Green Bacterium Chloroflexus Aurantiacus Research Articles

The triplet state of the primary donor of the green photosynthetic bacterium Chloroflexus aurantiacus

The technique of absorbance-detected electron spin resonance in zero magnetic field (ADMR) was applied to investigate the structure of the reaction center of the facultatively aerobic green bacterium Chloroflexus aurantiacus. The triplet-minus-singlet absorbance difference spectrum thus obtained at 1.2K shows a clear resemblance to those earlier reported for Rhodopseudomonas viridis and Rps. sphaeroides R-26. The most prominent features are the bleaching of the Q y band of the primary electron donor at 887 nm and the appearance of a narrow band at 807 nm upon triplet formation. We conclude that the primary electron donor P-865 of Chloroflexus aurantiacus is a BChl a dimer with Q y and Q x absorbance bands at 887 and 606 nm, respectively, at 1.2 K; apparently the triplet state is localized on an optical time scale on one of the constituent pigments of the dimer. The zero field splitting parameters | D| and, | E| of P-865 are 197.7 (± 0.7) × 10 −4 cm −1 and 47.3 (± 0.7) × 10 −4 cm −1, respectively. Decay rates of 12 660 (± 750) s −1, 14 290 (± 800) s −1 and 1690 (± 50) s −1 were observed for the x, y and z triplet sublevels, respectively.

Optical properties of the photosynthetic reaction center of Chloroflexus aurantiacus at low temperature

A study was made of the optical properties of isolated reaction centers of the gliding green bacterium Chloroflexus aurantiacus. The absorption spectrum measured at 4 K indicates that these reaction centers contain bacteriochlorophyll a and bacteriopheophytin a, but no other pigments. The fluorescence excitation spectrum at 4 K showed efficient energy transfer from all pigment molecules to P-865, the primary electron donor. Absorption, linear and circular dichroism spectra of reduced and oxidized samples were measured at 77 K in order to obtain information about the dipole strengths, orientations and interactions of the transition dipoles. The results agree with a model for the reaction center that involves three bacteriopheophytin and four closely interacting bacteriochlorophyll molecules. The three bacteriopheophytins do not seem to be closely coupled to the primary donor. The primary electron donor consists of a bacteriochlorophyll dimer absorbing at 887 nm at 77 K, while the other two bacteriochlorophylls are responsible for the absorption around 800 nm and are also strongly coupled as indicated by their exciton splitting.

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.

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.

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.

Isolation and development of chlorosomes in the green bacterium Chloroflexus aurantiacus.

Freeze-fracture electron microscopy was used to study further the changes in chlorosome structure during the development of the photosynthetic apparatus in Chloroflexus aurantiacus J-10-fl. During development, in response to decreased light intensity or lower oxygen tension, the number of chlorosomes per cell increased. The same conditions also led to a general thickening of chlorosomes but did not affect their length or width. The thickening of the chlorosomes paralleled increases in the bacteriochlorophyll c/bacteriochlorophyll a ratio. Semiaerobic induction of the photosynthetic apparatus did not produce a synchronous assembly of chlorosomes in all cells of a given culture. Even adjacent cells of a single filament showed great variations in the rate and extent of response. Parallel appearance of (i) approximately 5-nm particles (in a lattice configuration) in the membrane attachment site, (ii) the crystalline baseplate material (with a periodicity of approximately 6 nm) adjacent to the membrane attachment site, and (iii) the chlorosome envelope layer preceded addition of longitudinally oriented, rodlike elements (diameter, congruent to 6 m) to the chlorosome core. It is estimated that each chlorosome can funnel energy into approximately 100 reaction centers. Chlorosomes could be isolated by a simple density gradient procedure only from cells grown at low light intensity. A bacteriochlorophyll a species absorbing at 790 nm was associated with isolated chlorosomes. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorosomes showed only a few low-molecular-weight polypeptides (less than 15,000).