Masamitsu Hirota

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

Abstract The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes.

SPECTRAL FORMS AND ORIENTATION OF BACTERIOCHLOROPHYLLS c AND α IN CHLOROSOMES OF THE GREEN PHOTOSYNTHETIC BACTERIUM Chloroflexus aurantiacus

Spectral forms of bacteriochlorophyll (Bchl) in chlorosomes were analyzed by linear dichroism, circular dichroism (CD), and deconvolution of these spectra. Isolated chlorosomes were embedded in polyacrylamide gels and compressed unidirectionally (along the x‐axis) while allowing the gel to stretch in another direction (along the z‐axis). The chlorosomes were aligned three‐dimensionally due to their flat oblong shape; the longest axis was presumed to parallel the z‐axis, its shortest axis was presumed to parallel the x‐axis, and the intermediate‐length axis was presumed to parallel the y‐axis. Degrees of polarization (AI− A1)/(AI+ A1) of Bchl c and a measured from the y‐axis with linearly polarized light were significantly different from those measured from the x‐axis. Deconvolution of spectra into components revealed the presence of two major forms of Bchl c with peaks at 744 nm and 727 nm. The degrees of polarization of the 744 and 727 nm spectral forms were 0.76 and 0.59 from the y‐axis and 0.48 and 0.39 from the x‐axis, respectively. The degrees of polarization of Bchl a794 were –0.21 from the y‐axis and 0.12 from the x‐axis. These values indicate that the direction of the Qy transition moment of Bchl c744 is almost completely parallel to the longest axis of chlorosomes and that of Bchl c727 is also nearly, but slightly less so, parallel to the longest axis of the chlorosomes. The Qy transition moment of the baseplate Bchl a peak at 794 nm is nearly perpendicular to the longest axis and parallel to the shortest axis: that is, it is perpendicular to the associated membrane plane in the cell. These alignments of Bchl transition moments in chlorosomes were lost after suspending the chlorosomes in a solution saturated with 1‐hexanol accompanying a shift in the peak position from 742 nm to 670 nm. The alignment recovered after the hexanol concentration was decreased. The presence of two major spectral forms of Bchl c was supported by the deconvolution of CD spectra and absorption spectra.

High degree of organization of bacteriochlorophyll c in chlorosome-like aggregates spontaneously assembled in aqueous solution

Pigment-lipid aggregates were formed in aqueous solution by diluting a chloroform/methanoll extract of chlorosomes of the green photosynthetic bacterium,3Chlorobium limicola. The aggregates showed absorption and fluorescence spectra very similar to those of intact chlorosomes. No proteins were detected in the aggregates. Electron micrographs showed that the pigment-lipid aggregates were ellipsoidal bodies with average size of 130 nm along the long axis and 86 nm along the short axis. The linear dichroism spectrum of bacteriochlorophyll c in the pigment-lipid aggregates oriented in a stretched polyacrylamide gel was as strong as that in chlorosomes. These results suggest that spontaneous assembly of the protein-free pigments and lipids extracted from chlorosomes restores not only direct chromophore-chromophore interactions of bacteriochlorophyll c molecules but also the chlorosome-like higher-order structures.

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox ≈ 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox ≈ 40) is CK-dependent.

Molecular organization of bacteriochlorophyll in chlorosomes of the green photosynthetic bacteriumChloroflexus aurantiacus: Studies of fluorescence depolarization accompanied by energy transfer processes

Examination was made of changes in fluorescence polarization plane by energy transfer in the chlorosomes of the green photosynthetic bacterium,Chloroflexus aurantiacus. Fluorescence anisotropy in the picosecond (ps) time region was analyzed using chlorosomes suspended in solution as well as those oriented in a polyacrylamide gel. When the main component of BChlc was preferentially excited, the decay of fluorescence anisotropy was found to depend on wavelength. In the chlorosome suspension, the anisotropy ratio of BChlc changed from 0.31 to 0.24 within 100 ps following excitation. In the baseplate BChla region, this ratio decreased to a negative value (−0.09) from the initial 0.14. In oriented samples, the degree of polarization remained at 0.68 for BChlc, and changed from 0.25 to −0.40 for the baseplate BChla by excitation light whose electric vector was parallel to the longest axis of chlorosomes. In the latter case, there was a shift from 0.30 to −0.55 by excitation perpendicular to the longest axis. Time-resolved fluorescence polarization spectra clearly indicated extensive changes in polarization plane accompanied by energy transfer. The directions of polarization plane of emission from oriented samples were mostly dependent on chlorosome orientation in the gel but not on that of the polarization plane of excitation light. Orientations of the dipole moment of fluorescence components was consistent with that of absorption components as determined by the linear dichroism (Matsuura et al. (1993) Photochem. Photobiol. 57: 92–97). A model for molecular organization of BChlc anda in chlorosomes is proposed based on anisotropic optical properties.

Quenching of bacteriochlorophyll fluorescence in chlorosomes from Chloroflexus aurantiacus by exogenous quinones.

Abstract The quenching of bacteriochlorophyll (BChl) c fluorescence in chlorosomes isolated from Chloroflexus aurantiacus was examined by the addition of various benzoquinones, naphthoquinones (NQ), and anthraquinones (AQ). Many quinones showed strong quenching in the micromolar or submicromolar range. The number of quinone molecules bound to the chlorosomes was estimated to be as small as one quinone molecule per 50 BChl c molecules. Quinones which exhibit a high quenching effect have sufficient hydrophobicity and one or more hydroxyl groups in the alpha positions of NQ and AQ. Chlorobiumquinone has been suggested to be essential for the endogenous quenching of chlorosome fluorescence in Chlorobium tepidum under oxic conditions. We suggest that the quenching effect of chlorobiumquinone in chlorosomes from Chl. tepidum is related to the 1′-oxo group neighboring the dicarbonyl group.

Pheophytinization of bacteriochlorophyll c and energy transfer in cells of Chlorobium tepidum.

Abstract Bacteriochlorophyll (BChl) c in whole cells of Chlorobium tepidum grown at 46 °C changed into bacteriopheophytin (BPhe) c within 10 days after reaching full growth. When a small amount of C. tepidum cells in which BChl c had been completely pheophytinized were transferred to a new culture medium, normal growth was observed after a short lag phase, and the absorption spectrum of the growing cells showed the presence of a normal amount of BChl c. During the growth of C. tepidum in the new culture, the BChl c concentration was nearly proportional to the cell density measured by turbidity (OD640). These results indicate that C. tepidum can survive even when BChl c has been completely pheophytinized and that BChl c is newly synthesized in such cells when transferred to a new culture medium. In partly pheophytinized cells, upon excitation of BPhe c at 550 nm the fluorescence emission spectrum showed maxima at 775 and 810 nm, which correspond to emissions from BChl c and BChl a, respectively. This indicates energy transfer from BPhe c to BChl c and BChl a. In cells in which BChl c was completely pheophytinized, fluorescence measurements were indicative of direct energy transfer from BPhe c to baseplate BChl a. These findings suggest that when BChl c in C. tepidum cells is pheophytinized, the product (BPhe c) remains in the chlorosomes and continues to work as a light-harvesting pigment.

Quenching of Energy Transfer in Chlorosomes from Chloroflexus by the Addition of Synthetic Quinones

The light-harvesting antennae of the green sulfur bacteria (Chlorobiaceae), known as chlorosomes, are ovoid structures on the inner side of the cytoplasmic membrane. The main component is bacteriochlorophyll (BChl) c,d or e, transferring excitation energy to a minor pool of BChl a in the chlorosomes. The excitation energy is then transferred via the Fenna-Matthews-Olson protein to the reaction center in the cytoplasmic membrane.