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.     

Triplet-triplet energy transfer in the major intrinsic light-harvesting complex of Amphidinium carterae as revealed by ODMR and EPR spectroscopies

We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae.Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet-triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching.     

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.     

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.     

Triplet–triplet energy transfer in fucoxanthin-chlorophyll protein from diatom Cyclotella meneghiniana: Insights into the structure of the complex

Although the major light harvesting complexes of diatoms, called FCPs (fucoxanthin chlorophyll a/c binding proteins), are related to the cab proteins of higher plants, the structures of these light harvesting protein complexes are much less characterized. Here, a structural/functional model for the “core” of FCP, based on the sequence homology with LHCII, in which two fucoxanthins replace the central luteins and act as quenchers of the Chl a triplet states, is proposed. Combining the information obtained by time-resolved EPR spectroscopy on the triplet states populated under illumination, with quantum mechanical calculations, we discuss the chlorophyll triplet quenching in terms of the geometry of the chlorophyll–carotenoid pairs participating to the process. The results show that local structural rearrangements occur in FCP, with respect to LHCII, in the photoprotective site.     

Singlet-triplet fission of carotenoid excitation in light-harvesting LH2 complexes of purple phototrophic bacteria

The current generally accepted structure of light-harvesting LH2 complexes from purple phototrophic bacteria conflicts with the observation of singlet-triplet carotenoid excitation fission in these complexes. In LH2 complexes from the purple bacterium Allochromatium minutissimum, a drop in the efficiency of carotenoid triplet generation is demonstrated, which correlates with the extent of selective photooxidation of bacteriochlorophylls absorbing at ～850 nm. We conclude that singlet-triplet fission of carotenoid excitation proceeds with participation of these excitonically coupled bacteriochlorophylls. In the framework of the proposed mechanism, the contradiction between LH2 structure and photophysical properties of carotenoids is eliminated. The possibility of singlet-triplet excitation fission involving a third mediator molecule was not considered earlier.     

Triplet exciton formation as a novel photoprotection mechanism in chlorosomes of Chlorobium tepidum

Chlorosomes comprise thousands of bacteriochlorophylls (BChl c, d, or e) in a closely packed structure surrounded by a lipid-protein envelope and additionally contain considerable amounts of carotenoids, quinones, and BChl a. It has been suggested that carotenoids in chlorosomes provide photoprotection by rapidly quenching triplet excited states of BChl via a triplet-triplet energy transfer mechanism that prevents energy transfer to oxygen and the formation of harmful singlet oxygen. In this work we studied triplet energy transfer kinetics and photodegradation of chlorosomes isolated from wild-type Chlorobium tepidum and from genetically modified species with different types of carotenoids and from a carotenoid-free mutant. Supporting a photoprotective function of carotenoids, carotenoid-free chlorosomes photodegrade approximately 3 times faster than wild-type chlorosomes. However, a significant fraction of the BChls forms a long-lived, triplet-like state that does not interact with carotenoids or with oxygen. We propose that these states are triplet excitons that form due to triplet-triplet interaction between the closely packed BChls. Numerical exciton simulations predict that the energy of these triplet excitons may fall below that of singlet oxygen and triplet carotenoids; this would prevent energy transfer from triplet BChl. Thus, the formation of triplet excitons in chlorosomes serves as an alternative photoprotection mechanism.     

On the role of ß-carotene in the reaction center chlorophyll a antennae of photosystem I

Absorption and low temperature fluorescence emission spectra were measured on chloroplast thylakoids and on purified reaction center chlorophyll a-protein complexes of photosystem I, CP-a₁. A clear association between the presence of ß-carotene and the occurrence of far red absorbing and emitting chlorophyll a components of the reaction center antennae of photosystem I was demonstrated. For this study chloroplasts and CP-a₁ were obtained from normal and carotenoid deficient plant material of various sources. The experimental material included 1) lyophilized pea chloroplasts extracted with petroleum ether, 2) the carotenoid deficient mutant C-6E of Scenedesmus obliquus and 3) wheat chloroplasts derived from normal and SAN-9789 treated plants. Removal of carotenoids, most likely principally ß-carotene, caused a loss of long wavelength absorbing chlorophylls in chloroplasts and purified CP-a₁, and the loss or diminution of the long wavelength peak seen in the low temperature fluorescence emission spectrum. This association between ß-carotene and special chlorophyll a forms may explain both the photoprotective and antenna functions ascribed to ß-carotene. In the absence of carotenoids in wheat and in the Scenedesmus mutant, the chlorophyll a antenna of photosystem I was extremely photosensitive. A triplet-triplet resonance energy transfer from chlorophyll a to ß-carotene and a singlet-singlet energy transfer from excited ß-carotene to chlorophyll would explain the photoprotective and antenna functions, respectively. The role of this association in determining some of the fluorescence properties of photosystem I is also discussed.     

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Biological functions of carotenoids--diversity and evolution

Carotenoids first emerged in archaebacteria as lipids reinforcing cell membranes. To serve this function their long molecules have extremely rigid backbone due to the linear chain of usually 10 to 11 conjugated C=C bonds in transconfiguration--the length corresponding the thickness of hydrophobic zone of membrane which they penetrate as "molecular rivets". Carotenoids retain their membrane-reinforcing function in some fungi and animals. The general structure of carotenoid molecule, originally having evolved for mechanical functions in membranes, possess a number of other properties that were later used for independent functions. The most striking fact is that these properties proved to fit some new functions to perfection. The polyene chain of 9-11 double bonds absorbs light precisely in the gap of chlorophyll absorption--function as accessory light-harvesting pigments in all plants; Unique arrangement of electronic levels owing to the by polyene chain structure makes carotenoids the only natural compounds capable of excitation energy transfer both (i) from carotenoid excited state to chlorophyll in the light-harvesting complex and (ii) from triplet chlorophyll or singlet oxygen to carotenoid in photosynthetic reaction centers--protection of RC from photodamage. The linear system of conjugated C=C bonds provides high reducing potential of carotenoid molecules making them potent antioxidants in lipid formations. Still, there is a lack of evidence of the chemical antioxidant function of carotenoids, especially in higher organisms; most data demonstrate an antioxidant ability rather than a function. Carotenoids have many other independent biological functions, including: specific coloration patterns in plants and animals, screening from excessive light and spectral filtering, defense of egg proteins from proteases in some invertebrates; the direct carotenoid derivative--retinal--acts as visual pigment in all animals and as chromophore in bacteriorhodopsin photosynthesis, retinoic acid in animals and abscisic acid in plants serve as hormones. All these functions utilize various properties (mechanical, electronic, stereospecific) of a single structure evolved in bacteria for a single membrane-reinforcing function, thus demonstrating an example of pure evolutionary preadaptation. One of the practical conclusions that can be reached by reviewing uniquely diverse properties and functions of carotenoids is that, when considering possible mechanisms of their effects in organisms (e.g., anticarcinogenic action), all their functional traits should be taken into account.     

Spectroscopic properties of the Chlorophyll a–Chlorophyll c 2–Peridinin-Protein-Complex (acpPC) from the coral symbiotic dinoflagellate Symbiodinium

Femtosecond time-resolved transient absorption spectroscopy was performed on the chlorophyll a–chlorophyll c ₂–peridinin-protein-complex (acpPC), a major light-harvesting complex of the coral symbiotic dinoflagellate Symbiodinium. The measurements were carried out on the protein as well on the isolated pigments in the visible and the near-infrared region at 77 K. The data were globally fit to establish inter-pigment energy transfer paths within the scaffold of the complex. In addition, microsecond flash photolysis analysis was applied to reveal photoprotective capabilities of carotenoids (peridinin and diadinoxanthin) in the complex, especially the ability to quench chlorophyll a triplet states. The results demonstrate that the majority of carotenoids and other accessory light absorbers such as chlorophyll c ₂ are very well suited to support chlorophyll a in light harvesting. However, their performance in photoprotection in the acpPC is questionable. This is unusual among carotenoid-containing light-harvesting proteins and may explain the low resistance of the acpPC complex against photoinduced damage under even moderate light conditions.     

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.     

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.     

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.     

Energy transfer dynamics in a red-shifted violaxanthin-chlorophyll a light-harvesting complex

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690?nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S₂ and hot S₁ states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.     

Characterization of unusual hydroxy- and ketocarotenoids in<Emphasis Type="Italic"> Rubrivivax gelatinosus</Emphasis>: involvement of enzyme CrtF or CrtA

Carotenoids are widely spread terpenoids found in photosynthetic organisms and a number of non-photosynthetic fungi and bacteria. The photosynthetic non-sulfur purple bacterium Rubrivivax gelatinosus produces carotenoids by both the spheroidene and the normal spirilloxanthin pathways. The characteristics of two carotenogenesis enzymes, spheroidene monooxygenase CrtA and O-methyltransferase CrtF, were investigated. Disruption of the corresponding genes by insertional mutagenesis affected carotenoid species in both pathways, and the genetic evidence indicated that both genes are involved in the two pathways. In these mutants, several unusual hydroxy- and ketocarotenoids were identified by spectroscopic and chemical methods. Moreover, the carotenoid analyses demonstrated that a large number of different carotenoid intermediates are accepted as substrates by the CrtA enzyme. The combined manipulation of crtF and crtA allowed new carotenoids to be produced and broadened the diversity of structurally different carotenoids synthesized by Rvi. gelatinosus. Methylated carotenoids, such as spheroidene and spirilloxanthin, are known to function as accessory pigments in the light-harvesting and reaction-center complexes of purple bacteria; the demethylated carotenoids described here were able to fulfill the same functions in the mutants.     

Rate of carotenoid triplet formation in solubilized light-harvesting complex II (LHCII) from spinach.

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.     

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.     

The spirilloxanthine-pathway carotenoid incorporation into light-harvesting complexes of Ectothiorhodospira haloalkaliphila in vitro

Carotenoidless light-harvesting complexes (DPA-complexes) LH1-RC and LH2 were isolated from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila in which carotenoid biosynthesis was suppressed with diphenylamine (DPA). Carotenoids of the spirilloxanthine series, which were isolated from the same bacterium, were incorporated into the DPA-complexes in vitro with an efficiency of 95–100%. The comparison of characteristics of the complexes with the incorporated carotenoids and the control complexes showed that the LH2 complexes with the incorporated carotenoids restored their absorption spectra, circular dichroism signals, and energy transfer from carotenoids to bacteriochlorophyll, which indicates that carotenoids were correctly incorporated into the structure of this complex.