Direct counting of submicrometer-sized photosynthetic apparatus dispersed in medium at cryogenic temperature by confocal laser fluorescence microscopy: estimation of the number of bacteriochlorophyll c in single light-harvesting antenna complexes chlorosomes of green photosynthetic bacteria

The number of pigments in single light-harvesting complexes (chlorosomes) were calculated by imaging single chlorosomes in a frozen buffer at cryogenic temperature with a confocal laser fluorescence microscope and pigment extraction. Chlorosomes were isolated from two types of green photosynthetic bacteria Chlorobium (Chl.) tepidum and Chloroflexus (Cfl.) aurantiacus and were individually imaged in the frozen medium. Each fluorescence spot observed mainly came from a single chlorosome and was ascribable to self-aggregates of bacteriochlorophyll (BChl) c molecules as core parts of chlorosomes. A three-dimensional distribution of fluorescence of single chlorosomes was analyzed, and the number of chlorosomes in a volume of 54,000 microm(3) was counted directly. On the basis of the results, averaged numbers of the BChl c molecules contained in a single chlorosome of Chl. tepidum and Cfl. aurantiacus were determined to be 1.4 x 10(5) and 9.6 x 10(4), respectively. The present numbers are almost comparable to those estimated by other methods (Martinez-Planells et al., Photosynth. Res. 2002, 71, 83 and Monta?o et al., Biophys. J. 2003, 85, 2560).     

Spectral heterogeneity in single light-harvesting chlorosomes from green sulfur photosynthetic bacterium chlorobium tepidum

The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c--chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.     

Qy-excitation resonance Raman spectra of chlorophyll a and bacteriochlorophyll c/d aggregates. Effects of peripheral substituents on the low-frequency vibrational characteristics

Low-frequency (80-700 cm-1) Qy-excitation resonance Raman (RR) spectra are reported for thin-solid-film aggregates of several chlorophyll (Chl) a and bacteriochlorophyll (BChl) c/d pigments. The pigments include Chl a, pyrochlorophyll a (PChl a), methylpyrochloropyllide a (MPChl a), methylbacteriochloropyllide d (MBChl d), [E,M] BChl cS, [E,E] BChl cF, and [P,E] BChl cF. The BChl c/d's are the principal constituents of the chlorosomal light-harvesting apparatus of green photosynthetic bacteria. Together, the various Chl a's and BChl c/d's represent a series in which the peripheral substituent groups on the chlorin macrocycle are varied in systematic fashion. All of the Chl a and BChl c/d aggregates exhibit rich low-frequency vibrational patterns. In the case of the BChl c/d's, certain modes in the very low-frequency region (100-200 cm-1) experience exceptionally strong Raman intensity enhancements. The frequencies of these modes are qualitatively similar to those of oscillations observed in femtosecond optical experiments on chlorosomes. The RR data indicate that the low-frequency vibrations are best characterized as intramolecular out-of-plane deformations of the chlorin macrocycle rather than intermolecular modes. The coupling of the out-of-plane modes in turn implies that the Qy electronic transition(s) of the aggregate have out-of-plane character. The RR spectra of the BChl c/d's also reveal that the nature of the alkyl substituents at the 8 and 12 positions of the macrocycle plays an important role in determining the detailed features of the low-frequency vibrational patterns. The frequencies of the modes are particularly sensitive to larger substituent groups whose conformations may be more easily perturbed in the tightly packed aggregates. These factors also make aggregates of pigments containing larger substituents more susceptible to structural, electronic, and vibrational inhomgeneities. Collectively, the RR studies of the various pigments delineate the factors which influence the low-frequency vibrational characteristics of chlorosomal aggregates.     

Supramolecular chlorophyll aggregates inspired from specific light-harvesting antenna “chlorosome”: Static nanostructure,dynamic construction process,and versatile application

Photosynthetic light-harvesting antennas possess a variety of supramolecular structures with the same function (collecting sunlight energy), dependent on their living habitats and environments of phototrophs. Notably, the main antenna in green photosynthetic bacteria called “chlorosome” is structurally unique. Its core is constructed solely from self-aggregates of chlorophyll molecules without the support of any protein scaffolds. The supramolecular structures of the chlorophyll aggregates were already estimated, but have not been determined completely due to the natural diversity of chlorosomes. The static structures of chlorosomes are somewhat difficult to be characterized, and also the determination of their dynamic construction processes in vivo, such as their biogenesis and growth, is more challenging. Consequently, the measurement and observation of a simplified chlorosome model prepared by in vitro self-assembly of native or non-native (semisynthetic) chlorophylls are important. The present review focuses on evaluation of the static and dynamic nanostructures of chlorosomal aggregates. The construction, observation, and analysis of such models could be translated into the supramolecular aggregates of native chlorosomes. Additionally, synthetic aggregates display remarkable properties, which would be valuable for the development of solar cells and artificial photosynthesis. Such systems could potentially also be applied as photofunctional materials.     

Efficient exciton transport in layers of self-assembled porphyrin derivatives

The photosynthetic apparatus of green sulfur bacteria, the chlorosome, is generally considered as a highly efficient natural light-harvesting system. The efficient exciton transport through chlorosomes toward the reaction centers originates from self-assembly of the bacteriochlorophyll molecules. The aim of the present work is to realize a long exciton diffusion length in an artificial light-harvesting system using the concept of self-assembled natural chlorosomal chromophores. The ability to transport excitons is studied for porphyrin derivatives with different tendencies to form molecular stacks by self-assembly. A porphyrin derivative denoted as ZnOP, containing methoxymethyl substituents ({meso-tetrakis[3,5-bis(methoxymethyl)phenyl]porphyrinato}zinc(II)) is found to form self-assembled stacks, in contrast to a derivative with tert-butyl substituents, ZnBuP ({meso-tetrakis[3,5-bis(tert-butyl)phenyl]porphyrinato}zinc(II)). Exciton transport and dissociation in a bilayer of these porphyrin derivatives and TiO2 are studied using the time-resolved microwave conductivity (TRMC) method. For ZnOP layers it is found that excitons undergo diffusive motion between the self-assembled stacks, with the exciton diffusion length being as long as 15 +/- 1 nm, which is comparable to that in natural chlorosomes. For ZnBuP a considerably shorter exciton diffusion length of 3 +/- 1 nm is found. Combining these exciton diffusion lengths with exciton lifetimes of 160 ps for ZnOP and 74 ps for ZnBuP yields exciton diffusion coefficients equal to 1.4 x 10(-6) m2/s and 1 x 10(-7) m2/s, respectively. The larger exciton diffusion coefficient for ZnOP originates from a strong excitonic coupling for interstack energy transfer. The findings show that energy transfer is strongly affected by the molecular organization. The efficient interstack energy transfer shows promising prospects for application of such self-assembled porphyrins in optoelectronics.     

Artificial Light-Harvesting Antennae: Singlet Excitation Energy Transfer from Zinc Chlorin Aggregate to Bacteriochlorin in Homogeneous Hexane Solution

Abstract— Zinc chlorins possessing 3¹-hydroxyl and 13¹-carbonyl groups self-assemble in nonpolar solvents, such as hexane, in a manner similar to bacteriochlorophyll c in the chlorosomes of green photosynthetic bacteria. Visible absorption and steady-state fluorescence measurements of zinc chlorin aggregates containing a small amount of the bacteriochlorin-zinc chlorin dyad molecules showed that singlet excitation energy transfer from the zinc chlorin aggregate to the bacteriochlorin moiety of the coaggre-gated dyad occurs in the homogeneous solution. In the coaggregated dyad, the bacteriochlorin moiety plays the role of an efficient energy trap and the chlorin moiety the role of an anchor to the donor aggregate. The artificial assembly thus mimics the structure and function of natural chlorosomes and can be considered as the first in vitro supramolecular light-harvesting antenna.     

Temperature and ionic strength effects on the chlorosome light-harvesting antenna complex

Chlorosomes, the peripheral light-harvesting antenna complex from green photosynthetic bacteria, are the largest and one of the most efficient light-harvesting antenna complexes found in nature. In contrast to other light-harvesting antennas, chlorosomes are constructed from more than 150,000 self-assembled bacteriochlorophylls (BChls) and contain relatively few proteins that play secondary roles. These unique properties have led to chlorosomes as an attractive candidate for developing biohybrid solar cell devices. In this article, we investigate the temperature and ionic strength effects on the viability of chlorosomes from the photosynthetic green bacterium Chloroflexus aurantiacus using small-angle neutron scattering and dynamic light scattering. Our studies indicate that chlorosomes remain intact up to 75 °C and that salt induces the formation of large aggregates of chlorosomes. No internal structural changes are observed for the aggregates. The salt-induced aggregation, which is a reversible process, is more efficient with divalent metal ions than with monovalent metal ions. Moreover, with treatment at 98 °C for 2 min, the bulk of the chlorosome pigments are undamaged, while the baseplate is destroyed. Chlorosomes without the baseplate remain rodlike in shape and are 30-40% smaller than with the baseplate attached. Further, chlorosomes are stable from pH 5.5 to 11.0. Together, this is the first time such a range of characterization tools have been used for chlorosomes, and this has enabled elucidation of properties that are not only important to understanding their functionality but also may be useful in biohybrid devices for effective light harvesting.     

Absorption linear dichroism measured directly on a single light-harvesting system: the role of disorder in chlorosomes of green photosynthetic bacteria

Chlorosomes are light-harvesting antennae of photosynthetic bacteria containing large numbers of self-aggregated bacteriochlorophyll (BChl) molecules. They have developed unique photophysical properties that enable them to absorb light and transfer the excitation energy with very high efficiency. However, the molecular-level organization, that produces the photophysical properties of BChl molecules in the aggregates, is still not fully understood. One of the reasons is heterogeneity in the chlorosome structure which gives rise to a hierarchy of structural and energy disorder. In this report, we for the first time directly measure absorption linear dichroism (LD) on individual, isolated chlorosomes. Together with fluorescence-detected three-dimensional LD, these experiments reveal a large amount of disorder on the single-chlorosome level in the form of distributions of LD observables in chlorosomes from wild-type bacterium Chlorobaculum tepidum . Fluorescence spectral parameters, such as peak wavelength and bandwidth, are measures of the aggregate excitonic properties. These parameters obtained on individual chlorosomes are uncorrelated with the observed LD distributions and indicate that the observed disorder is due to inner structural disorder along the chlorosome long axis. The excitonic disorder that is also present is not manifested in the LD distributions. Limiting values of the LD parameter distributions, which are relatively free of the effect of structural disorder, define a range of angles at which the excitonic dipole moment is oriented with respect to the surface of the two-dimensional aggregate of BChl molecules. Experiments on chlorosomes of a triple mutant of Chlorobaculum tepidum show that the mutant chlorosomes have significantly less inner structural disorder and higher symmetry, compatible with a model of well-ordered concentric cylinders. Different values of the transition dipole moment orientations are consistent with a different molecular level organization of BChl's in the mutant and wild-type chlorosomes.     

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

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.     

Excitation energy transfer in isolated chlorosomes from Chlorobaculum tepidum and Prosthecochloris aestuarii

Excitation energy transfer in chlorosomes from photosynthetic green sulfur bacteria, Chlorobaculum (Cba.) tepidum and Prosthecochloris (Pst.) aestuarii, have been studied at room temperature by time-resolved femtosecond transient absorption spectroscopy. Bleach rise times from 117 to 270 fs resolved for both chlorosomes reflect extremely efficient intrachlorosomal energy transfer. Bleach relaxation times, from 1 to 3 ps and 25 to 35 ps, probed at 758 nm were tentatively assigned to intrachlorosomal energy transfer based on amplitude changes of the global fits and model calculations. The anisotropy decay constant of about 1 ps resolved at 807 nm probe wavelength for the chlorosomes from Chloroflexus aurantiacus, Pst. aestuarii and Cba. tepidum was related to energy transfer between bacteriochlorophyll a molecules of the baseplate and partly to intrachlorosomal energy transfer. The longer anisotropy components 6.6, 8.8 and 12.1 ps resolved for the three chlorosomes, respectively, were assigned to chlorosome to baseplate energy transfer. Global fits of magic-angle data also revealed longer chlorosome to baseplate energy transfer components from 95 to 135 ps, in accord with results from simulations.     

Temperature-dependent spectral changes of self-aggregates of zinc chlorophylls esterified by different linear alcohols at the 17-propionate

Systematically synthetic zinc 3-hydroxymethyl-13¹-oxo-chlorins esterified by different linear alcohols (methanol, 1-propanol, 1-hexanol, 1-dodecanol and 1-octadecanol) at the 17-propionate were self-assembled in the presence of cetyltrimethylammonium bromide in an aqueous solution. These zinc chlorins exhibited red-shifted Q _y absorption bands and circular dichroism (CD) signals in the corresponding Q _y region after incubation for 17 h, indicating that the zinc chlorins formed self-aggregates like those in natural chlorosomes of green photosynthetic bacteria. Visible absorption and CD spectra of self-aggregates of the zinc chlorins depended on the length of their esterifying alcohols. Zinc chlorins esterified by shorter alcohols gave larger changes in their visible absorption and CD spectra after incubation above 40°C, whereas zinc chlorins esterified by longer alcohols afforded smaller changes. These results indicate that hydrophobic interaction among esterifying chains of chlorin molecules as well as that between the esterifying chains and peripheral surfactants or lipids play an important role in the stability of chlorosomal self-aggregates.     

Molecular Requirement of Chlorosomal Chlorophylls. Self-Organization of a Chlorophyll Derivative Possessing a Hydroxyl Group at Ring II

Aggregation of zinc 7¹-hydroxyl-13²-demethoxycarbonyl-pheophytin a (Zn-7¹-OH-Chl) was examined in relation to the structure and function of the self-aggregates of 3¹-OH-type chlorophylls (Chi) in chlorosomes of green photosynthetic bacteria. The Zn-7¹-OH-Chl aggregates yielded a Q_y absorption band at 712 nm with a 1.2-fold larger width (full width at half maximum, 500 cm⁻¹) than the monomer's (420 cm⁻¹). Infrared and NMR spectroscopies revealed that each molecule in the aggregate links together with simultaneous coordination (C7¹-OH…Zn) and hydrogen bonding (C7¹-OH … O=C13¹). A nonlinear alignment of the constituent molecules in the oligomeric structure was assumed. Despite the similar molecular linkages, linearly aligned Q_y, moments in the Zn-3¹-OH-Chl aggregate gave a chlorosome-like broader, more redshifted Q_y band (740 nm; 670 cm⁻¹, 2.1-fold larger than the monomer's). Because it is advantageous for efficient light harvesting and energy transfer to have several Q_y, spectral components, spread over a wide spectral range, that can act as the energy gradient, it is concluded that not only the intermolecular linkages but the linear locations of OH, C=0 and Mg in the molecule are crucial for photosynthetic antenna of the self-assembled chiorosomal Chl.     

Limitations of Linear Dichroism Spectroscopy for Elucidating Structural Issues of Light-Harvesting Aggregates in Chlorosomes

Linear dichroism (LD) spectroscopy is a widely used technique for studying the mutual orientation of the transition-dipole moments of the electronically excited states of molecular aggregates. Often the method is applied to aggregates where detailed information about the geometrical arrangement of the monomers is lacking. However, for complex molecular assemblies where the monomers are assembled hierarchically in tiers of supramolecular structural elements, the method cannot extract well-founded information about the monomer arrangement. Here we discuss this difficulty on the example of chlorosomes, which are the light-harvesting aggregates of photosynthetic green-(non) sulfur bacteria. Chlorosomes consist of hundreds of thousands of bacteriochlorophyll molecules that self-assemble into secondary structural elements of curved lamellar or cylindrical morphology. We exploit data from polarization-resolved fluorescence-excitation spectroscopy performed on single chlorosomes for reconstructing the corresponding LD spectra. This reveals that LD spectroscopy is not suited for benchmarking structural models in particular for complex hierarchically organized molecular supramolecular assemblies.     

Hexanol-induced order-disorder transitions in lamellar self-assembling aggregates of bacteriochlorophyll c in Chlorobium tepidum chlorosomes

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.     

Immobilization of light-harvesting chlorophyll a/b complex (LHCIIb) studied by surface plasmon field-enhanced fluorescence spectroscopy

The major light-harvesting chlorophyll a/ b complex (LHCIIb) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that engage in rapid excitation energy transfer. This property makes LHCIIb potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Such applications would require the immobilization of LHCIIb (or similar dye-protein complexes) on a solid surface. In this work, the immobilization of recombinant LHCIIb is tested and optimized on functionalized gold surfaces via a histidine 6 tag (His tag) in the protein moiety. Immobilization efficiency and kinetics are analyzed by using surface plasmon resonance (SPR) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The latter was also used to assess the integrity of immobilized LHCIIb by recording Chl b-sensitized Chl a emission spectra. Since His tags have been included in a substantial number of recombinant proteins, the immobilization technique developed here for LHCIIb presumably can be extended to a large range of other membrane and water-soluble proteins.     

CHLOROPHYLL-PROTEINS AND REACTION CENTER PREPARATIONS FROM PHOTOSYNTHETIC BACTERIA,ALGAE AND HIGHER PLANTS*,‡

Abstract— The use of sodium dodecyl sulfate to dissociate photosynthetic membranes followed by standard fractionation techniques yields chlorophyll-proteins and reaction center complexes with molecular weights of 500,000 or less. Much about the structure and function of photo-synthetic units in vivo can be deduced from the properties of the isolated complexes. The Bchl-protein from green bacteria is approximated by an incompletely filled sphere ? 80 Â in diameter consisting of four identical subunits. The five Bchl molecules in each subunit are 14 to 20Â apart. The related Chl a-proteins from a blue-green alga and various eukaryotic plants may have similar structural characteristics. The Chl a-protein from a blue-green alga contains one molecule of P700 per 60–90 Chl a molecules. The quantum requirement for P700 oxidation is 2.6 or less. The midpoint potential in various preparations ranges from 0.38 V to 0.42 V. Green algae and higher plants yield a Chl a-protein similar to that from the blue-green alga; in addition they yield another Chl-protein (mol. wt. = 2–3×10⁴) which contains an equal amount of Chl a and Chl b. These two Chl-proteins account for most of the chlorophyll in these organisms. Two photosynthetic bacteria (Rhodopseudomonas viridis and Chromatium) yield protein complexes containing Bchl, carotenoid, and bound cytochromes. The reaction center complex from R. viridis contains P960 (E_m, ₈= 0.39 to 0.42 V), cytochrome 558 (E_m,8= 0.33 V) and cytochrome 553 (E_m,7=— 0.02 V). Quantum requirements for P960 and C558 oxidation are ?2.2 and 3.0, respectively. Complex A from Chromatium contains Bchl 890, P883, cytochrome 556 (E_m,8= 0.34 V) and cytochrome 552 (E_m,7=?0.04 V). The quantum requirement for C556 oxidation is about 15. Both high- and low-potential cytochromes can donate electrons to the reaction center chlorophyll present in either complex. This fact supports the idea that only one kind of photochemical reaction center functions in photosynthetic bacteria. An hypothesis about the nature of the photosynthetic unit in purple bacteria is outlined.     

In vitro self-assembly of bacteriochlorophyll c derivatives monoesterified with α,ω-diols isolated from the green sulfur photosynthetic bacterium Chlorobaculum tepidum

Esterifying chains of chlorophyllous pigments play important roles in the formation of photosynthetic supramolecules, but their effects have not thoroughly been unraveled yet. Substitution of the esterifying chains in these pigments will be one possible strategy to elucidate this enigma. Recently, unnatural bacteriochlorophylls (BChls) c possessing a hydroxy group at the terminus of the esterifying chains were successfully biosynthesised in the green sulfur bacterium Chlorobaculum (Cba.) tepidum grown by supplementation of α,ω-diols. In this paper, in vitro assembling behaviours of unnatural BChls c isolated from Cba. tepidum grown with 1,8-octanediol, 1,12-dodecanediol and 1,16-hexadecanediol were characterised in aqueous Triton X-100 micelles to investigate the effects of the terminal hydroxy group in the esterifying chains of BChls c on self-aggregates such as chlorosomes, major antenna complexes in green photosynthetic bacteria. The bacteriochlorophyll (BChl) c derivatives monoesterified with α,ω-diols formed chlorosomal self-aggregates, but their formations were much slower than those of natural BChl c. The Q_y absorption bands of the residual monomers of these BChl c derivatives were larger than those of natural BChl c. These suggest that the esterifying α,ω-diols in these unnatural BChls c somewhat interfered with formation of chlorosome-like aggregates compared with the natural esterifying farnesol.     

PROPERTIES OF ZINC AND MAGNESIUM METHYL BACTERIOPHEOPHORBIDE d AND THEIR AGGREGATES

Abstract— Light-harvesting bacteriochlorophylls are believed to he aggregated in oligomcric forms in chlorosomes of green photosynthetic bacteria. Zn and Mg methyl bacteriopheophorhides d (MBPd) were synthesized from chlorophyll a and studied as model compounds for bacteriochlorophyll d . Monomeric Zn and Mg MBPd in methanol have Q y absorption maxima at 650 nm and 657 nm and fluorescence decay lifetimes of 5.1 ns and 5.4 ns, respectively, comparcd to 5.6 ns for bactcriochlorophyll d . Zn and Mg MBPd both form oligomers in nonpolar solvents and exhibit Q, absorption maxima at 728 nm and 731 nm and fluorescence decay lifetimes of 14 ps and 19 ps, respectively, compared to 730 nm and 9 ps for similar bacteriochlorophyll d aggregates. One of the diastereomers at the 3^l position, R-Mg MBPd, forms intermediate-sized aggregated species that are equivalent to the dimer and a highly fluorescent species formed by bacteriochlorophylls c and d . The similarities of quantitative propcrtics between the model compounds, and the antenna pigments bacteriochlorophyll c and d indicate that Mg and Zn MBPd are good models for studying pigment interactions in chlorosomes and that the long hydrocarbon tail in the natural pigment is not required for oligomer formation. The dimer and the highly fluorescent species do not appear to be the building blocks of the oligomcr.     

THE CHLOROPHYLL a AGGREGATE ABSORBING NEAR 685 nm IS SELECTIVELY FORMED IN AQUEOUS TETRAHYDROFURAN*

An aqueous solution of 2–12% (vol/vol) tetrahydrofuran (THF) induced the selective aggregation of chlorophyll a (Chl a) to form a novel species, A-685, absorbing near 685 nm. The formation of A-685 was closely correlated with a decrease in water activity of the solution. A Raman spectrum of the Chl a species formed in the presence of 6% THF suggests a unique interaction among Chl a, solvent THF and water molecules to give a stacked aggregate (Chl a.THF.H₂O.THF.Chl a). The circular dichroic spectrum of the Chl a species formed in the 6% THF aqueous solution showed an intense signal that had negative and positive wings with about 100-fold larger molar ellipticity for the A-685 than for monomer. However, Chl a', the C10 epimer of Chl a, and chlorophyllide, with a phytyl chain replaced by an ethyl group, did not form A-685 in 6% THF. These clearly indicate that 10-methylcarboxylate and the phytyl chain have a significant role in stabilizing A-685. A possible structure for A-685 is proposed as a novel in vitro model for the P-680 Chl a dimer.     

Effect of carotenoid biosynthesis inhibition on the chlorosome organization in Chlorobium phaeobacteroides strain CL1401

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl-supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and beta-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30-40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from approximately 0.24 for native chlorosomes to approximately 0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.