Christoph Theiss

Excitation energy transfer in intact cells and in the phycobiliprotein antennae of the chlorophyll d containing cyanobacterium Acaryochloris marina.

The cyanobacterium Acaryochloris marina is unique because it mainly contains Chlorophyll d (Chl d) in the core complexes of PS I and PS II instead of the usually dominant Chl a. Furthermore, its light harvesting system has a structure also different from other cyanobacteria. It has both, a membrane-internal chlorophyll containing antenna and a membrane-external phycobiliprotein (PBP) complex. The first one binds Chl d and is structurally analogous to CP43. The latter one has a rod-like structure consisting of three phycocyanin (PC) homohexamers and one heterohexamer containing PC and allophycocyanin (APC). In this paper, we give an overview on the investigations of excitation energy transfer (EET) in this PBP-light-harvesting system and of charge separation in the photosystem II (PS II) reaction center of A. marina performed at the Technische Universität Berlin. Due to the unique structure of the PBP antenna in A. marina, this EET occurs on a much shorter overall time scale than in other cyanobacteria. We also briefly discuss the question of the pigment composition in the reaction center (RC) of PS II and the nature of the primary donor of the PS II RC.

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.

Excited State Dynamics in Recombinant Water-Soluble Chlorophyll Proteins (WSCP) from Cauliflower Investigated by Transient Fluorescence Spectroscopy

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.

Excitonic energy level structure and pigment-protein interactions in the recombinant water-soluble chlorophyll protein. II. Spectral hole-burning experiments.

Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ~658 and ~673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm(-1) for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ~150 cm(-1) and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B2008, 112, 13951).

Water soluble chlorophyll binding protein of higher plants: A most suitable model system for basic analyses of pigment–pigment and pigment–protein interactions in chlorophyll protein complexes

This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.

Results of a round-robin experiment in multiple-pulse LIDT measurement with ultrashort pulses

For the development of standard measurement procedures in optics characterization, comparative measurement campaigns (Round-robin experiments) are indispensable. Within the framework of the CHOCLAB project in the mid-90s, several international Round-robins were successfully performed qualifying procedures for e. g. 1 on 1-LIDT, laser-calorimetry and total scattering. During the recent years, the demand for single pulse damage investigations has been overtaken by the more practically relevant S on 1-LIDT. In contrast to the industrial needs, the comparability of the multiple-pulse LIDT has not been proven by Round-robin experiments up to now. As a consequence of the current research activities on the interaction of ultra-short pulses with matter as well as industrial applications, numerous fs-laser systems become available in universities and research institutes. Furthermore, special problems for damage testing may be expected because of the intrinsic effects connected with the interaction of ultrashort pulses with optical materials. Therefore, a Round-robin experiment on S on 1-damage testing utilizing fs-pulses was conducted within the framework of the EUREKA-project CHOCLAB II. For this experiment, seven parties investigated different types of mirrors and windows. Most of the partners were guided by the International Standard ISO 11254-2, but one partner employed his own damage testing technique. In this presentation, the results of this comparative experiment are compiled demonstrating the problems induced by special effects of damage testing in the ultra-short pulse regime.

Photoacoustic imaging of fluorophores using pump-probe excitation.

A pump-probe technique for the detection of fluorophores in tomographic PA images is introduced. It is based on inducing stimulated emission in fluorescent molecules, which in turn modulates the amount of thermalized energy, and hence the PA signal amplitude. A theoretical model of the PA signal generation in fluorophores is presented and experimentally validated on cuvette measurements made in solutions of Rhodamine 6G, a fluorophore of known optical and molecular properties. The application of this technique to deep tissue tomographic PA imaging is demonstrated by determining the spatial distribution of a near-infrared fluorophore in a tissue phantom.

Femtosecond-laser induced ablation of silicate glasses and the intrinsic dissociation energy

The relation between ablation threshold fluence upon femtosecond laser pulse irradiation and the average dissociation energy density of silicate based multicomponent glass is studied. A simple model based on multiphoton absorption quantifies the absorbed energy density at the ablation threshold fluence. This energy density is compared to a calculated energy density which is necessary to decompose the glass compound into its atomic constituents. The results confirm that this energy density is a crucial intrinsic material parameter for the description of the femtosecond laser ablation threshold fluence of dielectrics.

Excitation Energy Transfer in the Phycobiliprotein Antenna of Acaryochloris marina Studied by Transient fs Absorption and Fluorescence Spectroscopy

We Investigated The Excitation Energy Transfer (Eet) In The Phycobiliprotein (Pbp) Antenna Of The Chl D-Containing Cyanobacterium Acaryochloris Marina. The Equilibration Of The Excitation Energy Over All Phycocyanin (Pc) And Allophycocyanin (Apc) Molecules Of The Pbpantenna In A. Marina Was Found To Occur With Time Constants Of 400 Fs, 3 Ps And 14 Ps. This Is More Than Ten Times Faster Than The Eet From The Pc Rod-Antenna To The Apc Core In Typical Cyanobacteria With Phycobilisomes As Synechococcus 6301 (Holzwarth 1991). These Results Are In Agreement With The Unique Structure Of The Pbp-Antenna Of A. Marina Which Consists Of Three Pc-Homohexamers And One Hetero-Hexamer Containing Pc And Apc (Marquardt Et Al. 1997). The Results Suggest That The Presence Of Apc And Pc Within One Hexamer, Which Has So Far Been Observed Only In A. Marina, Enables A Very Fast 3 Ps Eet From Pc To Apc, Thereby Facilitating A Fast Energy Transfer From Phycobiliproteins To Ps Ii With A Time Constant Of 70 Ps (Petrasek Et Al. 2005).

Investigation of the excited states dynamics in the Chl d-containing cyanobacterium Acaryochloris marina by time-and wavelength correlated single-photon counting

The phototrophic cyanobacterium Acaryochloris marina discovered in 1996 has a unique composition of the light harvesting system. The chlorophyll (Chl) antenna contains mainly Chl d instead of the usually dominant Chl a and the Phycobiliprotein (PBP) antenna has a simpler rod shaped structure than in typical cynobacteria [1]. The interaction of the photosynthetic subunits and especially the mechanisms regulating the energy transfer under different stress conditions are presently interesting and open fields in photosynthesis research. In this study we use time- and wavelength-resolved single photon counting to investigate the excited states dynamics in living cells of A.marina. The fluorescence dynamics is synchronistically monitored in the visible and near infrared spectrum with high signal to noise ratio and short data acquisition times while using low excitation light intensities. These attributes are necessary to investigate photosynthetic processes in sensitive biological samples, when the light emission varies due to metabolic changes. The results suggest a fast excitation energy transfer kinetics of 20-30 ps along the PBP antenna of A.marina followed by a transfer of about 60 ps to the Chl d antenna. Cells of A. marina which are stored at 0°C for some time show a decoupling of the PBP antenna, which is partially reversible when the sample is kept at 25 °C for a short time. Decoupling effects appearing after strong illumination with white light (1600 W/m2) suggest a mechanism which removes the PBP antenna at different stress conditions to avoid photo damage of the reaction center of Photosystem II (PS II).