Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

Molecular adaptation of photoprotection: triplet states in light-harvesting proteins

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis.     

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.     

How carotenoids protect bacterial photosynthesis

The essential function of carotenoids in photosynthesis is to act as photoprotective agents, preventing chlorophylls and bacteriochlorophylls from sensitizing harmful photodestructive reactions in the presence of oxygen. Based upon recent structural studies on reaction centres and antenna complexes from purple photosynthetic bacteria, the detailed organization of the carotenoids is described. Then with specific reference to bacterial antenna complexes the details of the photoprotective role, triplet triplet energy transfer, are presented.     

光合细菌H3菌株色素分析

H3菌株系由盐田微生物层中分离获得的光合细菌株。具有丰富的天然色素。经活细胞色素光谱吸收峰值测定,色素经有机溶剂提取、硅胶薄板层析、SDS－PAGE电泳等,结果表明H3菌株的主要色素包括细菌叶绿素a、细菌脱镁叶绿素（Bacteriophaeophytin）和三种类胡萝卜素。总胡萝卜素含量占细胞于重的0．6％,胡萝卜素蛋白复合体的分子量约11,000.培养条件的差异对色素形成及相对含量有不同程度的影响。     

Magnetophotoselection applied to the triplet state observed by EPR in photosynthetic bacteria.

Magnetophotoselection (MPS) techniques have been used to study the triplet state observed by electron paramagnetic resonance (epr) in photosynthetic bacteria. Intact $\text{R.}\text{rubrum}$ chromatophores and systems in which the effects of energy transfer via antenna chlorophyll molecules have been minimized were examined. These preliminary results indicate that there is order in the bacteriochlorophyll antenna system, that the optical transition at 890 nm appears to be along the triplet y axis of the bacteriochlorophyll special pair (Bchl_sp), and that the resultant transition moment associated with 800 nm is approximately parallel to the long wavelength transition moment of the Bchl_sp.     

Protein modifications affecting triplet energy transfer in bacterial photosynthetic reaction centers.

The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer.     

Purification and properties of two succinate semialdehyde dehydrogenases from human brain

The changes in the in vivo bacteriochlorophyll fluorescence induced by a Xenon flash at low temperatures (77--200 K) with the "primary" acceptor X chemically prereduced have been examined in whole cells of several species of photosynthetic bacteria which contain carotenoids absorbing in the visible part of the absorption spectrum. Two groups of species with different behaviour could be distinguished. In both cases a flash-induced rise of the fluorescence yield was observed with X prereduced at 77 k; as the temperature was increased the ratio of the maximum fluorescence (FM) and the basal fluorescence (F0) decreased and the kinetics of the decay of the high fluorescent state, as observed during the tail of the flash, apparently accelerated. Of the species examined the flash-induced changes in fluorescence-yield kinetics appeared to occur at higher temperatures in the members of one group (Chromatium vinosum, Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris) than in the members of the other (Rhodopseudomonas palustris) than in the members of the other (Rhodopseudomonas sphaeroides and Rhodospirillum rubrum). These effects are interpreted in terms of the light-induced generation of triplet states within the reaction centre. It is suggested that the species-dependent differences may reflect differences in the molecular organisation of the reaction centre. It was found that in all species the reaction centre carotenoid triplet does not act as a fluorescence quencher under these conditions.     

Horizontal Transfer of the Photosynthesis Gene Cluster and Operon Rearrangement in Purple Bacteria

A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the beta subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus. The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes. The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF-bchCXYZ-puf and bchFNBHLM-lhaA-puhA, were conserved. Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi. gelatinosus was originated from those of the species belonging to the alpha subclass of purple bacteria. It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the alpha subclass to that of the beta subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster.     

What is beta-carotene doing in the photosystem II reaction centre?

During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Redox functions of carotenoids in photosynthesis

Carotenoids are well-known as light-harvesting pigments. They also play important roles in protecting the photosynthetic apparatus from damaging reactions of chlorophyll triplet states and singlet oxygen in both plant and bacterial photosynthesis. Recently, it has been found that beta-carotene functions as a redox intermediate in the secondary pathways of electron transfer within photosystem II and that carotenoid cation radicals are transiently formed after photoexcitation of bacterial light-harvesting complexes. The redox role of beta-carotene in photosystem II is unique among photosynthetic reaction centers and stems from the very strongly oxidizing intermediates that form in the process of water oxidation. Because of the extended pi-electron-conjugated system of carotenoid molecules, the cation radical is delocalized. This enables beta-carotene to function as a "molecular wire", whereby the centrally located oxidizing species is shuttled to peripheral redox centers of photosystem II where it can be dissipated without damaging the system. The physiological significance of carotenoid cation radical formation in bacterial light-harvesting complexes is not yet clear, but may provide a novel mechanism for excitation energy dissipation as a means of photoprotection. In this paper, the redox reactions of carotenoids in photosystem II and bacterial light-harvesting complexes are presented and the possible roles of carotenoid cation radicals in photoprotection are discussed.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Hexacoordination of bacteriochlorophyll in photosynthetic antenna LH1

The ability of chlorophylls to coordinate ligands is of fundamental structural importance for photosynthetic pigment-protein complexes, where in virtually all cases the pigment is thought to be in a pentacoordinated state. In this study, the correlation of the Q(X) transition energy with the coordination state of the central metal in bacteriochlorophyll is applied in investigating the pigment coordination state in bacterial photosynthetic antenna LH1. To facilitate a detailed spectral analysis in the Q(X) region, carotenoid-depleted forms of LH1 are prepared and model LH1 are constructed with non-native carotenoids having blue-shifted absorption. The deconvolution of the Q(X) envelope in LH1 reveals that the band is the sum of two transitions, which peak near 590 and 607 nm, showing that a significant fraction (up to 25%) of hexacoordinated bacteriochlorophyll is present in the complex. The hexacoordination can be seen also in LH1 antennae from other species of purple photosynthetic bacteria. It seems correlated with the LH1 aggregation state and probably is a consequence of the structural flexibility of the assembled complex. The sixth ligand probably originates from the apoprotein and seems not to affect the chromophore core size. These findings show that in light-harvesting complexes a hexacoordinated state of bacteriochlorophyll is not uncommon. Its presence may be relevant to a correct assembly of the antenna and have functional consequences, as it results in a splitting of the pigment S2 excited state (Q(X)), i.e., the carotenoid excitation acceptor state, what might affect intracomplex carotenoid-to-bacteriochlorophyll energy transfer.     

Triplet excited state spectra and dynamics of carotenoids from the thermophilic purple photosynthetic bacterium <Emphasis Type="Italic">Thermochromatium tepidum</Emphasis>

Light-harvesting complex 2 from the anoxygenic phototrophic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption, fluorescence and flash photolysis spectroscopy. Steady-state absorption and fluorescence measurements show that carotenoids play a negligible role as supportive energy donors and transfer excitation to bacteriochlorophyll-a with low energy transfer efficiency of ~30%. HPLC analysis determined that the dominant carotenoids in the complex are rhodopin and spirilloxanthin. Carotenoid excited triplet state formation upon direct (carotenoid) or indirect (bacteriochlorophyll-a Q_x band) excitation shows that carotenoid triplets are mostly localized on spirilloxanthin. In addition, no triplet excitation transfer between carotenoids was observed. Such specific carotenoid composition and spectroscopic results strongly suggest that this organism optimized carotenoid composition in the light-harvesting complex 2 in order to maximize photoprotective capabilities of carotenoids but subsequently drastically suppressed their supporting role in light-harvesting process.     

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.     

Roles of bacteriochlorophyll and carotenoid synthesis in formation of intracytoplasmic membrane systems and pigment-protein complexes in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Synthesis of bacteriochlorophyll and carotenoids was inhibited in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114, by alpha, alpha'-dipyridyl and diphenylamine. Formation of two pigment-protein complexes, reaction center-B870 (RC-B870) and B806, and development of the intracytoplasmic membranes of the cells were studied by spectral analysis and electron microscopy. Inhibition of bacteriochlorophyll synthesis by alpha, alpha'-dipyridyl, which was accompanied by a decrease in carotenoid synthesis, suppressed formation of intracytoplasmic membranes in the cells. Growth under illumination had a similar effect on formation of pigments and membranes. On the other hand, inhibition of carotenoid synthesis by diphenylamine did not suppress either development of the membrane system or bacteriochlorophyll synthesis. Formation of RC-B870 and B806 complexes, however, was differentially affected by blockage of carotenoid synthesis. In the presence of diphenylamine, the B806 complex was formed in a much smaller amount than the RC-B870 complex. These results suggest that, in Erythrobacter sp. strain OCh114, bacteriochlorophyll plays an essential role in intracytoplasmic membrane development, and carotenoids are important for assembly of pigment-protein complexes.     

Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77 K

Photoinhibition in Lemna gibba L. was studied by interpreting chlorophyll fluorescence characteristics at 77 K on the basis of the bipartite model of Butler and co-workers (Butler 1978). Application of this analysis to chloroplasts (isolated from plants before and after exposure to a photosynthetic photon flux density of 1 750 μmol m⁻² s⁻¹ at 3°C for 2 h) revealed that photoinhibition had the following effect on primary events in photosynthesis. Firstly, the fluorescence of PS II increased (44%) in the state of open traps (F_o) and decreased (32%) in the state of closed traps (F_m). It is suggested, that the F_o-decrease reflects increased quenching by radiationless decay, both effects occurring at PS II reaction centers. Secondly, the rate constant for transfer of excitation energy from PS II to PS I (k_T(μ→J)) increased by 34%. However, in the state of closed traps, the flux of excitation energy via this transfer process decreased, most likely because of increased quenching by radiationless decay at PS II reaction centers. Thirdly, the probability for fluorescence from PS I decreased (19%). This indicates increased quenching by radiationless decay.     

Carotenoid triplet states in reaction centers from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Purified photochemical reaction centers from three strains of Rhodopseudomonas sphaeroides and two of Rhodospirillum rubrum were reduced with Na₂S₂O₄ so as to block their photochemical electron transfer reactions. They then were excited with flashes lasting 5–30 ns. In all cases, absorbance measurements showed that the flash caused the immediate formation of a transient state (P^F) which had been detected previously in reaction centers from Rps. sphaeroides strain R26. Previous work has shown that state P^F is an intermediate in the photochemical electron transfer reaction in the reaction centers of that particular strain, and the present work generalizes that conclusion.

In the reaction centers from two strains that lack carotenoids (Rps. sphaeroides R26 and R. rubrum G9), the decay of P^F yields a longer-lived state (P^R) which is probably a triplet state of the bacteriochlorophyll of the reaction center. In the R26 preparation, the decay of P^F was found to have a half-time of 10±2 ns. The decay kinetics rule out the identification of P^F as the fluorescent excited singlet state of the reaction center.

In the reaction centers from three strains that contain carotenoids (Rps sphaeroides 2.4.1 and Ga, and R. rubrum S1), state P^R was not detected, and the decay of P^F generated triplet states of carotenoids. The efficiency of the coupling between the decay of P^F and the formation of the carotenoid triplet appeared to be close to 100% at room temperature, but somewhat lower at 77 °K. Taken with previous results, this suggests that the coupling is direct and does not require the intermediate formation of state P^R. This conclusion would be consistent with the view that P^F is a biradical which can be triplet in character.     

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.