Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

Contrasting packing modes for tubular assemblies in chlorosomes.

The largest light-harvesting antenna in nature, the chlorosome, is a heterogeneous helical BChl self-assembly that has evolved in green bacteria to harvest light for performing photosynthesis in low-light environments. Guided by NMR chemical shifts and distance constraints for Chlorobaculum tepidum wild-type chlorosomes, the two contrasting packing modes for syn-anti parallel stacks of BChl c to form polar 2D arrays, with dipole moments adding up, are explored. Layered assemblies were optimized using local orbital density functional and plane wave pseudopotential methods. The packing mode with the lowest energy contains syn-anti and anti-syn H-bonding between stacks. It can accommodate R and S epimers, and side chain variability. For this packing, a match with the available EM data on the subunit axial repeat and optical data is obtained with multiple concentric cylinders for a rolling vector with the stacks running at an angle of 21° to the cylinder axis and with the BChl dipole moments running at an angle ߠ∼ 55° to the tube axis, in accordance with optical data. A packing mode involving alternating syn and anti parallel stacks that is at variance with EM appears higher in energy. A weak cross-peak at -6 ppm in the MAS NMR with 50 kHz spinning, assigned to C-181, matches the shift of antiparallel dimers, which possibly reflects a minor impurity-type fraction in the self-assembled BChl c.

Nature of S-States in the Oxygen-Evolving Complex Resolved by High-Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy.

Photosystem II, the water splitting enzyme of photosynthesis, utilizes the energy of sunlight to drive the four-electron oxidation of water to dioxygen at the oxygen-evolving complex (OEC). The OEC harbors a Mn4CaO5 cluster that cycles through five oxidation states Si (i = 0-4). The S3 state is the last metastable state before the O2 evolution. Its electronic structure and nature of the S2 → S3 transition are key topics of persisting controversy. Most spectroscopic studies suggest that the S3 state consists of four Mn(IV) ions, compared to the Mn(III)Mn(IV)3 of the S2 state. However, recent crystallographic data have received conflicting interpretations, suggesting either metal- or ligand-based oxidation, the latter leading to an oxyl radical or a peroxo moiety in the S3 state. Herein, we utilize high-energy resolution fluorescence detected (HERFD) X-ray absorption spectroscopy to obtain a highly resolved description of the Mn K pre-edge region for all S-states, paying special attention to use chemically unperturbed S3 state samples. In combination with quantum chemical calculations, we achieve assignment of specific spectroscopic features to geometric and electronic structures for all S-states. These data are used to confidently discriminate between the various suggestions concerning the electronic structure and the nature of oxidation events in all observable catalytic intermediates of the OEC. Our results do not support the presence of either peroxo or oxyl in the active configuration of the S3 state. This establishes Mn-centered storage of oxidative equivalents in all observable catalytic transitions and constrains the onset of the O-O bond formation until after the final light-driven oxidation event.

On the PsbS-induced quenching in the plant major light-harvesting complex LHCII studied in proteoliposomes.

Non-photochemical quenching (NPQ) in photosynthetic organisms provides the necessary photoprotection that allows them to cope with largely and quickly varying light intensities. It involves deactivation of excited states mainly at the level of the antenna complexes of photosystem II using still largely unknown molecular mechanisms. In higher plants the main contribution to NPQ is the so-called qE-quenching, which can be switched on and off in a few seconds. This quenching mechanism is affected by the low pH-induced activation of the small membrane protein PsbS which interacts with the major light-harvesting complex of photosystem II (LHCII). We are reporting here on a mechanistic study of the PsbS-induced LHCII quenching using ultrafast time-resolved chlorophyll (Chl) fluorescence. It is shown that the PsbS/LHCII interaction in reconstituted proteoliposomes induces highly effective and specific quenching of the LHCII excitation by a factor ≥ 20 via Chl-Chl charge-transfer (CT) state intermediates which are weakly fluorescent. Their characteristics are very broad fluorescence bands pronouncedly red-shifted from the typical unquenched LHCII fluorescence maximum. The observation of PsbS-induced Chl-Chl CT-state emission from LHCII in the reconstituted proteoliposomes is highly reminiscent of the in vivo quenching situation and also of LHCII quenching in vitro in aggregated LHCII, indicating a similar quenching mechanism in all those situations. The PsbS mutant lacking the two proton sensing Glu residues induced significant, but much smaller, quenching than wild type. Added zeaxanthin had only minor effects on the yield of quenching in the proteoliposomes. Overall our study shows that PsbS co-reconstituted with LHCII in liposomes represents an excellent in vitro model system with characteristics that are reflecting closely the in vivo qE-quenching situation.

Characterization of fluorescent chlorophyll charge-transfer states as intermediates in the excited state quenching of light-harvesting complex II.

Light-harvesting complex II (LHCII) is the major antenna complex in higher plants and green algae. It has been suggested that a major part of the excited state energy dissipation in the so-called "non-photochemical quenching" (NPQ) is located in this antenna complex. We have performed an ultrafast kinetics study of the low-energy fluorescent states related to quenching in LHCII in both aggregated and the crystalline form. In both sample types the chlorophyll (Chl) excited states of LHCII are strongly quenched in a similar fashion. Quenching is accompanied by the appearance of new far-red (FR) fluorescence bands from energetically low-lying Chl excited states. The kinetics of quenching, its temperature dependence down to 4 K, and the properties of the FR-emitting states are very similar both in LHCII aggregates and in the crystal. No such FR-emitting states are found in unquenched trimeric LHCII. We conclude that these states represent weakly emitting Chl-Chl charge-transfer (CT) states, whose formation is part of the quenching process. Quantum chemical calculations of the lowest energy exciton and CT states, explicitly including the coupling to the specific protein environment, provide detailed insight into the chemical nature of the CT states and the mechanism of CT quenching. The experimental data combined with the results of the calculations strongly suggest that the quenching mechanism consists of a sequence of two proton-coupled electron transfer steps involving the three quenching center Chls 610/611/612. The FR-emitting CT states are reaction intermediates in this sequence. The polarity-controlled internal reprotonation of the E175/K179 aa pair is suggested as the switch controlling quenching. A unified model is proposed that is able to explain all known conditions of quenching or non-quenching of LHCII, depending on the environment without invoking any major conformational changes of the protein.

Five-coordinate MnIV intermediate in the activation of nature's water splitting cofactor.

Nature's water splitting cofactor passes through a series of catalytic intermediates (S0-S4) before O-O bond formation and O2 release. In the second last transition (S2 to S3) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all MnIV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S4 How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S3') in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (>50%) in (i) chemical-modified cofactors in which Ca2+ is exchanged with Sr2+; the Mn4O5Sr cofactor remains active, but the S2-S3 and S3-S0 transitions are slower than for the Mn4O5Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S3' electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S3 signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S2 state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S3' cofactor is similar to the high spin form of the preceding S2 state, which contains a cuboidal Mn3O4Ca unit tethered to an external, 5-coordinate Mn ion (Mn4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn4. The interaction of ammonia with the cofactor is also discussed.

"Super-quenching" state protects Symbiodinium from thermal stress - Implications for coral bleaching.

The global rise in sea surface temperatures causes regular exposure of corals to high temperature and high light stress, leading to worldwide disastrous coral bleaching events (loss of symbiotic dinoflagellates (Symbiodinium) from reef-building corals). Our picosecond chlorophyll fluorescence experiments on cultured Symbiodinium clade C cells exposed to coral bleaching conditions uncovered the transformations of the alga's photosynthetic apparatus (PSA) that activate an extremely efficient non-photochemical "super-quenching" mechanism. The mechanism is associated with a transition from an initially heterogeneous photosystem II (PSII) pool to a homogeneous "spillover" pool, where nearly all excitation energy is transferred to photosystem I (PSI). There, the inherently higher stability of PSI and high quenching efficiency of P(700)(+) allow dumping of PSII excess excitation energy into heat, resulting in almost complete cessation of photosynthetic electron transport (PET). This potentially reversible "super-quenching" mechanism protects the PSA against destruction at the cost of a loss of photosynthetic activity. We suggest that the inhibition of PET and the consequent inhibition of organic carbon production (e.g. sugars) in the symbiotic Symbiodinium provide a trigger for the symbiont expulsion, i.e. bleaching.

Effect of the LHCII pigment-protein complex aggregation on photovoltaic properties of sensitized TiO2 solar cells.

A modified dye-sensitized solar cell consisting of a thin TiO2 barrier layer sensitized with natural trimeric light-harvesting complex II (LHCII) from spinach was used as a biomimetic model to study the effects of LHCII aggregation on the photovoltaic properties. The aggregation of individual trimers induced molecular reorganization, which dramatically increased the photocurrent. The morphology of small- and large-size LHCII aggregates deposited on a surface was confirmed by atomic force microscopy. Enhanced LHCII immobilization was accomplished via electrostatic interaction with amine-functionalized photoanodes. The photocurrent responses of the assembled solar cells under illumination at three characteristic wavelength bands in the UV-Vis absorption spectra of LHCII solutions confirmed that a significant photocurrent was generated by LHCII photosensitizers. The enhanced photocurrent by large aggregated LHCII is shown to correlate with the quenching in the far-red fluorescence deriving from chlorophyll-chlorophyll charge transfer states that are effectively coupled with the TiO2 surface and thus inject electrons into the TiO2 conduction band. The large aggregated LHCII with more chlorophyll-chlorophyll charge transfer states is a much better sensitizer since it injects electrons more efficiently into the conduction band of TiO2 than the small aggregated LHCII mostly consisting of unquenched chlorophyll excited state. The assembled solar cells demonstrated remarkable stability in both aqueous buffer and acetonitrile electrolytes over 30 days.

Charge-transfer character of the low-energy Chl a Q(y) absorption band in aggregated light harvesting complexes II.

One of the key functions of the major light harvesting complex II (LHCII) of higher plants is to protect Photosystem II from photodamage at excessive light conditions in a process called "non-photochemical quenching" (NPQ). Using hole-burning (HB) spectroscopy, we investigated the nature of the low-energy absorption band in aggregated LHCII complexes - which are highly quenched and have been established as a good in vitro model for NPQ. Nonresonant holes reveal that the lowest energy state (located near 683.3 nm) is red-shifted by ~4 nm and significantly broader (by a factor of 4) as compared to nonaggregated trimeric LHCII. Resonant holes burned in the low-energy wing of the absorption spectrum (685-710 nm) showed a high electron-phonon (el-ph) coupling strength with a Huang-Rhys factor S of 3-4. This finding combined with the very low HB efficiency in the long-wavelength absorption tail is consistent with a dominant charge-transfer (CT) character of the lowest energy transition(s) in aggregated LHCII. The value of S decreases at shorter wavelengths (<685 nm), in agreement with previous studies (J. Pieper et al., J. Phys. Chem. B 1999, 103, 2422-2428), proving that the low-energy excitonic state is strongly mixed with the CT states. Our findings support the mechanistic model in which Chl-Chl CT states formed in aggregated LHCII are intermediates in the efficient excited state quenching process (M. G. Müller et al., Chem. Phys. Chem. 2010, 11, 1289-1296; Y. Miloslavina et al., FEBS Lett. 2008, 582, 3625-3631).

Two different mechanisms cooperate in the desiccation-induced excited state quenching in Parmelia lichen.

The highly efficient desiccation-induced quenching in the poikilohydric lichen Parmelia sulcata has been studied by ultrafast fluorescence spectroscopy at room temperature (r.t.) and cryogenic temperatures in order to elucidate the quenching mechanism(s) and kinetic reaction models. Analysis of the r.t. data by kinetic target analysis reveals that two different quenching mechanisms contribute to the protection of photosystem II (PS II). The first mechanism is a direct quenching of the PS II antenna and is related to the characteristic F740 nm fluorescence band. Based on the temperature dependence of its spectra and the kinetics, this mechanism is proposed to reflect the formation of a fluorescent (F740) chlorophyll-chlorophyll charge-transfer state. It is discussed in relation to a similar fluorescence band and quenching mechanism observed in light-induced nonphotochemical quenching in higher plants. The second and more efficient quenching process (providing more than 70% of the total PS II quenching) is shown to involve an efficient spillover (energy transfer) from PS II to PS I which can be prevented by a short glutaraldehyde treatment. Desiccation causes a thylakoid-membrane rearrangement which brings into direct contact the PS II and PS I units. The energy transferred to PS I in the spillover process is then quenched highly efficiently in PS I due to the formation of a long-lived P700(+) state in the dried state in the light. As a consequence, both PS II and PS I are protected very efficiently against photodestruction. This dual quenching mechanism is supported by the low temperature kinetics data.

An NMR comparison of the light-harvesting complex II (LHCII) in active and photoprotective states reveals subtle changes in the chlorophyll a ground-state electronic structures.

To protect the photosynthetic apparatus against photo-damage in high sunlight, the photosynthetic antenna of oxygenic organisms can switch from a light-harvesting to a photoprotective mode through the process of non-photochemical quenching (NPQ). There is growing evidence that light-harvesting proteins of photosystem II participate in photoprotection by a built-in capacity to switch their conformation between light-harvesting and energy-dissipating states. Here we applied high-resolution Magic-Angle Spinning Nuclear Magnetic Resonance on uniformly (13)C-enriched major light-harvesting complex II (LHCII) of Chlamydomonas reinhardtii in active or quenched states. Our results reveal that the switch into a dissipative state is accompanied by subtle changes in the chlorophyll (Chl) a ground-state electronic structures that affect their NMR responses, particularly for the macrocycle (13)C4, (13)C5 and (13)C6 carbon atoms. Inspection of the LHCII X-ray structures shows that of the Chl molecules in the terminal emitter domain, where excited-state energy accumulates prior to further transfer or dissipation, the C4, 5 and 6 atoms are in closest proximity to lutein; supporting quenching mechanisms that involve altered Chl-lutein interactions in the dissipative state. In addition the observed changes could represent altered interactions between Chla and neoxanthin, which alters its configuration under NPQ conditions. The Chls appear to have increased dynamics in unquenched, detergent-solubilized LHCII. Our work demonstrates that solid-state Nuclear Magnetic Resonance is applicable to investigate high-resolution structural details of light-harvesting proteins in varied functional conditions, and represents a valuable tool to address their molecular plasticity associated with photoprotection.

Structural variability in wild-type and bchQ bchR mutant chlorosomes of the green sulfur bacterium Chlorobaculum tepidum.

The self-aggregated state of bacteriochlorophyll (BChl) c molecules in chlorosomes belonging to a bchQ bchR mutant of the green sulfur bacteria Chlorobaculum tepidum, which mostly produces a single 17(2)-farnesyl-(R)-[8-ethyl,12-methyl]BChl c homologue, was characterized by solid-state nuclear magnetic resonance (NMR) spectroscopy and high-resolution electron microscopy. A nearly complete (1)H and (13)C chemical shift assignment was obtained from well-resolved homonuclear (13)C-(13)C and heteronuclear (1)H-(13)C NMR data sets collected from (13)C-enriched chlorosome preparations. Pronounced doubling (1:1) of specific (13)C and (1)H resonances revealed the presence of two distinct and nonequivalent BChl c components, attributed to all syn- and all anti-coordinated parallel stacks, depending on the rotation of the macrocycle with respect to the 3(1)-methyl group. Steric hindrance from the 20-methyl functionality induces structural differences between the syn and anti forms. A weak but significant and reproducible reflection at 1/0.69 nm(-1) in the direction perpendicular to the curvature of cylindrical segments observed with electron microscopy also suggests parallel stacking of BChl c molecules, though the observed lamellar spacing of 2.4 nm suggests weaker packing than for wild-type chlorosomes. We propose that relaxation of the pseudosymmetry observed for the wild type and a related BChl d mutant leads to extended domains of alternating syn and anti stacks in the bchQ bchR chlorosomes. Domains can be joined to form cylinders by helical syn-anti transition trajectories. The phase separation in domains on the cylindrical surface represents a basic mechanism for establishing suprastructural heterogeneity in an otherwise uniform supramolecular scaffolding framework that is well-ordered at the molecular level.

Organization of bacteriochlorophylls in individual chlorosomes from Chlorobaculum tepidum studied by 2-dimensional polarization fluorescence microscopy.

Chlorosomes are the largest and most efficient natural light-harvesting systems and contain supramolecular assemblies of bacteriochlorophylls that are organized without proteins. Despite a recent structure determination for chlorosomes from Chlorobaculum tepidum (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525), the issue of a possible large structural disorder is still discussed controversially. We have studied individual chlorosomes prepared under very carefully controlled growth condition by a novel 2-dimensional polarization single molecule imaging technique giving polarization information for both fluorescence excitation and emission simultaneously. Contrary to the existing literature data, the polarization degree or modulation depth (M) for both excitation (absorption) and emission (fluorescence) showed extremely narrow distributions. The fluorescence was always highly polarized with M ≈ 0.77, independent of the excitation wavelength. Moreover, the fluorescence spectra of individual chlorosomes were identical within the error limits. These results lead us to conclude that all chlorosomes possess the same type of internal organization in terms of the arrangement of the bacteriochlorophyll c transition dipole moments and their total excitonic transition dipole possess a cylindrical symmetry in agreement with the previously suggested concentric multitubular chlorophyll aggregate organization (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525).

First solid-state NMR analysis of uniformly ¹³C-enriched major light-harvesting complexes from Chlamydomonas reinhardtii and identification of protein and cofactor spin clusters.

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in ¹³C-¹³C dipolar homonuclear correlation spectra on a uniformly ¹³C-labeled sample. In particular, we were able to resolve several chlorophyll 13¹ carbon resonances that are sensitive to hydrogen bonding to the 13¹-keto carbonyl group. The data show that ¹³C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.

On the nature of the "dark S*" excited state of ß-carotene.

Femtosecond transient absorption spectroscopy has been applied to the isolated carotenoid ß-carotene under a large variety of experimental conditions regarding solvent, temperature, excitation wavelength, and intensity to study the excited state relaxation dynamics in order to elucidate the origin of the so-called "dark S* state", which has been discussed very controversially in the literature. The results are analyzed in terms of lifetime density maps, and various kinetic models are tested on the data. The sample purification was found to be critical. The appearance of a component with a lifetime longer than that of the relaxed S(1) state (i.e., τ > 10 ps), which has been associated previously with the S* (or S(‡)) state is due to the presence of an impurity. For pure samples, four lifetimes are typically observed (all ≤10 ps at room temperature). Consideration of the large body of experimental data leads us to exclude relaxation schemes implying a separate "dark S* state" in ß-carotene formed in parallel to the normal S(2) → S(1) relaxation scheme. Vibrational cooling in the S(1) state can explain fully all the features of the transient spectra on the picosecond time scale within a S(2) → S(1v) → S(1v') → S(1) → S(0) relaxation scheme without invoking any additional electronic or distinctly different conformational states. Thus, we exclude assignments of the previously reported "S* state" signals in ß-carotene (i) to require the postulate of a separate electronic state, (ii) to require the postulate of a large conformational change and/or a partial cis configuration formed in the relaxation pathway, or (iii) to require a vibrationally excited ground state (GS) species. High intensity excitation leads in part to a two-photon excitation to the S(2N) state which upon relaxation gives rise to a different vibrational excitation pattern in the initially created hot S(1) state(s). The spectral changes in the S(1v) state observed upon both short wave excitation as well as high intensity excitation can be explained well by such a modified vibrational excitation pattern. In contrast, the variations in the difference spectra of the partially (S(1v')) and fully vibrationally relaxed S(1) states (S(1)) are minor. The data do not provide any evidence that would require one to postulate the existence of a separate "S* state".

Singlet energy dissipation in the photosystem II light-harvesting complex does not involve energy transfer to carotenoids.

The energy dissipation mechanism in oligomers of the major light-harvesting complex II (LHC II) from Arabidopsis thaliana mutants npq1 and npq2, zeaxanthin-deficient and zeaxanthin-enriched, respectively, has been studied by femtosecond transient absorption. The kinetics obtained at different excitation intensities are compared and the implications of singlet-singlet annihilation are discussed. Under conditions where annihilation is absent, the two types of LHC II oligomers show distributive biexponential (bimodal) kinetics with lifetimes of approximately 5-20 ps and approximately 200-400 ps having transient spectra typical for chlorophyll excited states. The data can be described kinetically by a two-state compartment model involving only chlorophyll excited states. Evidence is provided that neither carotenoid excited nor carotenoid radical states are involved in the quenching mechanism at variance with earlier proposals. We propose instead that a chlorophyll-chlorophyll charge-transfer state is formed in LHC II oligomers which is an intermediate in the quenching process. The relevance to non-photochemical quenching in vivo is discussed.

Alternating syn-anti bacteriochlorophylls form concentric helical nanotubes in chlorosomes.

Chlorosomes are the largest and most efficient light-harvesting antennae found in nature, and they are constructed from hundreds of thousands of self-assembled bacteriochlorophyll (BChl) c, d, or e pigments. Because they form very large and compositionally heterogeneous organelles, they had been the only photosynthetic antenna system for which no detailed structural information was available. In our approach, the structure of a member of the chlorosome class was determined and compared with the wild type (WT) to resolve how the biological light-harvesting function of the chlorosome is established. By constructing a triple mutant, the heterogeneous BChl c pigment composition of chlorosomes of the green sulfur bacteria Chlorobaculum tepidum was simplified to nearly homogeneous BChl d. Computational integration of two different bioimaging techniques, solid-state NMR and cryoEM, revealed an undescribed syn-anti stacking mode and showed how ligated BChl c and d self-assemble into coaxial cylinders to form tubular-shaped elements. A close packing of BChls via pi-pi stacking and helical H-bonding networks present in both the mutant and in the WT forms the basis for ultrafast, long-distance transmission of excitation energy. The structural framework is robust and can accommodate extensive chemical heterogeneity in the BChl side chains for adaptive optimization of the light-harvesting functionality in low-light environments. In addition, syn-anti BChl stacks form sheets that allow for strong exciton overlap in two dimensions enabling triplet exciton formation for efficient photoprotection.

Far-red fluorescence: a direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching.

Time-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll-chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl-Chl charge transfer state which plays a crucial part in the quenching.

Long-range organization of bacteriochlorophyll in chlorosomes of Chlorobium tepidum investigated by cryo-electron microscopy.

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryo-electron microscopy to study the long-range organization of bacteriochlorophyll (BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multi-layered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed.

Structural changes upon excitation of D1-D2-Cyt b559 photosystem II reaction centers depend on the beta-carotene content.

Different preparations of D1-D2-Cyt b559 complexes from spinach with different beta-carotene (Car) content [on average from <0.5 to 2 per reaction center (RC)] were studied by means of laser-induced optoacoustic spectroscopy. phiP680(+)Pheo(-) does not depend on the preparation (or on the Car content) inasmuch as the magnitude of the prompt heat (produced within 20 ns) does not vary for the different samples upon excitation at 675 and 620 nm. The energy level of the primary charge-separated state, P680(+)Pheo(-), was determined as EP680(+)Pheo(-) = 1.55 eV. Thus, an enthalpy change accompanying charge separation from excited P680 of deltaH*P680Pheo-->P680(+)Pheo(-) = -0.27 eV is obtained. Calculations using the heat evolved during the time-resolved decay of P680(+)Pheo(-) (< or = 100 ns) affords a triplet (3[P680Pheo]) quantum yield phi3[P680Pheo] = 0.5 +/- 0.14. The structural volume change, deltaV1, corresponding to the formation of P680(+)Pheo(-), strongly depends on the Car content; it is ca. -2.5 A3 molecule(-1) for samples with <0.5 Car on average, decreases (in absolute value) to -0.5 +/- 0.2 A3 for samples with an average of 1 Car, and remains the same for samples with two Cars per RC. This suggests that the Car molecules induce changes in the ground-state RC conformation, an idea which was confirmed by preferential excitation of Car with blue light, which produced different carotene triplet lifetimes in samples with 2 Car compared to those containing less carotene. We conclude that the two beta-carotenes are not structurally equivalent. Upon blue-light excitation (480 nm, preferential carotene absorption) the fraction of energy stored is ca. 60% for the 9Chl-2Car sample, whereas it is 40% for the preparations with one or less Cars on average, indicating different paths of energy distribution after Car excitation in these RCs with remaining chlorophyll antennae.