Kai Griebenow

Pigment organization and energy transfer in green bacteria. 2. Circular and linear dichroism spectra of protein-containing and protein-free chlorosomes isolated from Chloroflexus aurantiacus strain Ok-70-fl

We have measured the circular dichroism (CD) and linear dichroism (LD) spectra of chlorosomes isolated from Chloroflexus aurantiacus strain Ok-70-fl obtained by two different isolation procedures. The gel-electrophoretic filtration procedure yields chlorosomes that are essentially free of BChl a 790 and proteins, while isolation by sucrose density gradient centrifugation yields the conventional chlorosome preparations. The LD spectra of the two kinds of preparation were very similar. In both cases the Q y LD signals correspond to an average angle between the BChl- c -Q y transition and the long axis of the chlorosome of approx. 15 ± 10°. In contrast to the LD spectra, the CD spectra of different preparations (membranes, BChl- a -free chlorosomes, BChl- a -containing chlorosomes) show pronounced differences both in the ellipticity as well as in the shape of the spectra and the number of maxima. However, these differences are not caused by the isolation procedure or the detergents used. We show that even freshly prepared membranes (of different, parallel grown batch cultures) give rise to very different CD spectra. The set of different CD spectra we obtained could be simulated well by linear combinations of two basic spectra. This strongly suggests that the variations in the CD spectra are caused by a variation in the relative amounts of two different species, two different types of chlorosome, or possibly by two different types of pigment aggregate within the chlorosomes.

Chlorosomes, photosynthetic antennae with novel self-organized pigment structures

Abstract The biochemical and spectroscopic evidence supporting the notion that chlorosomes, the main antenna complexes of the green photosynthetic bacterium Chloroflexus aurantiacus represent a novel type of self-organizing protein-free pigment structure are reviewed. The spectroscopic data from two different chlorosome preparations are compared with those of artificial pigment aggregates and the striking similarities in all spectroscopic aspects are discussed. Thus, chlorosomes represent the first known natural antenna structure where pigment-protein interactions do not determine the pigment arrangement.

Pigment organization and energy transfer in green bacteria 1. Isolation of native chlorosomes free of bound bacteriochlorophyll a from Chloroflexus aurantiacus by gel-electrophoretic filtration

A newmethod, employing a combination of native gel electrophoresis and gel filtration which we call gel-electrophoretic filtration, has been used for the isolation of native chlorosomes from the green bacterium Chloroflexus aurantiacus . The chlorosome preparation does not contain a BChl a protein complex in contrast to chlorosomes isolated according to previous preparation methods. Due to the lack of the BChl a complex these chlorosome preparations lack also absorption and emission bands at 792–795 nm and approx. 805 nm, respectively. The implications of a chlorosome preparation without a BChl a protein complex for the understanding of the structural organization of pigments in chlorosomes are discussed.

Primary processes in isolated bacterial reaction centers from Rhodobacter sphaeroides studied by picosecond fluorescence kinetics

Abstract Picosecond to nanosecond fluorescence kinetics as a function of emission wavelength is reported for isolated reaction centers of the purple bacterium Rb. sphaeroides . New kinetic components of 12 ps and about 100 ps lifetime have been found in the fluorescence decay kinetics of the primary donor P*. Various kinetic models of the primary and secondary electron transfer processes are discussed which could possibly explain these new components. The data indicate either the necessity for more complex electron transfer schemes than assumed so far or a substantial heterogeneity in the rates of both the primary and secondary electron transfer steps.

Picosecond energy transfer kinetics between pigment pools in different preparations of chlorosomes from the green bacterium Chloroflexus aurantiacus Ok-70-fl

Abstract Decay-associated (time-resolved) fluorescence spectra of isolated bacteriochlorophyll (BChl) a-containing and BChl a-free chlorosomes were measured using a picosecond single-photon timing apparatus in the detection wavelength range 730 – 820 nm. Two different relaxation steps can be resolved in the energy transfer chain. The first relaxation step occurs within the main BChl c pigment pool and the other is due to the energy transfer from one BChl c pool to the BChl a pool in the so-called baseplate. The corresponding equilibration times are approximately 5 ps and approximately 14 ps respectively.

Characterization of light-harvesting pigments of Chloroflexus aurantiacus. Two new chlorophylls: oleyl (octadec-9-enyl) and cetyl (hexadecanyl) bacteriochlorophyllides-c

The bacteriochlorophyll-c pigment contained in the chlorosomes of the phototrophic and thermophilic bacterium Chloroflexus aurantiacus is shown to comprise, in the least, 10 constituents, viz. the 2a-epimeric pairs [i.e., the (2aR,7S,8S)- and (2aS,7S,8S)-diastereoisomers in 2:1 ratio] of five bacteriochlorophyllide-c esters. All compounds were fully characterized by 1H and 13C NMR spectroscopy, and by mass spectra. They include the bacteriochlorophyllides-c of geranylgeranio, phytol, and stearol(octadecan-1-ol), which have been reported previously but have not yet been completely characterized, and two novel bacteriochlorophylls-c, viz. the bacteriochlorophyllides-c of the non-isoprenoid alcohols ‘cetol’(hexadecan-1-ol) and ‘oleol’(octadec-9-en-1-ol).

Primary processes in isolated photosynthetic bacterial reaction centres from Chloroflexus aurantiacus studied by picosecond fluorescence spectroscopy

Abstract The stationary and time-resolved fluorescence emission spectra of reaction centres (RCs) isolated from the thermophilic phototrophic bacterium Chloroflexus aurantiacus strain Ok-70-fl have been studied. Several lifetime components in the picosecond and nanosecond time range have been resolved at room temperature. A short-lived approx. 3 ps component is related to an energy-transfer process from the excited accessory BChl-αL to the special pair P. This is demonstrated by the existence of positive and negative amplitudes of this component, depending on the emission wavelength. A conformational relaxation of P∗ causing a bathochromic shift is excluded for this time constant due to the absence of this component at an excitation wavelength of 850 nm (P) and also by the temperature dependence of the stationary fluorescence. Two further short-lived components with positive amplitudes and lifetimes of about 7 ps and about 18 ps were also resolved. Their decay-associated spectra (DAS) and emission maxima at about 910–920 nm are similar, showing that their origin is fluorescence from P∗. Their relative amplitudes change strongly, depending on the excitation intensity. They represent the charge separation times of open RCs (7 ps from P∗HQA) and closed RCs (18 ps from P∗ HQA−). The time constant for open RCs is in full agreement with the value reported recently from transient absorption measurements (Becker et al. (1991) Biochim. Biophys. Acta 1057, 299–312). The presence of “heterogeneity” in the primary rates of charge separation in RC preparations proposed recently is discussed. It is suggested that the “heterogeneity” reported by other authors is possibly an open/closed RC heterogeneity.

Resonance Raman Spectroscopic Evidence for the Identity of the Bacteriochlorophyll c Organization in Protein-Free and Protein-Containing Chlorosomes from Chloroflexus auvantiacus

Protein-free and protein-containing chlorosomes from Chloroflexus aurantiacus, strain Ok-70 fl, were studied by resonance Raman (RR) spectroscopy. Both preparations gave the same spectra of the bacteriochlorophyll c (BChl c) chromophores in the range of 1200-1750 cm-1. This strongly corroborates previous evidence [Griebenow et al., Z. Naturforsch. 45c, 823-828 (1990), and references therein] that the three-dimensional structure of the antenna complexes is not determined by direct interaction with protein but rather is due to BChl c selforganization. The analysis of the coordination-sensitive marker bands of the chlorin macrocycle reveals a mixed six- and fivefold ligation of the Mg ion. Based on two C = O stretching vibrations originating from a free and a Mg-bound C-9 keto group, it is concluded that only in the six-coordinated state the keto group serves as an axial ligand to the Mg ion of a neighbouring chlorin. The second permanently bound axial ligand is attributed to the C-2a hydroxyl group.

Pigment organization and energy transfer in green bacteria. 3. Picosecond energy transfer kinetics within the B806-866 bacteriochlorophyll a antenna complex isolated from Chloroflexus aurantiacus*

The B806-866 antenna protein complex from Chloroflexus aurantiacus has been isolated using the detergents lauryldimethylamine N-oxide (LDAO) and Deriphat-160. These preparations have been characterized spectroscopically. While the B806-866 complex was unstable for longer periods in 0.1% LDAO a high stability was observed when 0.1% Deriphat-160 was used as a detergent. In order to study the energy-transfer processes between the 806 and 866 nm absorbing excited states, fluorescence decays were measured with picosecond resolution in the detection wavelength range from 800 to 910 nm upon excitation at 790 nm. A fast energy transfer from the 806 nm to the 866 nm absorbing states was resolved, and was found to have an equilibration time of 5 ± 1 ps. This equilibration time is in line with previous estimates from steady-state fluorescence. Due to the fast equilibration time, resolution of this energy-transfer process has not been achieved in the past when measuring whole cells of C. aurantiacus.

Charge separation kinetics in isolated photosynthetic reaction centers of Chloroflexus aurantiacus (with QA reduced) at low temperatures

Abstract The picosecond fluorescence kinetics of closed (quinone acceptor QA reduced) reaction centers isolated from the phototrophic bacterium Chloroflexus aurantiacus shows time constants of ≈ 20 and ≈ 300 ps (amplitude ratio ≈ 1:1), which are nearly independent of temperature (7 to 80 K). Assuming a three-state kinetic model, we tested various assignments of the kinetics to the electron transfer processes. Only two of these assignments seem to be physically reasonable. One of these includes a fast reversible electron transfer between P* and HM, the pheophytin(s) in the M branch of the reaction center which up to now has been considered virtually inactive in electron transfer. The other possible model involves the formation of a monomeric bacteriochlorophyll anion.