Carotenoid Excitation Research Articles

Selective Excitation of Carotenoids of the Allochromatium vinosum Light-Harvesting LH2 Complexes Leads to Oxidation of Bacteriochlorophyll.

The mechanism of bacteriochlorophyll photooxidation in light-harvesting complexes of a number of purple photosynthetic bacteria when the complexes are excited into the carotenoid absorption bands remains unclear for many years. Here, using narrow-band laser illumination we measured action spectrum of this process for the spectral ranges of carotenoid and bacteriochlorophyll. It is shown that bacteriochlorophyll excitation results in almost no photooxidation of these molecules, while carotenoid excitation leads to oxidation with quantum yield of about 0,0003. Low value of the yield enabled an assumption that the studied process is initiated by the triplet states of the main carotenoids of the complexes with the number of conjugated double-bond chain length of N = 11. Interaction of these states with oxygen facilitates formation, though with low efficiency, of the excited singlet oxygen, which oxidizes bacteriochlorophylls. The carotenoid triplet states are formed in the process of the earlier studied singlet-triplet fission. The obtained results point at the necessity of reconsidering the functions of carotenoids in the light-harvesting complexes of purple bacteria.

A different perspective for nonphotochemical quenching in plant antenna complexes

Light-harvesting complexes of plants exert a dual function of light-harvesting (LH) and photoprotection through processes collectively called nonphotochemical quenching (NPQ). While LH processes are relatively well characterized, those involved in NPQ are less understood. Here, we characterize the quenching mechanisms of CP29, a minor LHC of plants, through the integration of two complementary enhanced-sampling techniques, dimensionality reduction schemes, electronic calculations and the analysis of cryo-EM data in the light of the predicted conformational ensemble. Our study reveals that the switch between LH and quenching state is more complex than previously thought. Several conformations of the lumenal side of the protein occur and differently affect the pigments’ relative geometries and interactions. Moreover, we show that a quenching mechanism localized on a single chlorophyll-carotenoid pair is not sufficient but many chlorophylls are simultaneously involved. In such a diffuse mechanism, short-range interactions between each carotenoid and different chlorophylls combined with a protein-mediated tuning of the carotenoid excitation energies have to be considered in addition to the commonly suggested Coulomb interactions.

Excitation energy transfer between monomolecular layers of light harvesting LH2 and LH1-reaction centre complexes printed on a glass substrate.

Light-harvesting 2 (LH2) and light-harvesting 1 - reaction centre (RCLH1) complexes purified from the photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were cross-patterned on glass surfaces for energy transfer studies. Atomic force microscopy (AFM) images of the RCLH1 and LH2 patterns show the deposition of monomolecular layers of complexes on the glass substrate. Spectral imaging and fluorescence life-time imaging microscopy (FLIM) revealed that RCLH1 and LH2 complexes, sealed under physiological conditions, retained their native light-harvesting and energy transfer functions. Measurements of the amplitude and lifetime decay of fluorescence emission from LH2 complexes, the energy transfer donors, and gain of fluorescence emission from acceptor RCLH1 complexes, provide evidence for excitation energy transfer from LH2 to RCLH1. Directional energy transfer on the glass substrate was unequivocally established by using LH2-carotenoid complexes and RCLH1 complexes with genetically removed carotenoids. Specific excitation of carotenoids in donor LH2 complexes elicited fluorescence emission from RCLH1 acceptors. To explore the longevity of this novel nanoprinted photosynthetic unit, RCLH1 and LH2 complexes were cross-patterned on a glass surface and sealed under a protective argon atmosphere. The results show that both complexes retained their individual and collective functions and are capable of directional excitation energy transfer for at least 60 days.

The Singlet–Triplet Fission of Carotenoid Excitation in Light-Harvesting Complexes from Thermochromatium tepidum

Abstract—Illumination of purple phototrophic bacteria in the carotenoid absorption band of light harvesting complexes often leads to low energy efficiency of absorbed light. This is due to singlet–triplet fission of carotenoid excitation. In the present study, the nature of this process was explored with a phototrophic bacterium Thermochromatium tepidum as an example. It was demonstrated by time-resolved EPR spectroscopy and magnetic field modulation of the fluorescence yield that the concept of intramolecular excitation fission that was developed in several publications was not confirmed experimentally. Evidence of the intermolecular character of excitation fission that involves two carotenoid molecules of the light-harvesting pigment–protein complexes, LH1-RC and LH2, has been obtained. The advantages of intermolecular excitation fission for application in photovoltaic solar energy converters are discussed.

Singlet-triplet Fission of Carotenoid Excitation in the Purple Phototrophic Bacteria Thermochromatium tepidum.

Singlet fission of carotenoid excitation is studied in purple phototrophic bacterium Thermochromatium tepidum. Using time-resolved EPR and magnetic field-induced modulation of fluorescence yield it is shown that the concept of intramolecular excitation fission developed in a number of publications is not supported by the experimental results. The obtained data favor intermolecular fission mechanism involving two carotenoid molecules.

Chlorophyll–carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

Shifted excitation resonance Raman difference spectroscopy system suitable for the quantitative in vivo detection of carotenoids in human skin

A system for shifted excitation resonance Raman spectroscopy (SERRDS) suitable for the application in medical practice for the in vivo detection of carotenoids in human skin is presented. This system comprises a miniaturized (150 mm × 27 mm × 12 mm) handheld probe and a wavelength-tunable diode laser-based 488 nm SHG light source. The diode laser provides two closely spaced excitation wavelengths. In parallel with the resonance excitation of carotenoids in the skin, SERRDS separates the fluorescence background from the Raman peaks. Inhomogeneities of human skin are averaged by the applied spot diameter of 3 mm. The implemented optics are designed for a detection of carotenoids over the whole excitation spot area. The system was calibrated using skin phantoms, resulting in a detection limit of 0.03 nmol g−1 (beta-carotene per gram of skin/tissue) which is more than one order of magnitude below the average beta-carotene concentration in human skin.

Spectroscopic properties of the S1 state of linear carotenoids after excess energy excitation

Properties of the S1 state of neurosporene, spheroidene and lycopene were studied after excess energy excitation in the S2 state. Excitation of carotenoids into higher vibronic levels of the S2 state generates excess vibrational energy in the S1 state. The vibrationally hot S1 state relaxes faster when carotenoid is excited into the S2 state with excess energy, but the S1 lifetime remains constant regardless of which vibronic level of the S2 state is excited. The S∗ signal depends on excitation energy only for spheroidene, which is likely due to asymmetry of the molecule, facilitating conformations responsible for the S∗ signal.

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium Rhodobacter sphaeroides

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S2 (11Bu+) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S2 (11Bu+) state to BChl a via the Qx state of the latter, accounting for 60% of the total transfer. The elevated S2 (11Bu+) → Qx transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S2 (11Bu+) → S0 (11Ag−) carotenoid emission and Qx absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.

Evaluating the Nature of So-Called S*-State Feature in Transient Absorption of Carotenoids in Light-Harvesting Complex 2 (LH2) from Purple Photosynthetic Bacteria.

Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation–bacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.

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.

Photoprotection in Plants Involves a Change in Lutein 1 Binding Domain in the Major Light-harvesting Complex of Photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

Unusual enhancement of triplet carotenoid formation in pigmentprotein complexes as revealed by femtosecond pump-probe spectroscopy

Ultrafast excited state dynamics of carotenoids in solution and bound to pigment-protein complexes have been investigated by means of femtosecond pump-probe spectroscopic measurements. Ultrafast triplet formation of carotenoids bound to pigmentprotein complexes was observed upon excitation of both carotenoids and bacteriochlorophyll. The anomalous reaction of triplet formation of carotenoids bound to pigment-protein complexes is discussed in terms of ultrafast triplet-triplet excitation energy transfer between carotenoid and bacteriochlorophyll.

The dependence of excitation energy transfer pathways on conjugation length of carotenoids in purple bacterial photosynthetic antennae

AbstractUltrafast excited state dynamics of carotenoids in solution and bound to pigment–protein complexes have been investigated by femtosecond pump‐probe spectroscopic measurements. Possible excitation energy transfer (EET) pathways between carotenoids and bacteriochlorophylls and their efficiency depend strongly on the conjugation length of carotenoids. In the case of Rhodobacter sphaeroides 2.4.1, dual EET channels from carotenoid to bacteriochlorophyll (S2 → Qx and S1 → Qy) upon excitation of carotenoid were observed. In the case of Rhodospirillum rubrum S1, on the other hand, the EET pathway of S1 → Qy upon excitation of carotenoid was closed, whereas reverse energy transfer from bacteriochlorophyll to carotenoid was clearly observed upon excitation of bacteriochlorophyll. The role of carotenoids in the light‐harvesting complexes is discussed in terms of their conjugation length.

Energy Transfer, Excited-State Deactivation, and Exciplex Formation in Artificial Caroteno-Phthalocyanine Light-Harvesting Antennas

We present results from transient absorption spectroscopy on a series of artificial light-harvesting dyads made up of a zinc phthalocyanine (Pc) covalently linked to carotenoids with 9, 10, or 11 conjugated carbon-carbon double bonds, referred to as dyads 1, 2, and 3, respectively. We assessed the energy transfer and excited-state deactivation pathways following excitation of the strongly allowed carotenoid S2 state as a function of the conjugation length. The S2 state rapidly relaxes to the S* and S1 states. In all systems we detected a new pathway of energy deactivation within the carotenoid manifold in which the S* state acts as an intermediate state in the S2-->S1 internal conversion pathway on a sub-picosecond time scale. In dyad 3, a novel type of collective carotenoid-Pc electronic state is observed that may correspond to a carotenoid excited state(s)-Pc Q exciplex. The exciplex is only observed upon direct carotenoid excitation and is nonfluorescent. In dyad 1, two carotenoid singlet excited states, S2 and S1, contribute to singlet-singlet energy transfer to Pc, making the process very efficient (>90%) while for dyads 2 and 3 the S1 energy transfer channel is precluded and only S2 is capable of transferring energy to Pc. In the latter two systems, the lifetime of the first singlet excited state of Pc is dramatically shortened compared to the 9 double-bond dyad and model Pc, indicating that the carotenoid acts as a strong quencher of the phthalocyanine excited-state energy.

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.

Ultrafast Dynamics of Carotenoid Excited States — From Solution to Natural and Artificial Systems

AbstractFor Abstract see ChemInform Abstract in Full Text.

Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems.

Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems.

Physiological Significance of Overproduced Carotenoids in Transformants of the Cyanobacterium Synechococcus PCC7942

Abstract The functional location of carotenoids in the photosynthetic apparatus of -crtB and -pys transformants of the cyanobacterium Synechococcus PCC7942 was studied and compared with a control strain -pFP 1-3. These transformants overproduce carotenoids due to the insertion of an additional foreign phytoene synthase gene. A higher carotenoid content was found for -crtB and -pys transformants both in whole cells and isolated membranes; the -crtB transformant was also enriched with chlorophyll. 77-K fluorescence emission and excitation spectra of the phycobilin-free membranes were examined for a possible location of overproduced carotenoids in pigment-protein complexes in situ. A similar ratio of the amplitudes of fluorescence bands at 716 and 695 nm emitted by photosystems I and II, found for the three strains, indicates that the stoichiometry between photosystems of the transformants was not changed. Overproduced carotenoids are not located in the core antenna of photosys­ tem I, since 77-K fluorescence excitation spectra for photosystem I of isolated membranes from the studied strains do not differ in the region of carotenoid absorption. When illuminated with light of the same intensity but different quality, absorbed preferentially by either carotenoids, chlorophylls or phycobilins, respectively, oxygen evolution was found always higher in the transformants -crtB and -pys than in -pFP 1-3 control cells. Identical kinetics of fluorescence induction of all strains under carotenoid excitation did not reveal a higher activity of photosystem II in cells enriched with carotenoids. It is suggested that overproduced carotenoids of the transformants are not involved in photosynthetic light-harvesting; rather they may serve to protect the cells and its membranes against photodestruction.

Probing Electron Transport through and around Photosystem II in vivo by the Combined Use of Photoacoustic Spectroscopy and Chlorophyll Fluorometry

AbstractPhotoacoustic spectroscopy and chlorophyll fluorometry were used in combination to monitor in vivo several aspects of the photosystem II (PSII) function (O2 evolution, electron transport, and energy storage) in leaves of various plant genotypes. Exposure of barley leaves to 45°C in low light induced a rapid and complete disruption of the O2‐evolving complex of PSII, as shown by the suppression of the photobaric component of the photoacoustic signal. The photoacoustically measured loss of O2 evolution during heat stress (i) was confirmed by polarographic measurements of O2 exchange by the leaf samples, (ii) was shown to be independent of the activity of the carbonic anhydrase, and (iii) was not correlated with a concomitant suppression of the photochemical energy storage and the electron transport activity of PSII. The maintenance of a significant PSII photochemical activity in the absence of O2 evolution is indicative of cyclic electron transport around PSII which bypasses the O2‐evolving complex. The modulation‐frequency dependence of the energy stored during cyclic PSII activity, its inhibition by the herbicide diuron, and its insensitivity to the plastoquinol antagonist DBMIB (2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone) indicated that QB, the secondary electron acceptor of PSII, is involved in the electron cycle and that the rate‐limiting step of the cycle is a reaction with a half‐time of ca. 1.5 ms. In the absence of heat stress, overreduction of the plastoquinone pool by strong illumination or by leaf infiltration with DBMIB was not sufficient to induce appreciable cyclic electron transport around PSII. The electron cycle was insensitive to preferential excitation of carotenoids with a blue‐green light and was not significantly affected by the operation of the xanthophyll cycle. Interruption of the electron cycle by diuron during prolonged heat treatment in moderate light was accompanied by a marked peroxidation of the chloroplast thylakoid membranes, as revealed by the appearance of chlorophyll thermoluminescence bands in the temperature range 70–120°C, demonstrating the photoprotective function of electron cycling around PSII.