Kristiane A. Schmidt

Composition and optical properties of reaction centre core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum.

Photosynthetically active reaction centre core (RCC) complexes were isolated from two species of green sulfur bacteria, Prosthecochloris (Ptc.) aestuarii strain 2K and Chlorobium (Chl.) tepidum, using the same isolation procedure. Both complexes contained the main reaction centre protein PscA and the iron–sulfur protein PscB, but were devoid of Fenna–Matthews–Olson (FMO) protein. The Chl. tepidum RCC preparation contained in addition PscC (cytochrome c). In order to allow accurate determination of the pigment content of the RCC complexes, the extinction coefficients of bacteriochlorophyll (BChl) a in several solvents were redetermined with high precision. They varied between 54.8 mM−1 cm−1 for methanol and 97.0 mM−1 cm−1 for diethylether in the QY maximum. Both preparations appeared to contain 16 BChls a of which two are probably the 132-epimers, 4 chlorophylls (Chls) a 670 and 2 carotenoids per RCC. The latter were of at least two different types. Quinones were virtually absent. The absorption spectra were similar for the two species, but not identical. Eight bands were present at 6 K in the BChl a QY region, with positions varying from 777 to 837 nm. The linear dichroism spectra showed that the orientation of the BChl a QY transitions is roughly parallel to the membrane plane; most nearly parallel were transitions at 800 and 806 nm. For both species, the circular dichroism spectra were dominated by a strong band at 807–809 nm, indicating strong interactions between at least some of the BChls. The absorption, CD and LD spectra of the four Chls a 670 were virtually identical for both RCC complexes, indicating that their binding sites are highly conserved and that they are an essential part of the RCC complexes, possibly as components of the electron transfer chain. Low temperature absorption spectroscopy indicated that typical FMO–RCC complexes of Ptc. aestuarii and Chl. tepidum contain two FMO trimers per reaction centre.

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.

Combined fluorescence and photovoltage studies on chlorosome containing bacteria

Energy transfer kinetics and primary charge separation were studied in whole cells and in chlorosome-depleted membranes of Chlorobium limicola by ps-fluorescence and ps-photovoltage as well as by stationary fluorescence spectroscopy. The fluorescence decay kinetics of whole cells indicate a sequential energy transfer from the chlorosomes via the baseplates and the Fenna–Matthews–Olson-protein (FMO) to the core-complexes with time constants of 35 ± 4 ps and 95 ± 10 ps, respectively. The quantitative analysis of fluorescence spectra and the occurrence of slow phases in the fluorescence decays reveal that in whole cells a significant fraction of BChl c in the chlorosome and of BChl a in the baseplate-FMO-protein is poorly connected to the core-complexes. The photovoltage kinetics of whole cells upon excitation in the chlorosome (λex = 532 nm) consisted of two electrogenic phases with time constants of 121 ± 10 ps and 575 ± 140 ps. The ≈ 120 ps phase is composed of energy transfer from the baseplate-FMO-protein to the core-complex and trapping from the core-complexes by P+A0- formation. The second phase (relative electrogenicity of 23%) is a charge stabilization step, probably from the first electron acceptor, A0, to the secondary electron acceptor FX. When the core-complexes were excited (λex = 840 nm) the photovoltage kinetics also consisted of two electrogenic phases, but the first phase showed a time constant of 23 ± 6 ps. This phase reflects exclusively trapping from the core-complexes by P+A0- formation. In dark-adapted whole cells the fluorescence yields of the peripheral antenna complexes increased strongly upon background illumination. This observation indicates the disappearance of endogenous quenchers, probably quinones.

Occurrence and Function of Quinones in the Reaction Center of Chlorobium Tepidum

In the photosynthetic reaction center (RC) from green sulfur bacteria and heliobacteria and photosystem I from cyanobacteria, algae and higher plants a light induced linear electron transfer leads to the reduction of NAD(P)+. In photosystem I it is well established that phylloquinone acts as the secondary electron acceptor of the electron transfer chain. However the occurrence and role of the quinone in the RC from green sulfur bacteria is discussed controversial. In membranes from Chlorobium limicola (1) and the isolated RC-complex from C. vibrioforme (2) a quinone can be photoreduced. In contrast thin layer chromatography analysis of extracted pigments of the purified photoactive RC-complex from C. tepidum did not show the presence of a quinone (3). This discrepancy may be due either to the use of different organisms or to the application of various procedures for purifying the RC-complex (RCC). Therefore we isolated the RCC from C. tepidum by three different methods and analyzed the content of quinones at each purification step.

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the ‘two-color’ measurements at 820 and 835 nm upon excitation at 800 nm, whereas the ‘one-color’ kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps.

Analysis of Interactions of Signaling Proteins with Phage- Displayed Ligands by Fluorescence Correlation Spectroscopy

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed. (Journal of Biomolecular Screening 2008:766-776)

Relaxation and Trapping In Reaction Centers of the Green Sulfur Bacterium Prosthecochloris Aestuarii

The reaction center of green sulfur bacteria resembles that of Photosystem I of green plants. The primary donor, P840, is a bacteriochlorophyll (BChl) a dimer and the primary electron acceptor, A0, is a pigment absorbing near 670 nm, (Chl a 670). In addition, there are about 15 antenna BChls a. Up to now, time-resolved absorption measurements in the picosecond domain had only been performed with membrane fragments, containing the Fenna-Matthews-Olson antenna complex in additon to the core complex (1). However, the development of a fast and simple method to isolate a purified reaction center core (RCC) complex in a photochemically active and relatively stable form (2) allowed us to study the excited state and electron transfer dynamics in more detail than had been possible before. This paper reports a study of the excited states of BChl a by pump-probe transient absorption spectroscopy in RCC complexes of the green sulfur bacterium Prosthecochloris aestuarii. A more detailed account of some of the experiments will be published elsewhere (3).

Electron Transfer in Reaction Centers from Green Sulfur Bacteria

The antenna system of green sulfur bacteria consists of three components: the chlorosome, the Fenna Matthews Olson (FMO) protein, and the reaction center core (RCC) complex. The RCC complex resembles the reaction centers of photosystem I and of heliobacteria. The primary donor, P840, is a bacteriochlorophyll (BChl) a dimer and the primary acceptor, A0, is a Chi a-like pigment absorbing near 670 nm. The role of a menaquinone as secondary electron acceptor, A1, is still a matter of debate. As in photosystem I, the further electron transfer involves three iron-sulfur (Fe-S) centers, Fx, FA and FB (1).

Reaction Centre Complexes of Green Sulfur Bacteria

Green sulfur bacteria contain reaction centre core (RCC) complexes of the iron-sulfur type. The RCC contains a homodimer of the 82 kD PscA protein, that binds the primary donor P840 (a BChl a dimer), the primary acceptor Chi a 670 and the Fe-S centre Fx. Two more Fe-S centres (FA, and FB) are bound by the 32 kD PscB protein (1–3). Earlier we described the isolation of photochemically active RCC complexes from Prosthecochloris aestuarii (4). Here we describe some of the biochemical and spectroscopic properties of this preparation and of a new preparation from Chlorobium tepidum.

Accumulated Photon Echo Studies on 3FMO-RCC and RCC from Prosthecochloris Aestuarii

Accumulated Photon Echo (APE) measurements (1) provide a tool to investigate the coherent lifetime of an initially excited state in pigment-protein complexes. As pure optical dephasing can be neglected at low temperatures, APE yields information on the mechanisms of energy tranfer in photosynthetic systems.