Green Photosynthetic Bacteria Research Articles

INTERCONVERSION OF BACTERIOCHLOROPHYLL c AGGREGATES IN SOLID FILMS UPON ORGANIC VAPOR TREATMENT

Abstract—Bacteriochlorophyll c (BChl c) solid films were prepared from a carbon tetrachloride solution on CaF2 plates as artificial aggregates. Effects of organic vapor such as acetone and tetrahydrofuran (THF) on the BChl c films were studied by absorption and Fourier‐transform infrared spectroscopy. Two major homologs (R[E,E]BChl cF and R[P,E]BChl cF) and one minor homolog (S[I,E]BChl c) isolated from the green photosynthetic bacterium Chlorobium limicola strain 6230 were examined for the experiments. The BChl c polymeric aggregates absorbing at739–753 nm similar to those in the chlorosome were induced for all homologs upon the treatment of BChl c solid film with acetone vapor. The 131‐keto C=O stretching band in the R[E,E]BChl cF solid film showed a downward shift from 1651 cm−1to 1643 cm−1 with a concomitant shift of the 31‐OH stretching bands from 3337 and 3238 cm−1 to 3163 cm−1. It was suggested that the lower aggregates brought about by Mg…O=C(131) and (31)O…O=C(131) bonds were transformed into the higher aggregates strongly hydrogen‐bonded in a Mg…(31)O‐H…O=C(13l) interaction. They were transformed to a monomer‐like form absorbing at 667 nm upon exposure to THF vapor and were reversibly converted to the higher aggregates upon removal of THF molecules in vacuo.

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c 551 and an Fe-S center (CFA/CFB) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t 1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CFA/CFB) which decayed with a t 1/2 of ∼400-650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em -360 mV) and methyl viologen (-440 mV), and less effectively by triquat (-540 mV). The difference spectrum of Cyt c had negative peaks at 551, ∼520 and ∼420 nm, with a positive rise at ∼440 to ∼500 nm. The difference spectrum of CFA/CFB resembled P430 of PSI, and had a broad negative peak at 430∼435 nm.

Femtosecond Spectroscopy of Chlorosome Antennas from the Green Photosynthetic Bacterium Chloroflexus aurantiacus

The antenna kinetics of bacteriochlorophyll (BChl) c- and a-containing chromosomes from the thermophilic filamentous green photosynthetic bacterium Chloroflexus aurantiacus were investigated using two independent pump-probe techniques with subpicosecond resolution. Isotropic one- and two-color absorption difference experiments using probe wavelengths between 710 and 770 nm reveal BChl c photobleaching (PB) and stimulated emission (SE) decay kinetics with major lifetime components of 50-100 fs, 1-2 ps, and 7-10 ps. Two-color PB.SE profiles pumped at 770 nm and probed at 800 nm (where BChl a pigments absorb) exhibit no detectable rise time. However, two-color experiments using 790 and 820 nm pump and probe wavelengths, respectively, yield PB/SE rise components of 100 fs, 2 ps, and 10 ps. Upon excitation at 720 nm, the BCHl c PB/SE spectrum observed using a broad-band probe continuum displays surprisingly little spectral evolution during the first 2 ps. 65 refs., 13 figs., 3 tabs.

Gene nomenclature recommendations for green photosynthetic bacteria and heliobacteria

Recommendations are given for naming of genes coding for reaction center, antenna and electron transport proteins in green photosynthetic bacteria and heliobacteria.

Effects of illumination intensity on bacteriochlorophyllc homolog distribution inChloroflexus aurantiacus grown under controlled conditions

Green photosynthetic bacteria contain a mixture of stereoisomers and homologs of their major light harvesting pigment, bacteriochlorophyll (BChl)c. We have determined the distribution of photosynthetic pigments in the green filamentous bacteriumChloroflexus aurantiacus grown in turbidostat culture under light-limited conditions at 5 different illumination intensities. Pigments were extracted from isolated cells, analyzed by HPLC, and the homologs of BChlc identified by their mass spectra. The ratio between BChlc, BChla and carotenoid remained constant at low illumination intensities; at higher intensities BChla and carotenoid increased in parallel compared to BChlc. The BChlc homolog distribution changed even under conditions where the ratio of the total amount to the other pigments was unchanged, but there were no evidence for a constant stoichiometric ratio between any pair of homologs.

Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC

A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.

Evolution of heliobacteria: Implications for photosynthetic reaction center complexes

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein shows rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

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.

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for (1)O2 quenching by BChl molecules (kq (M)) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the kq (M) values coincide with the real values for (1)O2 quenching rate constants by BChl molecules. Aggregation weakly influenced the kq (M) values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the kq (M) value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus (1)O2 quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of (1)O2 formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as (1)O2 physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.

Structural differences in chlorosomes from Chloroflexus aurantiacus grown under different conditions support the BChl c-binding function of the 5.7 kDa polypeptide

Structurally different chlorosomes were isolated from the green photosynthetic bacterium Chloroflexus aurantiacus grown under different conditions. They were analysed with respect to variable pigment-protein stoichiometries in view of the presumed BChI c-binding function of the 5.7 kDa chlorosome polypeptide. Under high-light conditions on substrate-limited growth medium the pigment-protein ratio of isolated chlorosomes was several times lower than under low-light conditions on complex medium. Proteolytic degradation of the 5.7 kDa polypeptide in high-light chlorosomes led to a 60% decrease of the absorbance at 740 nm. The CD spectrum of high-light chlorosomes exhibited a sixfold lower relative intensity at 740 nm ( ΔA/A 740) than low-light chlorosomes, but it showed a fivefold increase in intensity upon degradation of the 5.7 kDa polypeptide compared to a twofold increase in low-light chlorosomes. It seems probable that BChl c in the chlorosomes is present as oligomers bound to the 5.7 kDa polypeptide. Our data suggest further that compared to low-light chlorosomes smaller oligomers or single BChl c molecules are bound to the 5.7 kDa polypeptide in high-light chlorosomes resulting in lower rotational strength.

Presence and significance of minor antenna components in the energy transfer sequence of the green photosynthetic bacterium Chloroflexus aurantiacus

Antenna components in the energy transfer processes of a green photosynthetic bacterium Chloroflexus aurantiacus were spectrally investigated by time-resolved fluorescence spectroscopy at −196°C on intact cells. Besides major antenna components so far reported, three minor components were resolved; those were Bchl c located at 785 nm, the baseplate Bchl a at 819 nm and Bchl a in the B808-866 complex at 910 nm. The last component was assigned to a longer wavelength antenna closely associated with a reaction center. An additional Bchl c fluorescence component was kinetically suggested to be present, which can be an energy donor to a major Bchl c. Presence of these minor components was signified in terms of (1) increase in the spectral overlap integral and (2) adjustment of the direction of dipole moments in the energy transfer sequence of intact cells.

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 Qy 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 3l 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.

Fast energy transfer in green photosynthetic bacteria Chlorobium limicola studied by spectral hole burning

Results of persistent hole burning fluorescence spectroscopy of green sulphur photosynthetic bacterium Chlorobium limicola are presented. Homogeneous spectral linewidths were determined from holewidths burned into inhomogeneously broadened NIR fluorescence spectra of chlorosomal bacteriochlorophyll-c at 1.8-4.2 K. The determined excited state relaxation time is strongly influenced by ultrafast energy transfer.

The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: properties of the triplet state and singlet oxygen photogeneration and quenching.

Measurements of pigment triplet-triplet absorption, pigment phosphorescence and photosensitized singlet oxygen luminescence were carried out on solutions containing monomeric bacteriochlorophylls (Bchl) c and d, isolated from green photosynthetic bacteria, and their magnesium-free and farnesyl-free analogs. The energies of the pigment triplet states fell in the range 1.29-1.34 eV. The triplet lifetimes in aerobic solutions were 200-250 ns; they increased to 280 +/- 70 microseconds after nitrogen purging in liquid solutions and to 0.7-2.1 ms in a solid matrix at ambient or liquid nitrogen temperatures. Rate constants for quenching of the pigment triplet state by oxygen were (2.0-2.5) x 10(9) M-1 s-1, which is close to 1/9 of the rate constant for diffusion-controlled reactions. This quenching was accompanied by singlet oxygen formation. The quantum yields for the triplet state formation and singlet oxygen production were 55-75% in air-saturated solutions. Singlet oxygen quenching by ground-state pigment molecules was observed. Quenching was the most efficient for magnesium-containing pigments, kq = (0.31-1.2) x 10(9) M-1 s-1. It is caused mainly by a physical process of singlet oxygen (1O2) deactivation. Thus, Bchl c and d and their derivatives, as well as chlorophyll and Bchl a, combine a high efficiency of singlet oxygen production with the ability to protect photochemical and photobiological systems against damage by singlet oxygen.

History of concepts of the comparative biochemistry of oxygenic and anoxygenic photosyntheses

Experiments of Hans Molisch in 1907 demonstrated that purple bacteria do not evolve molecular oxygen during photosynthetic metabolism, and can use organic compounds as sources of cell carbon for anaerobic 'photoheterotrophic' growth. Molisch's conclusion that he discovered a new photosynthetic growth mode was not accepted for some 30 years because of the prevailing definition of photosynthesis as light-dependent conversion of carbon dioxide and inorganic reductants to cell materials. Meanwhile, during the decade of the 1930s, Cornelis van Niel formulated the 'comparative biochemical watercleavage hypothesis' of photosynthesis, which enjoyed great popularity for about 20 years. According to this concept, photolysis of water yielded 'H' and 'OH', the former acting as the hydrogen donor for CO2 reduction in all modes of photosynthesis. Oxygenic organisms were presumed to contain a unique biochemical system capable of converting 'OH' to water and O2. To explain the absence of O2 formation by purple and green photosynthetic bacteria, it was supposed that such organisms lacked the oxygen-forming system and, instead, 'OH' was disposed of by reduction with an inorganic H(e) donor (other than water) according to the general equation:[Formula: see text] where H2A is H2 or an inorganic sulfur compound.Critical tests of van Niel's hypothesis could not be devised, and his proposal was abandoned soon after the discovery of in vitro photophosphorylation by green plant chloroplasts and membranes of purple bacteria in 1954. Photophosphorylation was then viewed as one key common denominator of oxygenic and anoxygenic photosyntheses. From later research it became clear that light-dependent phosphorylation of adenosine diphosphate was a consequence of photochemical charge separation and electron flow in reaction centers embedded in membranes of all photosynthetic organisms. The similarities, as well as the differences, in fine structure and function of reaction centers in anoxygenic and oxygenic organisms are now believed to reflect the course of evolution of oxygenic organisms from anoxygenic photosynthetic precursors. Thus, with the acquisition of new knowledge, concepts of the comparative biochemistry of photosynthetic processes have been radically altered during the past several decades. This paper describes highpoints of the history of these changes.

Redox regulation of energy transfer efficiency in antennas of green photosynthetic bacteria.

The efficiency of energy transfer from the peripheral chlorosome antenna structure to the membrane-bound antenna in green sulfur bacteria depends strongly on the redox potential of the medium. The fluorescence spectra and lifetimes indicate that efficient quenching pathways are induced in the chlorosome at high redox potential. The midpoint redox potential for the induction of this effect in isolated chlorosomes from Chlorobium vibrioforme is -146 mV at pH 7 (vs the normal hydrogen electrode), and the observed midpoint potential (n = 1) decreases by 60 mV per pH unit over the pH range 7-10. Extraction of isolated chlorosomes with hexane has little effect on the redox-induced quenching, indicating that the component(s) responsible for this effect are bound and not readily extractable. We have purified and partially characterized the trimeric water-soluble bacteriochlorophyll a-containing protein from the thermophilic green sulfur bacterium Chlorobium tepidum. This protein is located between the chlorosome and the membrane. Fluorescence spectra of the purified protein indicate that it also contains groups that quench excitations at high redox potential. The results indicate that the energy transfer pathway in green sulfur bacteria is regulated by redox potential. This regulation appears to operate in at least two distinct places in the energy transfer pathway, the oligomeric pigments in the interior of the chlorosome and in the bacteriochlorophyll a protein. The regulatory effect may serve to protect the cell against superoxide-induced damage when oxygen is present. By quenching excitations before they reach the reaction center, reduction and subsequent autooxidation of the low potential electron acceptors found in these organisms is avoided.

A synthetic zinc chlorin aggregate as a model for the supramolecular antenna complexes in the chlorosomes of green bacteria

UV—visible spectra of synthetic zinc chlorins in non-polar organic solvents indicate a supramolecular structure similar to the aggregates of bacteriochlorophylls- c in the extramembranous antenna complexes of green photosynthetic bacteria.

Chlorosomes, photosynthetic antennae with novel self-organized pigment structures

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.

Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus.

Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

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,3 Chlorobium 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.