx, changing the solvent polarity showed no significant effects on the absorption spectrum and the excited state dynamics were hardly influenced. In contrast, a solvent dependence is observed in the absorption spectrum and excited state dynamics of fx. The S1 lifetime depends strongly on the solvent polarity and an additional broad excited state absorption band red shifted compared to the S1 excited state absorption appears. The occurrence of the described features can be explained with an intramolecular charge transfer state, which is stabilized in a polar environment and appears only in carotenoids with a conjugated carbonyl group. Despite its rather short excited state lifetimes of less than 200 fs (S2) and 30-60 ps (S1), fx acts as a very efficient energy donor in the FCPs. The ultrafast energy transfer dynamics of the isolated proteins FCPa and FCPb were investigated in a comprehensive study using transient absorption in the visible and NIR spectral region complemented with polarized transient absorption spectroscopy. The excitation energy transfer was not influenced significantly by changing the light conditions during the growth, which yields an altered amount of ddx and dtx. It can be concluded that the contribution of the xanthophyll cycle pigments to the energy transfer is not significant. The altered oligomerization state results in a more efficient energy transfer for the trimeric FCPa, which is also reflected in different Chl a fluorescence quantum yields. Thus, an increased quenching in the higher oligomers of FCPb can be assumed. The observed dynamics change drastically for two different excitation wavelengths λ = 500 nm and λ = 550 nm, which both lead to the population of the S2 excited state of individual carotenoids, namely blue and red absorbing fx molecules. The differing absorption maxima result from distinct microenvironments within the protein. For FCPa an additional slow time constant of 25 ps was found after excitation at 500 nm. By means of polarized transient absorption spectroscopy applied to FCPa different transition dipole moments for the S1 and the ICT state of fx could be identified. Based on the presented studies a detailed model explaining the excitation energy transfer pathways could be developed. In agreement with the faster overall transfer rate which is also evident in the anisotropy data in case of 550 nm excitation, upon excitation at 500 nm one slow transfer channel is active. It can be attributed to a blue absorbing fx not strongly associated with a Chl a molecule. Most likely excitation energy transfer takes place between the S1/ICT states of two different fx molecules before the energy is transferred to Chl a. Additional transient absorption experiments with an improved time resolution were performed to investigate the oscillations observed. These coherent effects superimposed the kinetics of isolated carotenoids as well as FCPs within the first 500 fs. The oscillations showed a very unusual damping behavior and vanished already after two oscillation periods. In case of fx, the solvent environment as well as the excitation wavelengths had an influence on the oscillations. The frequencies of the oscillations were 70-100 cm^-1 for fx in solvents with varying polarity and 50-80 cm^-1 for the FCPs. These results could further confirm the assumption that the red absorbing fx molecules are located in a more polar environment within the protein compared to the blue absorbing fx. To clarify the origin of the oscillations in more detail, further experiments with a controlled chirp of the applied pulses and comparison between different carotenoids in various solvents are required. This approach promises to give further insight in the excited state dynamics and to answer the question whether dark states are involved. Right now, the coherent excitation of the strongly coupled excited states 1Bu+ (S2) and 1Bu- resulting in electronic quantum beats and the existence of an additional short lived excited state absorption (S2-SN2) in the visible spectral region are the most reasonable explanations for the occurrence of the coherent effects in the transient absorption spectra of carotenoids.
The enzyme quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes is a membrane protein complex that couples the catalysis of the oxidation of menaquinol to menaquinone to that of the reduction of fumarate to succinate. This is the terminal step in fumarate respiration, a form of anaerobic respiration in which oxygen is replaced by fumarate as the terminal electron acceptor in many anaerobic microorganisms. In QFR, both the heme groups (low-potential distal and high-potential proximal heme b group in transmembrane subunit C) are part of the electron transport chain between the two catalytic sites of the redox enzyme. Although the reduction of fumarate by menaquinol is exergonic, it is not exergonic enough to support the generation of a transmembrane electrochemical proton potential delta p. Evidence has previously shown that this reaction is catalysed by a novel mechanism, involving the facilitation of transmembrane electron transfer by transmembrane proton transfer via an essential compensatory transmembrane proton transfer pathway ("E-pathway") which is inactive in the oxidized state of the enzyme. The two key constitutents of the the pathway are the amino acid residue Glu C180 of the transmembrane helix V (located in subunit C) and the ring C propionate of the distal heme bD. The aim of the project was to obtain, by employing a combination of time-resolved as well as static spectroscopic approaches, a detailed insight of the transmembrane electron coupled proton transfer mechanism. Minute changes in both the oxidized and reduced states of a redox protein system can be selectively and sensitively monitored by static Fourier Transformed Infrared (FTIR) difference spectroscopy. The technique employed in this context, electrochemically induced FTIR difference spectroscopy, is complemented by computer-based electrostatic calculations. In order to elucidate the catalytic mechanism of the important reactions in QFR, it is necessary to investigate these in a time-resolved manner. Rapid scan FTIR difference spectroscopy is a suitable technique that allows the course of the reaction to be monitored in a time dependent fashion. The techniques employed in this context are time-resolved (tr-FTIR) and transient absorption spectroscopy. In the following, the details of individual sub-projects are discussed in brief. ...