Thermophilic Green Bacterium Chloroflexus Research Articles

Coherent intradimer dynamics in reaction centers of photosynthetic green bacterium Chloroflexus aurantiacus

Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140 cm−1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945 nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ΔA spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.

Crystallization and X-ray analysis of the reaction center from the thermophilic green bacterium Chloroflexus aurantiacus

The photochemical reaction center of Chloroflexus (Cf.) aurantiacus, a membrane bound pigment-protein complex, has been crystallized in the presence of monodisperse polyoxyethylene detergents. The crystals possessed a pronounced polymorphism. Three different crystal forms belonging to triclinic, monoclinic and orthorhombic space groups have been characterized by X-ray analysis. The triclinic crystal form, with unit cell dimensions of a=88 A ̊ , b=115 A ̊ and c=151 A ̊ , diffracts up to 3.2 Å in two directions and to 4.0 Å in the third direction.

Malate dehydrogenase from the thermophilic green bacterium Chloroflexus aurantiacus: purification, molecular weight, amino acid composition, and partial amino acid sequence.

Malate dehydrogenase (MDH; EC 1.1.1.37) from the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus was purified by a two-step procedure involving affinity chromatography and gel filtration. The enzyme consists of identical subunits which had molecular weights of approximately 35,000. In its active form at 55 degrees C, it formed tetramers. At lower temperatures, inactive dimers and trimers existed. Antibodies against the purified enzyme were produced, and immunotitration and enzyme-linked immunosorbent assays showed that there was an immunochemical homology between the MDH from C. aurantiacus and MDHs from several other bacteria. The amino acid composition of C. aurantiacus MDH was similar to those of other MDHs. The N-terminal amino acid sequence was enriched with hydrophobic amino acids, which showed a high degree of functional similarity to amino acids at the N-terminal ends of both Escherichia coli and Thermus flavus MDHs. The activity of the native enzyme was inhibited by high concentrations of substrate and had temperature and pH optima consistent with the optimal growth conditions for the organism.

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Qy transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Qy transition of BChl c and the γ-carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Qy transition is close to the magic angle (55°).

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30-35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808-866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Qy transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Qy transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808-866 complex of C. aurantiacus and the B800-850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 Å was derived.

Partial purification, subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides ( M r = 30000 and 28000) in a reaction center complex of M r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.