Low-temperature time-resolved spectroscopic study of the major light-harvesting complex of Amphidinium carterae

The major light-harvesting complex of Amphidinium (A.) carterae, chlorophyll-a–chlorophyll-c ₂–peridinin–protein complex (acpPC), was studied using ultrafast pump-probe spectroscopy at low temperature (60 K). An efficient peridinin–chlorophyll-a energy transfer was observed. The stimulated emission signal monitored in the near-infrared spectral region was stronger when redder part of peridinin pool was excited, indicating that these peridinins have the S₁/ICT (intramolecular charge-transfer) state with significant charge-transfer character. This may lead to enhanced energy transfer efficiency from “red” peridinins to chlorophyll-a. Contrary to the water-soluble antenna of A. carterae, peridinin–chlorophyll-a protein, the energy transfer rates in acpPC were slower under low-temperature conditions. This fact underscores the influence of the protein environment on the excited-state dynamics of pigments and/or the specificity of organization of the two pigment–protein complexes.     

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.     

Amphidinol C,a major polyketide from an Irish strain of the dinoflagellate Amphidinium carterae

An Irish strain of the dinoflagellate Amphidinium carterae was previously shown to produce antibacterial amphidinol derivatives of unknown masses. Inspection of the major metabolites present in a bulk culture of this strain led to the isolation and structure elucidation of a new amphidinol derivative named amphidinol C featuring an unprecedented tetrahydropyran ring between the positions C-7 and C-11. The structure was determined using extensive analyses of NMR and MS data and comparison with data of analogues. The new ring was proposed to stem from a nucleophilic substitution of the sulphate present on the side chain of Amphidinol B. The major metabolites isolated were tested for their antibacterial and antifungal activities and Amphidinol C showed moderate fungicidal activity against yeast and filamentous fungi at 8–16 µg mL⁻¹.     

Energy transfer pathways in the CAC light-harvesting complex of Rhodomonas salina

Photosynthetic organisms had to evolve diverse mechanisms of light-harvesting to supply photosynthetic apparatus with enough energy. Cryptophytes represent one of the groups of photosynthetic organisms combining external and internal antenna systems. They contain one type of immobile phycobiliprotein located at the lumenal side of the thylakoid membrane, together with membrane-bound chlorophyll a/c antenna (CAC). Here we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the CAC proteins of cryptophyte Rhodomonas salina. The major CAC carotenoid, alloxanthin, is a cryptophyte-specific carotenoid, and it is the only naturally-occurring carotenoid with two triple bonds in its structure. In order to explore the energy transfer pathways within the CAC complex, three excitation wavelengths (505, 590, and 640 nm) were chosen to excite pigments in the CAC antenna. The excitation of Chl c at either 590 or 640 nm proves efficient energy transfer between Chl c and Chl a. The excitation of alloxanthin at 505 nm shows an active pathway from the S₂ state with efficiency around 50%, feeding both Chl a and Chl c with approximately 1:1 branching ratio, yet, the S₁-route is rather inefficient. The 57 ps energy transfer time to Chl a gives ~25% efficiency of the S₁ channel. The low efficiency of the S₁ route renders the overall carotenoid-Chl energy transfer efficiency low, pointing to the regulatory role of alloxanthin in the CAC antenna.     

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.     

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the F?rster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.     

Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

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.     

Crystallization and preliminary X-ray analysis of a peridinin-chlorophyll a protein from Amphidinium carterae

Crystals of a water-soluble (Mr approximately 39,000) peridinin-chlorophyll a protein from Amphidinium carterae are reported. The crystals diffract to 2.2 A and belong to a monoclinic (B2) and a triclinic (P1) space group. Spectra of the protein in the crystal and in solution are almost identical.     

Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness

The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae (Plymouth 450)

The marine dinoflagellate Amphidinium carterae (Plymouth 450) releases several water-soluble peridinin-chlorophyll a proteins after freezethawing. These chromoproteins have a molecular weight of 39.2 × 10³ and are comprised of noncovalently bound peridinin and chlorophyll a and a nonoligomeric protein. They have distinct isoelectric points and may be resolved into six components by either isoelectric focusing on polyacrylamide gel or ion exchange chromatography. The predominant chromoprotein, which has a pI of 7.5, constitutes about 90% of the extractable peridinin-chlorophyll a protein. It consists of an alanine-rich apoprotein of molecular weight 31.8 × 10³ stoichiometrically associated with 9 peridinin and 2 chlorophyll a molecules. Additionally, the peridinin-chlorophyll a proteins with pI values of 7.6 and 6.4 were purified and found to have amino acid and chromophore composition essentially identical with the pI 7.5 protein. Peridinin-chlorophyll a protein, pI 7.5, after treatment at alkaline pH was transformed into several more acid pI forms of the protein, strongly suggesting that the naturally occurring proteins are deamidation products of a single protein. Fluorescence excitation and emission spectra demonstrate that light energy absorbed by peridinin induces chlorophyll a fluorescence presumably by intramolecular energy transfer. The peridinin-chlorophyll a proteins presumably function in vivo as photosynthetic light-harvesting pigments.     

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl--D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.Abbreviations LHC II light harvesting chlorophyll protein complex associated with photosystem II - LHCP1 and LHCP3 monomeric and oligomeric forms of LHC II, respectively, observed on non-denaturing gels - LiDS lithium dodecylsulphate - PMSF phenylmethylsulfonyl fluoride     

Identification by time-resolved EPR of the peridinins directly involved in chlorophyll triplet quenching in the peridinin-chlorophyll a-protein from Amphidinium carterae

The mechanism of triplet-triplet energy transfer in the peridinin-chlorophyll-protein (PCP) from Amphidinium carterae was investigated by time-resolved EPR (TR-EPR). The approach exploits the concept of spin conservation during triplet-triplet energy transfer, which leads to spin polarization conservation in the observed TR-EPR spectra. The acceptor (peridinin) inherits the polarization of the donor (chlorophyll) in a way which depends on the relative geometrical arrangement of the donor-acceptor couple. Starting from the initially populated chlorophyll triplet state and taking the relative positions among Chls and peridinins from the X-ray structure of PCP, we calculated the expected triplet state polarization of any peridinin in the complex. Comparison with the experimental data allowed us to propose a path for triplet quenching in the protein. The peridinin-chlorophyll pair directly involved in the triplet-triplet energy transfer coincides with the one having the shortest center to center distance. A water molecule, which is coordinated to the central Mg atom of the Chl, is also placed in close contact with the peridinin. The results support the concept of localization of the triplet state mainly in one specific peridinin in each of the two pigment subclusters related by a pseudo C2 symmetry.     

Pigment-pigment interactions in PCP of Amphidinium carterae investigated by nonlinear polarization spectroscopy in the frequency domain

Peridinin-chlorophyll a-protein (PCP) is a unique antenna complex in dinoflagellates that employs peridinin (a carotenoid) as its main light-harvesting pigment. Strong excitonic interactions between peridinins, as well as between peridinins and chlorophylls (Chls) a, can be expected from the short intermolecular distances revealed by the crystal structure. Different experimental approaches of nonlinear polarization spectroscopy in the frequency domain (NLPF) were used to investigate the various interactions between pigments in PCP of Amphidinium carterae at room temperature. Lineshapes of NLPF spectra indicate strong excitonic interactions between the peridinin's optically allowed S(2) (1Bu(+)) states. A comprehensive subband analysis of the distinct NLPF spectral substructure in the peridinin region allows us to assign peridinin subbands to the two Chls a in PCP having different S(1)-state lifetimes. Peridinin subbands at 487, 501, and 535 nm were assigned to the longer-lived Chl, whereas a peridinin subband peaking at 515 nm was detected in both clusters. Certain peridinin(s), obviously corresponding to the subband centered at 487 nm, show(s) specific (possibly Coulombic?) interaction between the optically dark S(1)(2A(g)(-)) and/or intramolecular charge-transfer (ICT) state and S(1) of Chl a. The NLPF spectrum, hence, indicates that this peridinin state is approximately isoenergetic or slightly above S(1) of Chl a. A global subband analysis of absorption and NLPF spectra reveals that the Chl a Q(y)-band consists of two subbands (peaking at 669 and 675 nm and having different lifetimes), confirmed by NLPF spectra recorded at high pump intensities. At the highest applied pump intensities an additional band centered at S(1)/ICT transition of peridinin.