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Evidence for long-range excitation migration in macroaggregates of the chlorophyll a/b light-harvesting antenna complexes
Abstract
We investigated the picosecond transient absorbance kinetics under singlet-singlet annihilation conditions and the steady-state spectroscopic features, absorbance, circular dichroism and low-temperature fluorescence spectra, in large, three-dimensional, stacked lamellar aggregates of the purified light-harvesting chlorophyll ab complexes (LHCII) and its form of small aggregates. Our data strongly suggest that the macroorganizational parameters significantly influence the spectroscopic properties and strongly affect the energy migration pathways in the aggregates. In small aggregates (d ≈ 100 nm) of LHCII trimers the excitation energy migration could be characterized with a percolation type of excitation migration in a small cluster of chromophores. In contrast, in chirally organized macroaggregates (d ≈ 2–4 μm) the annihilation kinetics were consistent with a model predicted for (infinitely) large three-dimensional aggregates, showing that LHCII macroaggregates can constitute a structural basis for long-range migration of the excitation energy.
... Recent evidence [1][2][3][4][5][6][7][8][9][10][11] suggests some individual light harvesting protein complexes (LHCs) and LHC subunits efficiently transfer excitons towards chemical reaction centers (RCs) via an interplay between excitonic quantum coherence, resonant protein vibrations, and thermal decoherence. The role of coherence in vivo is unclear however, where excitons are transferred through multi-LHC/RC aggregates over distances typically large compared with intra-LHC scales [12][13][14]. Here we assess the possibility of long-range coherent transfer in a simple chromophore network with disordered site and transfer coupling energies. ...
... The bath Hamiltonian H B = q ω(q)a(q) † a(q) is the sum over harmonic vibrational modes with wave vectors q and frequencies ω(q). The system-bath interaction is H SB = i qĜ (12) (q) a(q) − a(−q) † , whereĜ (12) We derive the system-bath coupling, g (12) (q), spectral density and a Markovian master equation for ρ I , the combined density matrix of the site populations in an interaction picture with respect to H S + H B (see Supplementary Information). The spectral density, J(ω) = 2π q δ(ω − ω(q))|g (12) (q)| 2 , which quantifies the strength of the system-bath coupling, is ...
... The bath Hamiltonian H B = q ω(q)a(q) † a(q) is the sum over harmonic vibrational modes with wave vectors q and frequencies ω(q). The system-bath interaction is H SB = i qĜ (12) (q) a(q) − a(−q) † , whereĜ (12) We derive the system-bath coupling, g (12) (q), spectral density and a Markovian master equation for ρ I , the combined density matrix of the site populations in an interaction picture with respect to H S + H B (see Supplementary Information). The spectral density, J(ω) = 2π q δ(ω − ω(q))|g (12) (q)| 2 , which quantifies the strength of the system-bath coupling, is ...
Photosynthetic light harvesting provides a natural blueprint for
bioengineered and biomimetic solar energy and light detection technologies.
Recent evidence suggests some individual light harvesting protein complexes
(LHCs) and LHC subunits efficiently transfer excitons towards chemical reaction
centers (RCs) via an interplay between excitonic quantum coherence, resonant
protein vibrations, and thermal decoherence. The role of coherence in vivo is
unclear however, where excitons are transferred through multi-LHC/RC aggregates
over distances typically large compared with intra-LHC scales. Here we assess
the possibility of long-range coherent transfer in a simple chromophore network
with disordered site and transfer coupling energies. Through renormalization we
find that, surprisingly, decoherence is diminished at larger scales, and
long-range coherence is facilitated by chromophoric clustering. Conversely,
static disorder in the site energies grows with length scale, forcing
localization. Our results suggest sustained coherent exciton transfer may be
possible over distances large compared with nearest-neighbour (n-n) chromophore
separations, at physiological temperatures, in a clustered network with small
static disorder. This may support findings suggesting long-range coherence in
algal chloroplasts, and provides a framework for engineering large chromophore
or quantum dot high-temperature exciton transfer networks.
... Singlet-singlet annihilation usually becomes significant under high light intensities when more than one excitation is present in the same domain. For LHCII aggregates the annihilation takes place from several picoseconds to many hundreds of picoseconds (Barzda et al., 1996Barzda et al., , 2001). Lifetime images obtained in this work show that large heterogeneity in the fluorescence lifetimes exists not only for different LHCII aggregates, but even within one single aggregate. ...
... Therefore, apart from the lifetime components of 0.5– 0.7 ns, a component with 3 ns becomes significant at higher repetition rates. During the time-gated lifetime imaging, counting of the photons starts after 0.7 ns, thus changes in the kinetics due to singlet-singlet annihilation do not significantly influence the imaging results (Barzda et al., 1996). InFig. ...
... The fluorescence lifetimes in the microvolumes varied over a broad range. Singlet-singlet annihilation cannot account for the large differences in the lifetimes on the 0.5 to 3 ns time scale because most of the annihilation in LHCII aggregates finishes after a few hundreds of picoseconds (Barzda et al., 1996). LHCII trimers show rather homogeneous lifetimes dominated by a 3.6-ns component (Connelly et al., 1997). ...
Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.
... Stacking of thylakoids [4,28] and aggregation of isolated LHCII [40] cause a decrease of absorbance in the Soret region due to an increase in light scattering and the flattening effect [41]. These scattering changes have been related to formation of multiaggregates of LHCII [40,41] and changes in intrinsic thylakoid membrane structure [4]. ...
... Stacking of thylakoids [4,28] and aggregation of isolated LHCII [40] cause a decrease of absorbance in the Soret region due to an increase in light scattering and the flattening effect [41]. These scattering changes have been related to formation of multiaggregates of LHCII [40,41] and changes in intrinsic thylakoid membrane structure [4]. On the other hand, a weak light-induced increase in absorption values at 400-520 and 690 nm is accompanied by a simultaneous decrease in light scattering associated with disaggregation of LHCII complexes [30]. ...
... Fig. 1). Similarly, both the red shift of bands at 680 and 490 nm and the decrease of absorbance in the Soret region of scan have been observed after the aggregations of LHCII [41]. On the other hand, the destacking of thylakoid membranes is associated with the blue shift of bands at 681 and 439 nm [4,28]. ...
The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.
... Gillbro et al. [18] followed the singletsinglet annihilation in LHCII aggregates by means of picosecond absorption thus confirming this minimal value for the domain size and a hopping time between trimers of 1-5 ps. These results are in good agreement with subsequent singlet-singlet annihilation studies of Barzda et al. [19,20], who estimated a hopping time of 4 ps. Barzda et al. [19] also calculated a value of 640 Å for the radius of excitation migration, which corresponds to a much larger number of Chl molecules. ...
... These results are in good agreement with subsequent singlet-singlet annihilation studies of Barzda et al. [19,20], who estimated a hopping time of 4 ps. Barzda et al. [19] also calculated a value of 640 Å for the radius of excitation migration, which corresponds to a much larger number of Chl molecules. However, Schödel et al. [21] clearly showed for LHCII that experimental data on fluorescence quenching due to high laser pulse energies in pigment-protein complexes containing strongly coupled pigments cannot always be interpreted by annihilation. ...
The dynamic properties of isobutanol are studied in a wide (90–350 K) temperature range by recording the relaxation shifts
in the steady-state luminescence spectra of eosin and erythrosine and by kinetic phosphorescent spectroscopy invoking the
Cole-Davidson model for dielectric relaxation. The characteristic dipole relaxation times and the activation energy of relaxation
processes in the matrix in the temperature range of 140–170 K are determined from the correlation function of the dynamic
Stokes shift of the phosphorescence spectrum of luminescent probe molecules.
... Gillbro et al. [18] followed the singletsinglet annihilation in LHCII aggregates by means of picosecond absorption thus confirming this minimal value for the domain size and a hopping time between trimers of 1-5 ps. These results are in good agreement with subsequent singlet-singlet annihilation studies of Barzda et al. [19,20], who estimated a hopping time of 4 ps. Barzda et al. [19] also calculated a value of 640 Å for the radius of excitation migration, which corresponds to a much larger number of Chl molecules. ...
... These results are in good agreement with subsequent singlet-singlet annihilation studies of Barzda et al. [19,20], who estimated a hopping time of 4 ps. Barzda et al. [19] also calculated a value of 640 Å for the radius of excitation migration, which corresponds to a much larger number of Chl molecules. However, Schödel et al. [21] clearly showed for LHCII that experimental data on fluorescence quenching due to high laser pulse energies in pigment-protein complexes containing strongly coupled pigments cannot always be interpreted by annihilation. ...
The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers-phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ(av) were quantified for a range of quencher concentrations. Applying the Stern-Volmer formalism, apparent quenching rate constants k(q) were determined from the dependencies on quencher concentration of the ratio τ(0)(av)/τ(av), where τ(0)(av) is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k(q)'/k(q), i.e., the apparent quenching rate constants determined in aggregates (or membranes), k(q)', and in detergent-solubilised LHCII trimers, k(q), respectively. In LHCII macroaggregates, the resulting values for the domain size were 15-30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12-24 LHCII trimers, corresponding to 500-1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment-protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.
... Moreover, different lifetimes occur even in the absence of annihilation due to quenching centers in the aggregates (Ide et al., 1987;Mullineaux et al., 1993;Barzda et al., 1998). An upper limit for the average hopping time of 6 ps was estimated for aggregates of LHCII by Barzda et al. (1996) but heterogeneity of the preparations was not taken into account during the analysis. Bittner et al. (1994) studied S-S annihilation in trimeric LHCII with the use of subpicosecond pump-probe spectroscopy. ...
... The estimates are in rather good agreement with the current findings. The obtained value for the average hopping time is smaller than the value of 6 ps that was obtained as an upper limit in recent S-S annihilation experiments where the excited dynamics on a picosecond time scale was also studied, but the heterogeneity of the samples was not taken into account (Barzda et al., 1996). Concomitantly, a lower value of ␣ was found in that study. ...
Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.
... A further consequence of MgCl 2 addition is an increase in absorption around 505 and 685 nm and a decrease around 430 and 470 nm (Fig. 4). It was reported that these changes reflect aggregation of LHCII trimers [36,38]. ...
... Addition of MgCl 2 results in a paracrystalline ordering. Similar MgCl 2 -induced structural changes were deduced from singulett – singulett annihilation measurements with LHCII aggregates [38]. The formation of hexagonal paracrystalline LHCII arrays in the presence of salt is well documented [3,13]. ...
The salt-induced aggregation of the light-harvesting complex (LHC) II isolated from spinach and its correlation with fluorescence quenching of chlorophyll a is reported. Two transitions with distinctly different properties were observed. One transition related to salt-induced fluorescence quenching takes place at low salt concentration and is dependent both on temperature and detergent concentration. This transition seems to be related to a change in the lateral microorganization of LHCII. The second transition occurs at higher salt concentration and involves aggregation. It is independent of temperature and of detergent at sub-cmc concentrations. During the latter transition the small LHCII sheets (approximately 100 nm in diameter) are stacked to form larger aggregates of approximately 3 microm diameter. Based on the comparison between the physical properties of the transition and theoretical models, direct and specific binding of cations can practically be ruled out as driving force for the aggregation. It seems that in vitro aggregation of LHCII is caused by a complex mixture of different effects such as dielectric and electrostatic properties of the solution and surface charges.
... A typical difference spectrum for a sample sonicated without MgCl 2 is shown in the inset of Figure 3. This spectrum is in good accordance with difference spectra that are associated with LHCII stacking (37,43). For monitoring MgCl 2 -dependent changes in grana stacking, the height of the characteristic absorption change at 505 nm is plotted against the salt concentration ( Figure 3). ...
... This is highly unlikely. From singletsinglet annihilation measurements on isolated LHCII aggregates it was estimated that the lateral diffusion radius for excitons is about 65 nm (43). Thus there would be a high probability that light energy harvested by the LHCII-only grana disks can never be used photochemically. ...
The excitation energy transfer between photosystem (PS) II complexes was studied in isolated grana disks and thylakoids using chlorophyll a fluorescence induction measurements in the presence of DCMU under stacked and destacked conditions. Destacking of grana was achieved using a sonication protocol in a buffer without MgCl(2). The degree of stacking was controlled and quantified by atomic force microscopy and by the concomitant absorption changes. As expected from the literature, intact thylakoids showed a strong dependency of the connectivity of PSII centers, the F(m)/F(o) ratio as well as the fraction of PSIIbeta centers on the MgCl(2) concentration. In contrast, these parameters did not change in isolated grana disks. In particular, the connectivity remained constantly high irrespective of the degree of destacking. These differences were explained by the high protein density in grana disks, which hinders separation and mixing of proteins sufficiently to change energy transfer properties. Due to the occurrence of stroma lamella in intact thylakoids, intermixing of PSII and PSI is possible and allows for changes in F(m)/F(o) ratio as is the separation of LHCII from PSII, thus leading to an increase in the fraction of PSIIbeta. Even if mixing and separation of proteins are impaired in isolated grana disks, destacking should lead to a decrease in connectivity if transversal excitation energy transfer between two opposite membranes is significant. Because the connectivity is constant over all degrees of destacking employed, we conclude that the energy transfer in granas is mainly lateral.
... This type of macro-organization of the complexes has been shown to be associated with the granal ultrastructure of chloroplasts, and to play a key role in the lateral segregation ('sorting') of the two photosystems between the stacked and unstacked regions (Garab et al. 1991;Garab and Mustárdy 1999). In isolated lamellar aggregates of LHC II, it has also been shown that in the macroarrays, the excitation energy can migrate for long distances (r > 64 nm) (Barzda et al. 1996b). ...
... The low-pH-induced increase in the psi-type CD in Chlorella is interpreted as a strengthening of the chiral macro-organization of the chromophores in the granum-like regions of the inner bands, and indicates an increase in the long-range order of the pigment dipoles. As inferred from the results obtained in LHC II (Barzda et al. 1996b), this facilitates the energy migration in the membranes over long distances. However, long range order alone may facilitate both the utilization and the quenching of the excitation energy. ...
We have used circular dichroism (CD) spectroscopy and chlorophyll fluorescence induction measurements in order to examine low-pH-induced changes in the chiral macro-organization of the chromophores and in the efficiency of non-photochemical quenching of the chlorophyll a fluorescence (NPQ) in intact, dark-adapted cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae). We found that: (i) high proton concentrations enhanced the formation of chiral macrodomains of the complexes, i.e. the formation of large aggregates with long-range chiral order of pigment dipoles; this was largely independent of the low-pH-induced accumulation of de-epoxidized xanthophylls; (ii) lowering the pH led to NPQ; however, efficient energy dissipation, in the absence of excess light, could only be achieved if a substantial part of violaxanthin was converted to zeaxanthin and antheraxanthin in Chlorella and Mantoniella, respectively; (iii) the low-pH-induced changes in the chiral macro-organization of pigments were fully reversed by titrating the cells to neutral pH; (iv) at neutral pH, the presence of antheraxanthin or zeaxanthin did not bring about a sizeable NPQ. Hence, low-pH-induced NPQ in dark adapted algal cells appears to be associated both with the presence of de-epoxidized xanthophylls and structural changes in the chiral macrodomains. It is proposed that the macrodomains, by providing a suitable structure for long-distance migration of the excitation energy, in the presence of quenchers associated with de-epoxidized xanthophylls, facilitate significantly the dissipation of unused excitation energy.
... The other part of the spectrum showed only minor changes. It is known that the chirally organized macrodomains show structural flexibility in the photosynthetic membranes, apparently providing photoprotective capability at the supramolecular level (Garab et al., 1988;Barzda et al., 1996;Ünnep et al., 2020). The thylakoids of diatom Phaeodactylum tricornutum have been shown to be arranged into loosely stacked, multilamellar membrane system, without having strictly distinguishable granal and stromal regions, but still exhibiting the intense psi-type CD signals (Szabó et al., 2008) that showed sensitivity to the light, temperature, and osmolarity. ...
The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.
... The former possibility can be aligned with the aggregation-induced conformational changes 24 , which allow singlet EET to lutein 6 . The latter possibility is supported by the observations of annihilation processes, which occur significantly more in aggregated LHCIIs than in solubilized LHCIIs [37][38][39][40] . Such enhanced annihilation is also observed more in dimeric photosystem II complexes than in the monomeric complexes 41 . ...
Understanding photosynthetic light harvesting requires knowledge of the molecular mechanisms that dissipate excess energy in thylakoids. However, it remains unclear how the physical environment of light-harvesting complex II (LHCII) influences the process of chlorophyll de-excitation. Here, we demonstrate that protein-protein interactions between LHCIIs affect the optical properties of LHCII and thus influence the total energy budget. Aggregation of LHCII in the dark altered its absorption properties, independent of the amount of prior light exposure. We also revisited the triplet excited state involved in light-induced fluorescence quenching and found another relaxation pathway involving emission in the green region, which might be related to triplet excited energy transfer to neighboring carotenoids and annihilation processes that result in photoluminescence. LHCII- containing liposomes with different protein densities exhibited altered fluorescence and scattering properties. Our results suggest that macromolecular reorganization affects overall optical properties, which need to be addressed to compare the level of energy dissipation.
... Thus, granal thylakoid membranes evidently possess a high propensity to undergo well discernible structural changes. The plasticity of grana appears to be based on the inherent property of lipid: LHCII macroassemblies, which are capable to self-assemble into large arrays and to undergo reversible reorganizations induced by light and/or by subtle changes in the physico-chemical environment (Barzda et al., 1996;Zer et al., 1999;Simidjiev et al., 2000;Janik et al., 2013;Hind et al., 2014;Lambrev and Akhtar, 2019). ...
The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I—light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects—in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular—pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.
... Gillbro et al. [138] investigated the singletsinglet annihilation in LHCII aggregates with picosecond absorption and confirmed this minimal value of 200-250 interconnected Chl molecules for the domain size. Barzda et al. [139,140] calculated 64 nm for the radius of excited state migration, which corresponds to a much larger number of Chl molecules. In a different approach the energetic connectivity was analyzed in aggregated and solubilised LHCII and native thylakoid membranes, employing artificial exogenous singlet quenchers, like dinitrobenzene and phenyl-p-benzoquinone. ...
Excitation energy transfer (EET) processes in different photosynthetic pigment-protein-complexes were analysed by time- and wavelength correlated single photon counting (TWCSPC). A new mobile 16-channel photomultiplier with flexible fiber optics, exchangeable light sources and temperature regulator (10 K – 350 K) was built up for the spectroscopy of samples in cuvettes, on surfaces or of whole leaves in vivo. The system represents a mobile setup of the powerful TCSPC technique with high optical throughput up to 106 counts/sec.
The theoretical description of the excited state dynamics in systems with pigment-pigment and pigment-protein interaction was performed by using rate equations that were applied on structures with increasing hierarchical complexity. The study started with a system consisting of two excitonically coupled Chl molecules in a tetrameric protein environment represented by the recombinant water soluble Chl binding protein (WSCP) of type IIa and it was completed with a study of the photosystem II (PSII) dynamics in whole leaves of the higher plant Arabidopsis thaliana. In this way a quantification of dissipative excited state relaxation processes as a function of increasing excitation light intensity was achieved. For parameter adaption in the corresponding systems of linear differential
equations a highly efficient algorithm was developed that allows the variation of parameters used to fit the time resolved optical data under any constraints on the coefficient matrix (e.g. invariance of thesymmetry). The approach permits the determination of selected parameter values, their probability and stability in any dynamical system. A way to calculate thermodynamic quantities (e.g. entropy) under
nonequilibrium conditions from rate equations is proposed.
The excited state dynamics observed in WSCP were explained by assuming an excitonically coupled Chl dimer that is modulated by the protein environment on different time scales. The dominating fluorescence decay component increases from 4.8 ns or 5.2 ns for Chl b or Chl a homodimers, respectively, at room temperature, to 7.0 or 6.2 ns, respectively, at 10 K. This temperature dependency is most probably caused by the pigment-pigment- interaction and the energies of the triplet states of the Chl molecules. A modulation of the electronic states of the coupled Chl dimer by the protein environment with a typical time constant of 100 ps at 10 K is inferred to be responsible for a fast and
strongly temperature dependent fluorescence component. This idea is qualitatively in line with refined theoretical models and results of complementary studies of hole burning and fluorescence line narrowing spectroscopy.
In the phycobiliprotein (PBP) antenna of the cyanobacterium Acaryochloris marina EET occurs with characteristic time constants of 400 fs, 3-5 ps and 14 ps inside trimeric phycocyanin (PC), from PC to allophycocyanin (APC) and from APC to the terminal emitter (TE) of the PBP antenna, respectively.
The TWCSPC spectra of whole cells and preparations of isolated PBP complexes exhibit a 20 ps component each that indicates the intact EET from PC to the TE in agreement with the results of transient fs absorption spectroscopy. The EET from the PBP antenna to the Chl d containing core antenna complexes of PS II represents an additional limiting transfer step of about 30-40 ps which leads to a time constant of the EET from PBP to Chl d in the range of 70 ps.
Coupled and temperature switch-able hybrid systems of surface treated CdSe/ZnS quantum dots (QDs) with 530 nm emission wavelength and the isolated PBP antenna complexes from A.marina were formed in aqueous solution by electrostatic self assembly. Based on the theory of Förster Resonance Energy Transfer (FRET) an average value of 3.2-3.5 nm was obtained for the distance between the neighbouring transition dipole moments in the QDs and the PBP antenna. It was shown that the functional coupling between QDs and PBP complexes is interrupted at temperatures below 0°C. This
effect enables the construction of switch-able molecular sensors, photosensibilisators or light harvesting devices with various applications in biochemistry, biomedicine and photovoltaics.
... Furthermore, the idea that changes in the aggregation state can control the efficiency of EET in the photosynthetic antenna was considered as a mechanism for NPQ in plants. In vitro studies demonstrated that aggregation of light-harvesting complex II (LHCII) antenna complexes leads to efficient energy dissipation (46)(47)(48)(49)(50)(51), although the significance of changes in LHCII aggregation state for energy dissipation in vivo is still debated (52,53). It is important to note a number of key differences between LHCII and PC. ...
In this paper we propose an energy dissipation mechanism that is
completely reliant on changes in the aggregation state of the
phycobilisome light-harvesting antenna components. All photosynthetic
organisms regulate the efficiency of excitation energy transfer
(EET) to fit light energy supply to biochemical demands. Not many do
this to the extent required of desert crust cyanobacteria. Following
predawn dew deposition, they harvest light energy with maximum
efficiency until desiccating in the early morning hours. In the
desiccated state, absorbed energy is completely quenched. Time
and spectrally resolved fluorescence emission measurements of the
desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced
EET between phycobilisome components, (ii) shorter fluorescence
lifetimes, and (iii) red shift in the emission spectra, compared
with the hydrated state. These changes coincide with a loss of the
ordered phycobilisome structure, evident from small-angle neutron
and X-ray scattering and cryo-transmission electron microscopy data.
Based on these observations we propose a model where in the hydrated
state the organized rod structure of the phycobilisome supports
directional EET to reaction centers with minimal losses due to
thermal dissipation. In the desiccated state this structure is lost, giving
way to more random aggregates. The resulting EET path will exhibit
increased coupling to the environment and enhanced quenching.
... It has been shown that aggregates of LHCII are energetically well interconnected and excitations can diffuse at long distances (38,39). Here we evaluated the connectivity, or functional domain size (the average number of sites that excitations can diffuse to before being deactivated), in LHCII aggregates, in reconstituted lipid membranes, and native LHCII-enriched membranes. ...
Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions - at (-)437 nm and (+)484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids - at (+)447 nm and ( )494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at ( )494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggrega-tion-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spec-tral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, while a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
... With respect to the importance of grana stacking for NPQ, it has been proposed that the macrodomain organization of LHCII, which is realized in the stacked grana regions, is essential for efficient NPQ . The long-range order of the photosynthetic pigments in the macrodomains, in conjunction with the delocalization of the excitation energy (Barzda et al., 1994(Barzda et al., , 1996, could provide the basis for efficient Z-dependent NPQ. The introduction of quenching sites into a system, whose excitonic coupling may span several adjacent grana membranes, would strongly enhance the efficiency of the quencher. ...
In their natural environment plants and algae are exposed to rapidly changing light conditions and light intensities. Illumination with high light intensities has the potential to overexcite the photosynthetic pigments and the electron transport chain and thus induce the production of toxic reactive oxygen species (ROS). To prevent damage by the action of ROS, plants and algae have developed a multitude of photoprotection mechanisms. One of the most important protection mechanisms is the dissipation of excessive excitation energy as heat in the light-harvesting complexes of the photosystems. This process requires a structural change of the photosynthetic antenna complexes that are normally optimized with regard to efficient light-harvesting. Enhanced heat dissipation in the antenna systems is accompanied by a strong quenching of the chlorophyll a fluorescence and has thus been termed non-photochemical quenching of chlorophyll a fluorescence, NPQ. The general importance of NPQ for the photoprotection of plants and algae is documented by its wide distribution in the plant kingdom. In the present review we will summarize the present day knowledge about NPQ in higher plants and different algal groups with a special focus on the molecular mechanisms that lead to the structural rearrangements of the antenna complexes and enhanced heat dissipation. We will present the newest models for NPQ in higher plants and diatoms and will compare the features of NPQ in different algae with those of NPQ in higher plants. In addition, we will briefly address evolutionary aspects of NPQ, i.e. how the requirements of NPQ have changed during the transition of plants from the aquatic habitat to the land environment. We will conclude with a presentation of open questions regarding the mechanistic basis of NPQ and suggestions for future experiments that may serve to obtain this missing information.
... Gillbro et al. [138] investigated the singletsinglet annihilation in LHCII aggregates with picosecond absorption and confirmed this minimal value of 200-250 interconnected Chl molecules for the domain size. Barzda et al. [139,140] calculated 64 nm for the radius of excited state migration, which corresponds to a much larger number of Chl molecules. In a different approach the energetic connectivity was analyzed in aggregated and solubilised LHCII and native thylakoid membranes, employing artificial exogenous singlet quenchers, like dinitrobenzene and phenyl-p-benzoquinone. ...
Excitation energy transfer (EET) processes in different photosynthetic pigment-protein-complexes were analysed by time- and wavelength correlated single photon counting (TWCSPC). A new mobile 16-channel photomultiplier with flexible fiber optics, exchangeable light sources and temperature regulator (10 K – 350 K) was built up for the spectroscopy of samples in cuvettes, on surfaces or of whole leaves in vivo. The system represents a mobile setup of the powerful TCSPC technique with high optical throughput up to 106 counts/sec. The theoretical description of the excited state dynamics in systems with pigment-pigment and pigment-protein interaction was performed by using rate equations that were applied on structures with increasing hierarchical complexity. The study started with a system consisting of two excitonically coupled Chl molecules in a tetrameric protein environment represented by the recombinant water soluble Chl binding protein (WSCP) of type IIa and it was completed with a study of the photosystem II (PSII) dynamics in whole leaves of the higher plant Arabidopsis thaliana. In this way a quantification of dissipative excited state relaxation processes as a function of increasing excitation light intensity was achieved. For parameter adaption in the corresponding systems of linear differential equations a highly efficient algorithm was developed that allows the variation of parameters used to fit the time resolved optical data under any constraints on the coefficient matrix (e.g. invariance of the symmetry). The approach permits the determination of selected parameter values, their probability and stability in any dynamical system. A way to calculate thermodynamic quantities (e.g. entropy) under nonequilibrium conditions from rate equations is proposed. The excited state dynamics observed in WSCP were explained by assuming an excitonically coupled Chl dimer that is modulated by the protein environment on different time scales. The dominating fluorescence decay component increases from 4.8 ns or 5.2 ns for Chl b or Chl a homodimers, respectively, at room temperature, to 7.0 or 6.2 ns, respectively, at 10 K. This temperature dependency is most probably caused by the pigment-pigment- interaction and the energies of the triplet states of the Chl molecules. A modulation of the electronic states of the coupled Chl dimer by the protein environment with a typical time constant of 100 ps at 10 K is inferred to be responsible for a fast and strongly temperature dependent fluorescence component. This idea is qualitatively in line with refined theoretical models and results of complementary studies of hole burning and fluorescence line narrowing spectroscopy. In the phycobiliprotein (PBP) antenna of the cyanobacterium Acaryochloris marina EET occurs with characteristic time constants of 400 fs, 3-5 ps and 14 ps inside trimeric phycocyanin (PC), from PC to allophycocyanin (APC) and from APC to the terminal emitter (TE) of the PBP antenna, respectively. The TWCSPC spectra of whole cells and preparations of isolated PBP complexes exhibit a 20 ps component each that indicates the intact EET from PC to the TE in agreement with the results of transient fs absorption spectroscopy. The EET from the PBP antenna to the Chl d containing core antenna complexes of PS II represents an additional limiting transfer step of about 30-40 ps which leads to a time constant of the EET from PBP to Chl d in the range of 70 ps. Coupled and temperature switch-able hybrid systems of surface treated CdSe/ZnS quantum dots (QDs) with 530 nm emission wavelength and the isolated PBP antenna complexes from A.marina were formed in aqueous solution by electrostatic self assembly. Based on the theory of Förster Resonance Energy Transfer (FRET) an average value of 3.2-3.5 nm was obtained for the distance between the neighbouring transition dipole moments in the QDs and the PBP antenna. It was shown that the functional coupling between QDs and PBP complexes is interrupted at temperatures below 0°C. This effect enables the construction of switch-able molecular sensors, photosensibilisators or light harvesting devices with various applications in biochemistry, biomedicine and photovoltaics.
... Numerous data show that such long-distance migra-tion in the 'lake' of pigments is possible (Paillotin 1976;Geacintov et al. 1984), and can lead to a connectivity of several PSII centers (Joliot et al. 1973;Lavergne and Trissl 1995). Direct evidence for long-distance migration of the excitation energy in macrodomains has been provided in lamellar aggregates of LHCII, in which singlet-singlet exciton annihilation experiments show that the excitation energy 'freely diffuses' probably in the entire domain, or at least with an estimated diffusion radius of >640 Å (Barzda et al. 1996b). It is inferred that chiral macrodomains in thylakoids function in a similar manner. ...
In higher plants and green algae two types of thylakoids are distinguished,
granum (stacked) and stroma (unstacked) thylakoids. They form a
three-dimensional (3D) network with large lateral heterogeneity: photosystem
II (PSII) and the associated main chlorophyll
a/b light-harvesting complex
(LHCII) are found predominantly in the stacked region, while PSI and LHCI are
located mainly in the unstacked region of the membrane. This picture emerged
from the discovery of the physical separation of the two photosystems
(Boardman and Anderson 1964). Granal chloroplasts possess significant
flexibility, which is essential for optimizing the photosynthetic machinery
under various environmental conditions. However, our understanding concerning
the assembly, structural dynamics and regulatory functions of grana is far
from being complete. In this paper we overview the significance of the
three-dimensional structure of grana in the absorption properties, ionic
equilibrations, and in the diffusion of membrane components between the
stacked and unstacked regions. Further, we discuss the role of chiral
macrodomains in the grana. Lateral heterogeneity of thylakoid membranes is
proposed to be a consequence of the formation of macrodomains constituted of
LHCII and PSII; their long range order permits long distance migration of
excitation energy, which explains the energetic connectivity of PSII
particles. The ability of macrodomains to undergo light-induced reversible
structural changes lends structural flexibility to the granum. In purified
LHCII, which has also been shown to form stacked lamellar aggregates with long
range chiral order, excitation energy migrates for large distances; these
macroaggregates are also capable of undergoing light-induced reversible
structural changes and fluorescence quenching. Hence, some basic properties of
grana appear to originate from its main constituent, the LHCII.
... However this stoichiometry cannot account for all LHCII present in the membrane [22]. There is substantial evidence that LHCII can also form ordered arrays of trimers, or LHCIIonly domains [18,19,22,23], in which trimers form close contacts between each other and can transfer excitation energy over a long distance [24]. It is very unlikely that isolated trimers, unconnected to the Photosystem II core, occur in the native membrane containing 70–80% protein [25]. ...
Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer–trimer contacts, has significant impact on the pigment interactions in the complexes.
... However this stoichiometry cannot account for all LHCII present in the membrane [22]. There is substantial evidence that LHCII can also form ordered arrays of trimers, or LHCIIonly domains [18,19,22,23], in which trimers form close contacts between each other and can transfer excitation energy over a long distance [24]. It is very unlikely that isolated trimers, unconnected to the Photosystem II core, occur in the native membrane containing 70-80% protein [25]. ...
... However this stoichiometry cannot account for all LHCII present in the membrane [22]. There is substantial evidence that LHCII can also form ordered arrays of trimers, or LHCIIonly domains [18,19,22,23], in which trimers form close contacts between each other and can transfer excitation energy over a long distance [24]. It is very unlikely that isolated trimers, unconnected to the Photosystem II core, occur in the native membrane containing 70-80% protein [25]. ...
... In the photosynthetic system light energy is absorbed by antenna chlorophylls and this energy is then passed onto a reaction centre chlorophyll dimer where charge separation occurs in less than 100 ps [1] and at about 95% efficiency [2]. Also, an organised connective light harvesting system is required for long range energy transfer [3]. As a matrix to stabilize the light harvesting systems we are using a combination of fatty acids and nitrous oxide, rather than conventional phospholipid-based combinations, which enables the production of small, elastic, artificial vesicles, called Pheroid TM , of 100 – 300 nm diameters. ...
Annual South African Institute of Physics (SAIP) Conference, University of Kwa-Zulu Natal (Westville Campus), Durban, 7-10 July, 2009 Currently the primary source of energy for industrial and domestic use is based on fossil fuels. The supplies of these fuels are limited and are becoming depleted. Thus there is a search for alternative and more sustainable energy sources. One such source is solar energy, which has many advantages over fossil fuels. Thus research into harvesting, transferring, and converting light energy is of great significance. The most abundant and efficient light harvesting, energy transfer and transduction systems are found in nature with the process of photosynthesis. Previous work has shown that photosynthetic light harvesting material can be incorporated into artificial vesicles called Pheroid. In this study researchers are characterising the level of organisation of the incorporated light harvesting and energy transfer systems using steady state optical techniques such as absorption spectroscopy. The incorporation ratio of photosynthetic material, Pheroid has been investigated and preliminary results indicate that the loading capacity of the Pheroid differs depending on the photosynthetic membrane fractions used.
... According to the theory of psi-type aggregates, in densely packed macroaggregates with long-range chiral order the excitation energy can delocalize over large domains . Comparative studies with nonlinear spectroscopic methods revealed significant differences in the energy migration patterns between small and large aggregates of LHCII (Lokstein et al., 1995;Barzda et al., 1996). However, these differences in the excitation energy of migration are highly unlikely to be responsible for the lightinduced reversible changes in the macrostructure of LHCII macroaggregates and granal thylakoid membranes. ...
In this paper, we show that stacked lamellar aggregates of the purified chlorophyll a/b light-harvesting antenna complexes (LHCII) and granal thylakoid membranes are capable of undergoing light-induced reversible changes in the chiral macroorganization of the chromophores as well as in the photophysical pathways. In granal thylakoids, the light-induced reversible structural changes, detected by circular dichroism (CD) measurements, are accompanied by reversible changes in the fluorescence yield that indicate an increased dissipation of the excitation energy. These changes become gradually more significant in excess light compared to nonsaturating light intensities, and can be eliminated by suspending the membranes in hypotonic, low-salt medium in which the chiral macroaggregates are absent. In lamellar aggregates of LHCII, the light-induced reversible changes of the main, nonexcitonic CD bands are also accompanied by reversible changes in the fluorescence yield. In small aggregates and trimers, no light-induced delta CD occurs, and the fluorescence changes are largely irreversible. It is proposed that the structural changes are induced by thermal effects due to the excess light energy absorbed by the pigments. Our data strongly suggest that the structure and function of the antenna system of chloroplasts can be regulated by the absorption of excess light energy with a mechanism independent of the operation of the photochemical apparatus.
... However, we did not find the DAS component with an emission maximum near 690 nm reported in the latter study probably because its lifetime is close to the lifetime of the 687 nm component. It has to be emphasized that the interpretation of the experimental data gathered at 80 K in terms of independent emitting species associated with single decay components seems to be rather crude for a description of the time-resolved data obtained with aggregated LHCII at 80 K due to the longrange excitation energy migration observed in macroaggregates (Barzda et al., 1996). At this temperature an uphill energy transfer between pigments with an energy gap of 2-5 nm will be only 2.2-6.6 times slower than the downhill process, and it will lead to a pronounced effect on the decay kinetics. ...
The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).
... The quality of the fits was evaluated by global 2 values, plots of residuals, and Studentised residuals (21). The relative triplet yield under exciton-exciton annihilation free conditions (22) was estimated from the slope at zero intensity of saturation curves of T-S signals recorded at the maximum of the Car triplet absorption (∼508 nm) (23). The Chl-to-Car triplet transfer efficiency was estimated by dividing the amount of Car triplets by the total amount of triplets on both Chl and Car. ...
The influence of aggregation on triplet formation in the light-harvesting pigment-protein complex of photosystem II of green plants (LHCII) has been studied with time-resolved laser flash photolysis. The aggregation state of LHCII has been varied by changing the detergent concentration. The triplet yield increases upon disaggregation and follows the same dependence on the detergent concentration as the fluorescence yield. The rate constant of intersystem crossing is not altered by disaggregation, and variations of the triplet yield appear to be due to aggregation-dependent quenching of singlet excited states. The efficiency of triplet transfer in LHCII aggregates from chlorophyll (Chl) to carotenoid (Car) is 92 +/- 7% at room temperature and 82 +/- 6% at 5 K, and does not change upon disaggregation. The Chl's that do not transfer their triplets to Car's seem to be bound to LHCII and are capable of transfering/accepting their singlet excitations to/from other Chl's. Two spectral contributions of Car triplets are observed: at 525 and 506 nm. Disaggregation of macroaggregates to small aggregates reduces by 10% the relative contribution of Car triplets absorbing at 525 nm. This effect most likely originates from a decreased efficiency of intertrimer Chl-to-Car triplet transfer. At the critical micelle concentration, at which small aggregates are disassembled into trimers, the interactions between Chl and Car are changed. At room temperature, this effect is much more pronounced than at 5 K.
... This implies that for the case that such structures are formed in the thylakoid membranes, they may be rather associated with the State 1-State II transition and migration of the major PS II antenna proteins within the thylakoid membranes. In fact the dimension of the LHC II lamellar aggregates reported by Garab and co-workers to be present in the photosynthetic apparatus [39,[47][48][49][50][51] corresponds well to the structures observed in our model system. The assembly of LHC II in aggregated structures is recognised to be the important mechanism of protection against decomposition of the photosynthetic apparatus under strong illumination [6][7][8]. ...
Monomolecular layers at the air-water interface were formed directly with isolated largest light-harvesting pigment-protein complex of Photosystem II (LHC II) or out of egg yolk lecithin (EYL) liposomes containing incorporated LHC II. Pure protein monolayers showed a mean area of 1400 A2 per molecule at the air-water interface. Monolayers were deposited onto glass slides by means of Langmuir-Blodgett (LB) technique. Chlorophyll fluorescence of LHC II-LB and EYL-LHC II-LB films proved energetic coupling of chlorophyll a and b, thus indicating native conformation of LHC II within the monolayers. Scanning force microscopy (SFM) revealed ring-like structures formed in monocomponent protein layers as well as in mixed protein-lipid films. These results suggest that a structural arrangement of LHC II is favoured in a lipid environment but that the protein has itself a strong tendency for structural complex rearrangement in our system.
Controlled by environmental factors, proteins in the grana thylakoid subcompartment in chloroplasts can rearrange into highly ordered semicrystalline arrays. The functional implications of these arrays are analyzed by using an Arabidopsis fatty acid desaturase (fad5) mutant as a model system, which constitutively forms these crystalline structures in thylakoid membranes. Stoichiometric analysis of the fad5 thylakoid membranes reveal the existence of two membrane domains in grana (domains with PSII crystals and LHCII-enriched domains). Probing the light-harvesting of PSII by chlorophyll fluorescence induction indicates a very efficient energy transfer between these domains in the mutant probably by transversal energy transfer across the aqueous stromal partition gap between adjacent grana discs. Furthermore, the protein mobility in fad5 measured by fluorescence recovery after photobleaching is higher compared to WT plants. This gives evidence for a high protein mobility in LHCII-enriched grana regions.
With reference to the recent achivements about the structure, spectra and kinetics of light-harvesting complex (LHC II) in PS Il of higher plants, a four-level model was provided to simulate the energy transfer process from LHC II to the reaction center. On the basis of this model, a set of rate equation was established. Analysis of its algebra solution led to a general picture of energy transfer process in LHC II of higher plants and the strong interaction among pigment molecules in this process. Based on the spectra, kinetics and biological structural data providing some information of energy transfer path and energy dissipation mechanism, it has been found that energy transfer mainly happened between the pigments whose energy level was most closely adjacent, the loss of energy had a close relation to the process of energy transfer and tended to increase with the decrease of energy level. The protective mechanism of antenna system was also discussed.
Earlier we have shown, mainly by polarization spectroscopic techniques, that the main chlorophyll a/b light harvesting complex of photosystem II, LHCII, forms chirally organized macroggregates with dimensions commensurate with the wavelength of the visible light. The high self-aggregation capability of LHCII and PSII particles (coated with LHCII) explains the lateral segregation of the two photosystems between the granum and stroma membranes, i.e. the sorting of complexes, a key structural attributum of higher plant thylakoids [1,2]. Lamellar aggregates of isolated LHCII have been shown to constitute the structural basis for the long-distance migration (or delocalization) of the excitation energy [3]. The chiral macrodomains in thylakoids as well as in isolated lamellar aggregates of LHCII have also been shown to possess a remarkable structural flexibility. Most notably, they have been shown to undergo light-induced reversible structural reorganizations [4-6]. In lamellar aggregates of LHCII, lipids have been shown to play a key role [7,8]. Prolonged illumination with intense light leads to similar but irreversible changes [9]. These structural changes are accompanied by fluorescence quenching transients, also in isolated LHCII [5,10]. These data suggest the involvement of these reorganizations in regulatory processes in excess light [11]. It is important to point out, however, that these light-induced reorganizations and fluorescence transients are largely independent of the photochemical activity of thylakoids, and occur in isolated LHCII. Further, the changes in LHCII and thylakoids are approximately linearly proportional to the light intensity, i.e. in thylakoids, they increase monotonically above the saturation of photosynthesis. Hence, simple redox or ΔpH feedback mechanisms cannot be held responsible for these changes. It is also important to stress that the apparently non-saturable nature of the reorganizations is of significant potential importance, a unique feature, with respect to protection of plants against excess excitation. These structural transitions have been proposed to be driven by a novel, thermo-optic mechanism: fast thermal transients arising from dissipated excitation energy, which lead to elementary structural transitions in the close vicinity of the dissipation centers, due to the presence of 'built-in' thermal instability in the (macro)assembly of complexes [12-14]. As concerns the nature of the structural changes in thylakoid membranes, it has been shown that light induces (i) unstacking of membranes, followed by (ii) a lateral desorganization of the macrodomains, and (iii) monomerization of the LHCII trimers [14]. Loosely stacked lamellar aggregates of isolated LHCII CELL. MOL. BIOL. LETT. Vol. 9, 2004, Supplement 22 exhibit very similar rearrangements, while tightly stacked lamellar aggregates or microcrystals do not possess this ability [15]. These structural changes, in addition to their role in the regulation of energy dissipation [see also 13, 16], might play an important role in photoprotection and different light-and temperature-adaptation processes. Reorganizations of similar nature have been shown to play a role in regulating the activity LHCII phosophorylation by light at the substrate level [17] and also in the proteolytic removal of the 'unneccessary' light harvesting complexes [18]. Thermo-optically driven rearrangements have also been detected in cyanobacteria, where energy migration from the phycobilisomes to the photosynthetic membranes can be regulated by heat or excess light. We would also like to point out, as a potential general significance of these type of reorganizations, that thermo-optically induced structural transitions lend a substantial local and temporal structural flexibility to molecular (macro)assemblies that otherwise possess large stability and rigidity.
{Zinc 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine} (ZnPC) exists as monomeric species in DMSO and is reasonably strong fluorescent. But ZnPC forms H-aggregates in water and hexafluoroisopropanol, which are strong hydrogen bonding solvents. Nanoaggregates are nearly nonemissive. Transient absorption spectroscopic technique has been used to investigate the excited state relaxation processes in both monomeric and aggregated forms of ZnPC. The lifetime of the S1 state of the monomoric form in DMSO is long (τ = 3.4 ns) but the excited states of ZnPC nanoaggregates show much faster ground state recovery (within 100 ps). The longest lifetime component, τ3, which is independent of excitation density, has been assigned to the unimolecular decay of the S1-exciton in the absence of annihilation reaction, while τ1 and τ2 are the lifetimes obtained by the two-component fit of the nonexponential decay arising due to the time-dependent decay rates of the S1-excitons because of diffusive migration controlled exciton - exciton annihilation reaction. Rates of the annihilation reaction (2.0 × 10–6 cm3s–1) and exciton migration (4.3 × 10–5 m2/s) as well as diffusion length (about 85 nm) of the S1-exciton created in the ZnPC nanoaggregates in HFIP have been determined.
The importance of the functional flexibility of the light-harvesting complexes
of photo-system II (LHCII) in accommodating the fluctuation in the balance
between light input and metabolic capacity in plants is emphasised. This
flexibility is provided for by a relatively complex assembly of protein
subunits, the interactions between them being controlled by protonation,
xanthophyll de-epoxidation and phosphorylation. It is suggested that the
3-dimensional order imposed upon this assembly of proteins by the grana is a
vital aspect of the modulation of LHCII function. Grana establish the LHCII
conformation needed for efficient light harvesting and help prevent the dense
array of proteins from collapsing into a highly dissipative state. The grana
then allow a controlled development of non-photochemical quenching under the
driving force of violaxanthin de-epoxidation and protonation. In plants grown
under different irradiances the different grana content and xanthophyll cycle
pool size together allow maximum quantum yield in limiting light and an
appropriate level of non-photochemical quenching in excess light.
Two-dimensional femtosecond spectroscopic studies have suggested some photosynthetic light harvesting protein complexes (LHCs) utilise quantum searches to improve the efficiency of exciton transport through networks of chromophores to chemical reaction centers (RCs) [1-4]. This has stimulated theoretical work in the quantum chemistry and quantum information communities, with particular focus on the so-called Frenkel Hamiltonian, which models chromophores as quantum dots. Findings indicate that high-efficiency exciton transport in individual LHCs is achieved via an interplay between excitonic quantum coherence, resonant vibrations in the surrounding protein matrix, and thermal decoherence [5-13].
We study the quantum dynamics of conversion of composite bosons into fermionic fragment species with increasing densities of bound fermion pairs using the open quantum system approach. The Hilbert space of N-state-functions is decomposed into a composite boson subspace and an orthogonal fragment subspace of quasi-free fermions that enlarges as the composite boson constituents deviate from ideal boson commutation relations. The tunneling dynamics of coupled composite bosons states in confined systems is examined, and the appearance of exceptional points under experimentally testable conditions (densities, lattice temperatures) is highlighted. The theory is extended to examine the energy transfer between macroscopically coherent systems such as multichromophoric macromolecules in photosynthetic light harvesting complexes.
The peripheral light-harvesting complex of the photosynthetic bacterium Rhodopseudomonas acidophila (LH2) and the major plant light-harvesting complex LHCII have a very similar function: to absorb solar photons and to transfer the electronic excitation to the pigments surrounding the reaction center, the so-called ‘core’. Nevertheless, their structures exhibit a dramatically different arrangement of the pigments. In LH2 the bacteriochlorophyll molecules are arranged in a highly symmetric ring, while in LHCII the positioning of the chlorophylls is very irregular. In both complexes the average distance between the pigments is 1 nm or less and, as a consequence, the electronic interaction between the pigments is strong ( > 100 cm−1). Therefore, the excitation transport in these photosynthetic light-harvesting systems can not be described by a simple Förster type transfer mechanism, but new or other transfer mechanisms may be operative, for instance a mechanism in which the excitation is to some extent delocalized. Crucial parameters are the strength of the electronic coupling, the amount of energetic disorder and/or heterogeneity and the nature and strength of the interactions of the pigments with the protein. Here we will discuss the current status of the field of photosynthetic energy transfer in particular for LH2. We will evaluate a few simple models that contain some of the essential ingredients to describe the process of energy transfer and finally we will discuss some of the perspectives in this scientific field.
We carried out a kinetic analysis of the light-induced fluorescence quenching (AF) of the light-harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) that was first observed by Jennings et at (Pho-tosynth. Res. 27, 57–64, 1991). We show that during a 2 min light, 2 min dark cycle, both the light and dark phases exhibit biexponential kinetics; this is tentatively explained by the presence of two types of light-induced quenchers in different domains of aggregated LHCII. Quantitative analysis could be carried out on the faster kinetic component; the slower component that was not completed during the measurement was not amenable for quantitative analysis. Our analysis revealed that the rate of the light-induced decrease of the fluorescence yield depended linearly on the light intensity, which shows that the generation of the quencher originates from a reaction that is first order with respect to the concentration of the excited domains. As shown by the estimated rate constant, pho-togeneration of the quencher is a fast reaction that can compete with other excitation-relaxation pathways. Both the decay and the recovery time constants of AF depended strongly on the temperature. Thermodynamic analysis showed that the fast light-induced decline in the fluorescence was determined by a low fraction of the excited states. Recovery was associated with large decrease in the entropy of activation that indicated the involvement of large structural rearrangements. Macroaggregated LHCII exhibited larger ΔF than small aggregates, which is consistent with the proposed role of aggregated LHCII in thy-lakoid membranes in nonphotochemical quenching.
Laser-induced changes in the absorption spectra of isolated light-harvesting chlorophyll a/b complex (LHC II) associated with photosystem II of higher plants have been recorded under anaerobic conditions and at ambient temperature by using multichannel detection with sub-microsecond time resolution. Difference spectra (ΔA) of LHC II aggregates have been found to differ from the corresponding spectra of trimers on two counts: (i) in the aggregates, the carotenoid (Car) triplet–triplet absorption band (ΔA>0) is red-shifted and broader; and (ii) the features attributable to the perturbation of the Qy band of a chlorophyll a (Chla) by a nearby Car triplet are more pronounced, than in trimers. Aggregation, which is known to be accompanied by a reduction in the fluorescence yield of Chla, is shown to cause a parallel decline in the triplet formation yield of Chla; on the other hand, the efficiency (100%) of Chla-to-Car transfer of triplet energy and the lifetime (9.3 μs) of Car triplets are not affected by aggregation. These findings are rationalized by postulating that the antenna Cars transact, besides light-harvesting and photoprotection, a third process: energy dissipation within the antenna. The suggestion is advanced that luteins, which are buried inside the LHC II monomers, as well as the other, peripheral, xanthophylls (neoxanthin and violaxanthin) quench the excited singlet state of Chla by catalyzing internal conversion, a decay channel that competes with fluorescence and intersystem crossing; support for this explanation is presented by recalling reports of similar behaviour in bichromophoric model compounds in which one moiety is a Car and the other a porphyrin or a pyropheophorbide.
In this work, dioxane-bound aggregates of chlorophyll a are prepared in 3-methylpentane. The properties of the aggregates are studied by using steady-state and time-resolved spectroscopies. The Qy-region absorption spectrum of the chlorophyll a-dioxane aggregate shows four clearly resolvable narrow bands with comparable intensities. The band maxima are located at 683, 689, 698 and 702 nm. The emission spectrum consists of two emission bands centred at 699 and 702 nm suggesting the presence of two types of aggregates. High degree of fluorescence polarization is detected yielding the angles between the absorption transition moments with respect to the 702 nm emission transition moment. The circular dichroism signal is strong and the sign sequence follows that of the polarization spectrum. ψ-type effect is discovered due to long-range interactions within the sizable aggregate frame. The insensitivity of the time-resolved emission kinetics to the temperature indicate the excitation delocalization length to be short. The rapid loss of time-resolved anisotropy at certain wavelengths supports the energy transfer to take place. The independence of the excitation spectrum over a broad range of emission wavelengths suggests that the two diverse spectral types are found within the same macro-aggregate and that they are coupled together by the energy transfer.
Absorption, linear dichroism and circular dichroism spectroscopy at 293 and 77 K have been used in order to further explore the process of energy quenching in LHC IIb, the main light-harvesting complex of photosystem II. Upon aggregation there was an enhancement of linear dichroism bands in the Q absorption region of chlorophyll b. The y absorption spectrum at 77 K of aggregates revealed new bands around 656 nm and 680 nm, characterised by positive linear dichroism and negative circular dichroism signals. In the circular dichroism spectrum of aggregates a characteristic change Ž . was seen in the carotenoid and chlorophyll b regions, an increase of the chlorophyll a transition at 438 nm y and decrease of the red most negative band at around 677 nm. The amplitude of this band was in a tight correlation with a fluorescence quenching occurring upon LHC II aggregation. A new transition appeared at 505 nm with positive linear dichroism signal. It is suggested that protein aggregation causes a change in conformation and association of some chlorophyll a, chlorophyll b and xanthophyll molecules. These features of the linear dichroism spectrum of the aggregates were also detected for thylakoids in which they were particularly enhanced at low pH, suggesting that at least part of the light harvesting complex in the thylakoid membrane is in an aggregated form and the extent of aggregation in vivo can be controlled by the thylakoid pH gradient. q 1997 Published by Elsevier Science B.V.
Abstract— Generation of the nonequilibrium distribution of excited vibrational modes stimulated by electronic energy relaxation in pigment-protein complexes of the light-harvesting antenna of some photosynthetic systems is discussed in this paper. It is shown that the simplest approach to this problem can be achieved by introducing a local temperature, which is a good parameter for describing the nonequilibrium distribution of the local vibrational modes of the pigment molecules and its nearest protein surroundings. Then the transient absorption kinetics is determined by the kinetics of the excitation relaxation as I well as the heating/cooling of the local vibrational modes. Experimentally, this process can be investigated in the i singlet-singlet annihilation conditions that create the i greatest amount of local heating. The systems under in-: vestigation are trimers of bacteriochlorophyll a contain- i ing pigment-protein complexes from the green sulfur i bacterium Chlorobium tepid urn (so-called FMO complexes) and aggregates of the light-harvesting complexes of photosystem II (LHC2) from higher plants containing chlorophyll alb. It was shown that at 77 K the heat redistribution kinetics in LHC2 is on the order of 3040 ps and in FMO it is approximately equal to 26 ps. The local heating effect at room temperature is less pronounced; however, by using longer pulses and at higher excitation energies (on the order of a magnitude higher), an additional kinetics of hundreds of ps, also related to the heating/cooling process, was observed.
The kinetics of a newly synthesized series of a first generation of polyphenylene dendrimers in which one phenyl in a dendritic arm was para-substituted by a peryleneimide chromophore are reported. One such peryleneimide chromophore is attached to 1, 3 or 4 arms. The results are compared to a series of polyphenylene dendritic compounds, which are identical except for the substitution at a meta-position of a phenyl ring. The para-substitution yields a better spatial definition of the peryleneimide units relative to one another and the influence of this aspect on the kinetics is studied. Four different kinetic components were resolved for both groups of dendrimers. An ultra-short component varying from 500 fs to 2 ps and attributed to intramolecular vibrational redistribution (IVR) is identical for both series. The decay time of a second component, which is comprized of the vibrational relaxation and a singlet–singlet annihilation process observed in both substituted dendrimer series, is shorter in the para-substituted dendrimers compared to the meta-substituted ones. It is also shown that the annihilation process, which is only present in the multi-chromophoric compounds of both the series and resolved with an excitation energy dependent study, has definitely a larger contribution in the partial amplitudes for the para-substituted compounds. This is related to the relative orientation of the transition dipoles of the chromophores in both the series.
Wavelength-selective photo-excitation of samples containing a detergent and LHCII (the main light-harvesting complex pertaining to photosystem II of green plants) is used for recording time-resolved triplet-minus-singlet (TmS) difference spectra, with a view to probing interactions between chlorophyll a (Chla) and chlorophyll b (Chlb), and between Chla and lutein (Lut). Once the detergent concentration (CD) exceeds a threshold, C©, the TmS spectrum becomes sensitive to λ⊗, the wavelength of excitation, and to t, the delay between excitation and observation. Each increment in CD brings about a diminution in the efficiency of a†→x† transfer (triplet–triplet transfer from Chla to Lut) and a rise in both the triplet formation yield and the fluorescence yield of Chla. What is more, b*→a* transfer (singlet–singlet transfer from Chlb to Chla) slackens to such an extent that Chlb*→Chlb† intersystem crossing, negligible when CD is below C©, begins to vie with transfer, for the deactivation of Chlb* (in the foregoing an asterisk/dagger denotes singlet/triplet excitation). The reduction in the efficiencies of the two transfers is easily understood by: (i) invoking the Kühlbrandt–Wang–Fujiyoshi model of LHCII, which posits each Chlb in contact with a Chla and each Chla in contact with a Lut, and (ii) assuming that the detergent severs contact between adjacent chromophores. That a growth in the triplet yield of Chla* accompanies the detergent-induced decrease in the efficiency of a†→x† transfer becomes intelligible if one assumes, further, that internal conversion in * is faster than that in , where under or over lining betokens the presence or absence of a carotenoid neighbour. When CD is close to C©, most Chla molecules are adjacent to a Lut, internal conversion dominates, and the overall triplet yield is low. As CD is gradually raised the transformation sets in, causing concomitant drops in the efficiencies of a†→x† transfer and internal conversion, and a consequent rise in the overall yields of Chla fluorescence and formation of Chla triplets.
Exciton–exciton annihilation (EEA) in J-aggregates of 3,3′-disulfopropyl-5,5′-dichloro-9-ethylthiacarbocyanine in solution at room temperature and at 77 K was related to incoherent exciton migration within large aggregate domains. Inside a domain, the rate constant for EEA was estimated to be at least 1.5 times smaller than that of exciton decay. The experimental results were incompatible with one-dimensional incoherent exciton transport. The intensity dependence of the fluorescence anisotropy decay was assigned to EEA. At 77 K and in the absence of EEA, the average angle of the transition dipole changed less than 25° during exciton migration over a domain.
The absorption spectrum of a suspension containing aggregates of LHC II, the light-harvesting chlorophyll complex associated with photosystem II, when corrected for distortions introduced by scattering and mutual shadowing of trimers within a single aggregate, turns out to be almost superposable on the absorption spectrum recorded after disrupting the aggregates by the addition of a detergent at a concentration close to its critical micelle concentration (CMC). The correction for scattering is effected by implementing a strategy proposed in 1962 by Latimer and Eubanks; that for shadowing, by using a relation derived by Duysens in 1956, which also furnishes an estimate of the aggregate size. The standard procedure for bringing down scattering-related distortions, namely the use of an opal-glass plate, is found to be unsatisfactory for LHC II samples. Extinction spectra (i.e. scattering-contaminated experimental absorption spectra), recorded over a limited range of the detergent concentration (lying between zero and the CMC), are found to pass through two isosbestic points, which differ from their counterparts in true absorption spectra: being points at which total extinction stays constant, their locations depend on the instrumental geometry as well as on the size of the aggregates.
How do chromophores interact when attached to the rim of a dendrimer? In order to approach this problem, a time-resolved fluorescence upconversion study with 250 fs resolution has been performed on a series of dendrimers, in which the number of chromophores varied between one and four. The dendrimers were first generation tetraphenylmethane core compounds, to which one to four peryleneimide chromophores were attached; four are shown colored red in 1. A very important process was found to be singlet-singlet annihilation taking place in the multichromophoric compounds.
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.
Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.
Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.
We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.
Steady-state absorption and fluorescence excitation spectra have been measured at 25 degrees C in order to elucidate the differences between isolated chloroplasts from pea (chilling-sensitive plant) and bean (chilling-tolerant plant) and their response to oxygen treatment. A weaker light harvesting in bean in comparison with pea chloroplasts is related to higher free fatty acids level and extended peroxidation activities of bean chloroplasts. Peroxidation of free fatty acids in bean chloroplasts results in an accumulation of oxygenated forms of fatty acids demonstrated by a large negative band around 400 nm in absorption difference spectra, while the excitation spectra are not significantly altered. Similar changes have been observed in the lipase-treated pea chloroplasts. In contrast, in both pea and bean chloroplasts exhibiting no peroxidation due to antimycin A treatment, oxygen induces a pronounced absorbance increase in the regions around 435, 470 and 674 nm indicating the chloroplast swelling. A decline of chlorophyll fluorescence excitation caused by oxygen, may result from a decrease in energy transfer from antennae complexes to chlorophyll species emitting at both 680 and 740 nm. The oxygen-induced changes are partially reversed upon restoration of anaerobic conditions. The presented data show for the first time, that in contrast to pea chloroplasts the peroxidation abolishes an oxygen-induced decrease in light harvesting in bean chloroplasts, i.e., a chilling-sensitive plant.
Energy transfer of the light harvesting complex LHC-II trimer, extracted from spinach, was studied in the Q(y) region at room temperature by femtosecond transient absorption spectroscopy. Configuration interaction exciton method [Linnanto et al. (1999) J Phys Chem B 103: 8739-8750] and 2.72 A structural information reported by Liu et al. was used to calculate spectroscopic properties and excitation energy transfer rates of the complex. Site energies of the pigments and coupling constants of pigment pairs in close contact were calculated by using a quantum chemical configuration interaction method. Gaussian random variation of the diagonal and off-diagonal exciton matrix elements was used to account for inhomogeneous broadening. Rate calculations included only the excitonic states initially excited and probed in the experiments. A kinetic model was used to simulate time and wavelength dependent absorption changes after excitation on the blue side of the Q(y) transition and compared to experimentally recorded rates. Analysis of excitonic wavefunctions allowed identification of pigments initially excited and probed into later. It was shown that excitation of the blue side of the Q(y) band of a single LHC-II complex results in energy transfer from chlorophyll b's of the lumenal side to chlorophyll a's located primarly on one of the monomers of the stromal side.
We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.
Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra were recorded for spinach thylakoids and for isolated, aggregated chlorophyll a/b light-harvesting pigment-protein complex, in random and magnetically aligned states of orientation at room and low temperatures. The shape and magnitude of the CD signal of most bands strongly depended on the orientation of the thylakoid membranes or the aggregated pigment-protein complex. In both thylakoids and aggregated light-harvesting complexes, however, the MCD spectra of the two different orientations were almost identical. Random and magnetically aligned samples exhibited anomalous, large CD signals outside the bands of pigment absorbance. Lack of similarity between the corresponding MCD and CD spectra showed that the large CD signals are not produced as a distortion of CD of absorbance by light scattering. Instead, these anomalous spectral features are believed to originate in differential selective scattering of circularly polarized light. The results lead to the conclusion that the light-harvesting pigment-protein complex in thylakoid grana forms a helical macroarray with dimensions commensurate with the wavelengths of the anomalous circular dichroism signals. A hypothesis is put forward suggesting a role for these macrodomains in granal organization.
The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes.
Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.
We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.
The structure of the light-harvesting chlorophyll a/b-protein complex, a membrane protein serving as the major antenna of solar energy in plant photosynthesis, has been determined at 6 A resolution by electron crystallography. Within the complex, three membrane-spanning alpha helices and 15 chlorophyll molecules are resolved. There is an intramolecular diad relating two of the alpha helices and some of the chlorophylls. The spacing of the chlorophylls suggests energy transfer by delocalized exciton coupling and Förster mechanisms.
Long-range chiral organization of the pigment-protein complexes in mature granal chloroplasts has been established by differential polarization imaging and local circular dichroism spectra. Linear and circular dichroism images of oriented chloroplasts were obtained in a confocal differential polarization microscope. The circular dichroism images display signals of opposite signs emerging from discrete regions with local dichroic values much larger than anticipated, indicating domains in the thylakoid membranes having long-range chiral organization. These domains are associated with positive and negative circular dichroism bands obtained at specific locations on the chloroplasts. Surprisingly, the local circular dichroism spectra do not display the excitonic shape of spectra obtained for macroscopic suspensions, but the latter can be produced by superposition of two local spectra of opposite sign. These data are evidence for the existence of long-range chiral order of the pigment-protein complexes in thylakoid membranes. The possible role of the long-range chiral domains in the efficiency of energy delocalization through the thylakoid membranes is discussed.
A highly purified chlorophyll a/b light-harvesting complex (chl a/b LHC; chl a/b ratio 1.2) was obtained from Triton-solubilized chloroplast membranes of pea and barley according to the method of Burke et al. (1978, Arch. Biochem. Biophys. 187: 252--263). Gel electrophoresis of the cation-precipitated chl a/b LHC from peas reveals the presence of four polypeptides in the 23- to 28-kdalton size range. Three of these peptides appear to be identical to those derived from re-electrophoresed CPII and CPII* bands. In freeze-fracture replicas, the cation-precipitated chl a/b LHC appears as a semicrystalline aggregate of membranous sheets containing closely spaced granules. Upon removal of the cations by dialysis, the aggregates break up into their constituent membranous sheets without changing their granular substructure. These membranous sheets can be resolubilized in 1.5% Triton X-100, and the chl a/b LHC particles then reconstituted into soybean lecithin liposomes. Freeze-fracture micrographs of the reconstituted chl a/b LHC vesicles suspended in a low salt medium reveal randomly dispersed approximately 80-A particles on both concave and convex fracture faces as well as some crystalline particle arrays, presumably resulting from incompletely solubilized fragments of the membranous sheets. Based on the approximately 80-A diameter of the particles, and on the assumption that one freeze-fracture particle represents the structural unit of one chl a/b LHC aggregate, a theoretical mol wt of approximately 200 kdalton has been calculated for the chl a/b LHC. Deep-etching and negative-staining techniques reveal that the chl a/b LHC particles are also exposed on the surface of the bilayer membranes. Addition of greater than or equal to 2 mM MgCl2 or greater than or equal to 60 mM NaCl to the reconstituted vesicles leads to their aggregation and, with divalent cations, to the formation of extensive membrane stacks. At the same time, the chl a/b LHC particles become clustered into the adhering membrane regions. Under these conditions the particles in adjacent membranes usually become precisely aligned. Evidence is presented to aupport the hypothesis that adhesion between the chl a/b LHC particles is mediated by hydrophobic interactions, and that the cations are needed to neutralize surface charges on the particles.
The theoretical relationships between the fluorescence and photochemical yields of PS II and the fraction of open reaction centers are examined in a general model endowed with the following features: i) a homogeneous, infinite PS II domain; ii) exciton-radical-pair equilibrium; and iii) different rates of exciton transfer between core and peripheral antenna beds. Simple analytical relations are derived for the yields and their time courses in induction experiments. The introduction of the exciton-radical-pair equilibrium, for both the open and closed states of the trap, is shown to be equivalent to an irreversible trapping scheme with modified parameters. Variation of the interunit transfer rate allows continuous modulation from the case of separated units to the pure lake model. Broadly used relations for estimating the relative amount of reaction centers from the complementary area of the fluorescence kinetics or the photochemical yield from fluorescence levels are examined in this framework. Their dependence on parameters controlling exciton decay is discussed, allowing assessment of their range of applicability. An experimental induction curve is analyzed, with a discussion of its decomposition into alpha and beta contributions. The sigmoidicity of the induction kinetics is characterized by a single parameter J related to Joliot's p, which is shown to depend on both the connectivity of the photosynthetic units and reaction center parameters. On the other hand, the relation between J and the extreme fluorescence levels (or the deviation from the linear Stern-Volmer dependence of 1/phi f on the fraction of open traps) is controlled only by antenna connectivity. Experimental data are consistent with a model of connected units for PS II alpha, intermediate between the pure lake model of unrestricted exciton transfer and the isolated units model.
Large molecular aggregates, condensed biological macromolecules, intact membrane systems, and cell organelles often exhibit intense anomalous circular dichroism (CD) bands which are absent in systems of lower structural complexity. Theory predicts that in dense, chirally organized macroaggregates the size of the aggregate controls the magnitude of the anomalous CD bands [Keller, D., & Bustamante, C. (1986) J. Chem. Phys. 84, 2972-2979]. Photosynthetic pigment-protein complexes in their native thylakoid membranes and in vitro were used to provide direct experimental evidence of the size dependency of CD in macroaggregates.
Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mm KCl and 10 mm MgCl(2) resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-Delta(3)-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII(1):LHCII(3)) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII(1):LHCII(3) of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LCHII observed in situ and in vitro.
It has been proposed that the increase in thermal dissipation of excitation energy by thylakoid membranes at high light intensity is dependent upon the formation of an aggregated state of the light-harvesting complexes of Photosystem II (LHCII) (Horton et al. (1991) FEBS Lett. 292, 1-4). Therefore, a study of the spectroscopic changes occurring upon LHCII aggregation in vitro has been undertaken. Aggregation of LHCII, brought about by the removal of detergent, is associated with quenching of chlorophyll fluorescence and the appearance of the major emission band at 700 nm at 77 K (F700). Aggregation is associated with absorption bands with maxima at approx. 510 nm, 660 nm and 690 nm; each of these bands preferentially sensitises F700. The temperature-dependence of the emissions at 680 nm and 700 nm is very different; whereas the yield of F680 saturated at 240 K, that of F700 was not saturated at 77 K. The results are discussed in terms of the unusual and unique properties of aggregated LHCII, providing the basis for energy dissipation in vivo.
The light-harvesting complex (LHC) of higher plants isolated using Triton X-100 has been studied during its transformation into a monomeric form known as CPII. The change was accomplished by gradually increasing the concentration of the detergent, sodium dodecyl sulfate (SDS). Changes in the red spectral region of the absorption, circular dichroism (CD), and linear dichroism spectra occurring during this treatment have been observed at room temperature. According to a current hypothesis the main features of the visible region absorption and CD spectra of CPII can be explained reasonably successfully in terms of an exciton coupling among its chlorophyll (Chl) b molecules. We suggest that the spectral differences between the isolated LHC and the CPII may be understood basically in terms of an exciton coupling between the Chl b core of a given CPII unit and at least one of the Chla's of either the same or the adjacent CPII. We propose that this Chl a-Chl b coupling existing in LHC disappears upon segregation into CPII, probably as a result of a detergent-related overall rotation of the strongly coupled Chl b core which changes the relative orientations of the two types of pigments and thus the nature of their coupling.
Spinach chloroplast chlorophyll fluorescence at 685 and 735 nm (F685 and F735) has been time-resolved with a low-jitter streak camera system. Measurements are reported largely for 77 K, using single 30-ps 532-nm excitation pulses with pulse fluences of 2 · 1012 to 2 · 1016 photons · cm−2. A slightly fluence-dependent delay (16 ± 3 ps) found for the rise of F735 relative to F685 is too small to correspond to transfer into the species emitting F735 from the species emitting F685. The rise of F735 is biphasic at lower intensities and monophasic at higher intensity. The delay in the rise of F685 is smaller than approx. 2 ps. The time-resolved F685 is fit with a three-component model in which energy is transferred from a large pool of antenna chlorophylls to a small pool closely connected to the reaction center, all exciton annihilation occurring in the small pool. The F735 biphasic rise and its sensitivity to excitation fluence is explained by a similar but independent model. Data at our lowest intensities are consistent with those obtained by a photon-counting method using very low intensity excitation.
Time-resolved picosecond absorption measurements were performed on aggregates of the light-harvesting chlorophyll (Chl) complexes from spinach thylakoids. Variation of the intensity of the exciting laser pulse showed that efficient excitation annihilation occurs in these aggregates. From the time-integrated absorption decays, the domain size of these aggregates was calculated to be at least 300 and probably about 1000 chlorophyll a molecules. From the time-resolved chlorophyll a excited state decays, the rate of excitation annihilation per pair of excitations, γ2, was calculated to be (2–3)·109s−1. It was shown that the annihilation competes with a 400 ps decay component in the mono-excitation decay, probably due to Chl aggregates. A second decay phase of 2–3 ns is not associated with annihilation. The calculated domain size and annihilation rate constants correspond to a nearest-neighbour transfer rate of at least 2·1011s−1 but probably closer to 1012s−1. Our results show that although these Chl complexes are dissociated from the membrane, their aggregation is such that fast energy transfer occurs over a large number of chlorophyll molecules.
Utilizing about 1 μs duration laser pulse excitation at 650 nm, it is demonstrated that the phenomenon of singlet-triplet exciton annihilation can provide information on the domain size of photosynthetic systems (number of chlorophyll molecules connected to each other by energy transfer). The domain sizes are estimated from the shapes of the fluorescence yield vs. excitation-energy curves, from the number of photon hits per molecule per pulse, and from the triplet quantum yield utilizing the master equation theory developed by Paillotin, Geacintov and Breton (Paillotin, G. et al. (1983) Biophys J. 44, 65–77). The photosynthetic systems investigated in this manner include light-harvesting chlorophyll-protein complexes (LHCP) isolated by sodium dodecyl sulfate solubilization of spinach chloroplasts followed by polyacrylamide gel electrophoresis, aggregates of light-harvesting complexes (LHC), and Photosystem I particles (both prepared by Triton X solubilization and sucrose gradient centrifugation), as well as chloroplasts and free chlorophyll a in pyridine solution or polyacrylamide gels. The LHCP particles are characterized by approx. five connected chlorophyll molecules per domain and a triplet quantum yield of approx. 0.2. The LHC complexes and chloroplasts are examples of large domains (more than 240 molecules) with triplet quantum yields of approx. 0.08 and 0.10–0.15, respectively. The Photosystem I particles similarly constitute large domains, but the fluorescence quenching is relatively inefficient, because of the apparently low triplet quantum yield of approx. 0.01.
A theory is developed for calculating the kinematic part of the exciton-exciton annihilation rate in molecular crystals. The spatial and spin motion of the excitons, as well as the annihilation process itself, is treated phenomenologically. Exciton propagation is assumed to take place as in the hopping model. The importance of the dimensionality of the exciton motion is pointed out; in nearly one- or two-dimensional cases, certain lifetime processes control the exciton collision rate, in contrast to the three-dimensional case. These lifetime processes include motion out of the one- or two-dimensional subspace and, for excitons with spin, spin relaxation. The theory leads to a description of magnetic field effects on the annihilation rate of triplet excitons at room temperature. When applied to triplet excitons in anthracene, this description gives a satsfactory fit to the observed effects and leads to the determination of the nearest-neighbor exciton annihilation rate, the singlet-channel annihilation rate constant, and the exciton diffusion constant for motion perpendicular to the ab plane of anthracene.
A theory of the polymer and salt induced (psi)‐type circular dichroism observed in DNA aggregates is presented. Using the main formalism developed in the previous paper to treat the interaction of light and large, dense molecular aggregates, it is shown that the anomalously large signals observed in the circular dichroism of certain molecular aggregates result from: (a) the presence of a long‐range chiral structure in the aggregate; (b) delocalization throughout the entire particle of the light‐induced excitations in the chromophores. This delocalization and the resulting ‘‘collective response’’ of the chromophores in the aggregate is favored in particles having a three‐dimensional packing. It is shown that to describe adequately the internal field in these aggregates, intermediate and radiation coupling mechanisms should be taken into account in addition to the regular dipole–dipole interactions. Furthermore, no dipole approximation in the exponentials of the form eik⋅x are made. It is shown that in these circumstances, one of the circular polarizations of the light can exchange energy more efficiently than the opposite polarization. This gives rise to a circular dichroism signal whose magnitude is proportional to the overall size and long‐range chiral nature of the aggregates. The theory is applied to two cases: (1) to the dimer ApA when it is shown (for the case of this small system) to reduce to the classical theories of DeVoe and Tinoco, and (2) for a toroidal aggregate of DNA of 3000 Å diameter with an internal chiral structure, as found by Haynes et al. in polylysine–DNA condensates. Good qualitative agreement with the observed spectra is found. The theory represents the first successful attempt to explain the physical origin of the psi‐type CD effect. Useful information regarding the chiral structure of the aggregates can be inferred from the theory.
Bimolecular and pseudo-unimolecular reactions of random walkers on large clusters are presented via simple, analytically soluble, rate equations. The rate coefficient has a simple dependence on time: K(t) ∝ t-h where 1 ≥ h ≥ 0 and h = 1-f where 2f is the effective fracton (phonon on fractal) dimension of Alexander and Orbach. h measures the degree of local heterogeneity. In the limit of local homogeneity h = 0. Triplet exciton homofusion rates on isotopic mixed naphthalene crystals are consistent with the formalism, giving classical diffusive behavior (h = 0) above the percolation threshold and nonclassical local heterogenous behavior below it (h = 0.48 for 4% C10H8 in C10D8).
The hypothesis that fluorescence quenching in aggregated light harvesting chlorophyll protein complex II is associated with the formation of minor spectral forms absorbing near 655 nm and between 680 nm-690 nm is examined. Using an homogeneous LHCII preparation, steady-state absorption changes measured at room temperature are quantitatively compared with the associated steady state fluorescence changes by means of the Stepanov relation. It is demonstrated that upon LHCII aggregation, the relative fluorescence yield is constant for chlorophyll forms absorbing between 650 nm and 690 nm. This indicates that the minor chlorophyll forms formed upon LHCII aggregation are not quenching species.
Time-resolved absorption difference profiles were obtained for LHC-II trimers, isolated from Photosystem II in spinach with n-dodecyl β-d-maltoside, using one-color and two-color pump-probe techniques. The one-color isotropic signals are predominantly excited state absorption at 640 nm, and a combination of photobleaching and stimulated emission at wavelengths ≥ 665nm. At intermediate wavelengths, dynamic red-shifting due to downhill energy transfer among the chlorophyll (Chl) spectral forms produces a bipolar signal, in which prompt photo-bleaching/stimulated emission is superseded at later times by excited state absorption. Triexponential analyses of these profiles yield the lifetime components 2–6 ps (associated with the spectral shifting), 14–36 ps (possibly due to energy transfer between LHC-II monomers), and several hundred picoseconds. The one-color anisotropy decays are resolvable at 665–675 nm, with lifetimes of 4.3 to 6.3 ps. They are unresolvably fast (i.e., exhibit subpicosecond lifetimes) at 640–650 nm. The two-color isotropic absorption difference signals show clear spectral evolution arising from equilibration among the LHC-II spectral components for excitation wavelengths shorter than 670 nm. However, most of this spectral evolution occurs within less than 2.5 ps. No resolvable anisotropy decay was observed in the two-color experiments. Taken together, the one-color and two-color experiments indicate that both picosecond and subpicosecond energy transfer steps occur in this antenna. The faster processes appear to dominate the spectral equilibration; slower processes occur in isoenergetic energy transfers among the longer-wavelength Chl a spectral forms that absorb between 665 and 675 nm. The values of the long-time anisotropic r(x), measured in the one-color and two-color experiments, are qualitatively consistent with static linear dichroism spectra of these preparations.
The major chlorophyll a/b light-harvesting complex LHC-II was isolated from spinach thylakoid membranes after solubilization with the non-ionic detergent n-dodecyl-β-d-maltoside. The complex is characterized by a chlorophyll a to b ratio of 1.5±0.1. The spectroscopic properties of this trimeric complex were analyzed and found to be very similar to those observed in the intact thylakoid membrane. By low-temperature absorption, linear dichroism and circular dichroism spectroscopy it is shown that the chlorophyll Qy(0−0) absorption region of the complex is characterized by at least six and perhaps nine chlorophyll transitions with significant differences in energy, orientation and rotational strength. Excitation of the pigments resulted in a sharp fluorescence band peaking near 680 nm at 77K. Presumably, this fluorescence originates from the main and red-most chlorophyll a Qy transition, which peaks at 676 nm and is oriented at a small angle with the plane of the particle. Fitting of polarized excitation spectra indicated that the polarization of Chl676 is about 0.1; this argues against a single emitting species, but is in line with a model in which efficient energy transfer occurs among several (>3)Chl676 molecules which form a circularly degenerate oscillator in a plane. Since the shapes of the linear dichroism and polarized excitation spectra were found to be virtually identical between 640 and 676 nm, this plane is probably the same as the plane of the particle. Above 676 nm, the polarization rises gradually to a value of about 0.2, which is interpreted as a result of inhomogeneous line broadening of the 676 nm transition. A model that describes the chlorophyll organization of LHC-II predominantly in terms of exciton interactions between several chlorophyll a and several chlorophyll b molecules is discussed.
A highly purified light-harvesting pigment--protein complex (LHC) was obtained by fractionation of cation-depleted chloroplast membranes using the nonionic detergent, Triton X-100. The isolated LHC had a chlorophyll a/b ratio of 1.2 and exhibited no photochemical activity. SDS-polyacrylamide gel electrophoresis of the LHC revealed three polypeptides in the molecular weight classes of 23, 25, and 30 x 10/sup 3/. Antibodies were prepared against the LHC and their specificity was established. The effect of the ..cap alpha..-LHC (antibodies to LHC) on salt-mediated changes in PS I and PS II photochemistry, Chl ..cap alpha.. fluorescence inductions, and 77/sup 0/K fluorescence emission spectra was investigated. The results show that: (i) The Mg/sup 2 +/-induced 20% decrease in photosystem I (PS I) quantum yield observed in control chloroplasts was blocked by the presence of the ..cap alpha..-LHC antibody. (ii) The Mg/sup 2 +/-induced 70% increase in photosystem II (PS II) quantum yield of control chloroplasts was reduced 35% for plastids in the presence of ..cap alpha..-LHC antibody. (iii) The Mg/sup 2 +/-induced increase in room-temperature variable fluorescence was reduced 60% by ..cap alpha..-LHC antibody. (iv) The Mg/sup 2 +/-induced increase in the F685/F730 emission peak ratio at 77/sup 0/K was inhibited 50% in the presence of ..cap alpha..-LHC antibody. These results provide direct evidence for the involvement of the light-harvesting complex in cation regulation of energy redistribution between the photosystems. The fact that the ..cap alpha..-LHC antibody does not fully block Mg/sup 2 +/-induced PS II increases or chlorophyll fluorescence increases supports the concept that Mg/sup 2 +/ has two mechanics of action: one effect on energy distribution and a second direct effect on photosystem II centers.
A new hypothesis is presented to explain the major molecular process that regulates the efficiency of light harvesting by chloroplast membranes. It is proposed that in excess light the decrease in the thylakoid lumen pH causes an increase in aggregation of the light harvesting complexes of photosystem II resulting in formation of an efficient pathway for non-radiative dissipation of excitation energy. The aggregation is potentiated by the conversion of violaxanthin to zeaxanthin. This hypothesis is based upon (i) similarity between the spectroscopic changes associated with energy dissipation and those observed upon aggregation of isolated light harvesting complex; and (ii) the link between changes in light scattering and increased energy dissipation.
The structure of the light-harvesting chlorophyll a/b-protein complex has been determined at 3.7 A resolution in projection by electron diffraction, electron microscopy and image analysis. Diffraction patterns and high-resolution spotscan images of two-dimensional crystals stabilized with tannin were recorded at low temperature. Phases of structure factors were obtained directly by image processing, after correction of the images for lattice distortions, defocus and beam tilt. Amplitudes were measured by electron diffraction. The projection map shows the detailed structure of the trimeric complex, suggesting the positions of two domains of potential structural and functional homology, of one membrane-spanning alpha-helix approximately perpendicular to the membrane plane and of several tightly bound lipid molecules.
The phenomenon of Duysens flattening of the absorption spectra resulting from the inhomogeneous distribution of the chromophores in the solution is analyzed. These inhomogeneities are treated as localized statistical fluctuations in the concentration of the absorbing species, by using the Gaussian distribution. A law of absorbance is obtained, and the effect of light scattering on the flattening is also characterized. The flattening in the circular dichroism spectra of particulate suspensions is then analyzed. It is shown that the degree of flattening of the circular dichroism of a suspension is, in general, different from the corresponding flattening of its absorption spectrum. A quantitative relationship between the two effects is established.
Angle dependence of circular intensity differential scattering (CIDS) and of nonpolarized scattering was determined in isolated spinach chloroplasts at 514.5 nm. CIDS between 0 degrees and 170 degrees was independent of the nonpolarized scattering and showed intense lobes of alternating signs, exhibiting the negative and positive maxima around 15 degrees and 70 degrees, respectively. These results provide experimental evidence for the existence of large helically organized macroaggregates of pigment-protein complexes in thylakoid membranes. Modeling of the CIDS data by a simple helical array of uniaxial polarizable groups suggests that the chiral structure is left-handed with pitch and radius of the order of 385 nm.
The non-exponential character of the fluorescence induction observed in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or at low temperature has been previously interpreted in terms of energy transfer between photosynthetic units. Alternative hypotheses have been recently proposed and this problem is discussed on the basis of new experimental results.Several independent methods are used to decrease the concentration of photoactive centers. If the inactive centres are blocked in a non-quenching form, one observes that the number of photons collected per active center increases up to a factor of 3 as the concentration of active centres decreases. However, if the inactive centres are blocked in a quenching form, the number of photons collected per active center remains independent of the concentration of the active centers.From the experiments described in this paper, one can conclude: 1.1. Each center includes only one photoactive chlorophyll.2.2. Energy transfer occurs between three and probably more connected photosynthetic units.3.3. One must assume that the photosynthetic units are not identical. This heterogeneity may be due to their size or some structural features.
The excitation energy transfer between chlorophyll b (Chl b) and chlorophyll a (Chl a) in the isolated trimeric chlorophyll-a/b-binding protein complex of spinach photosystem 2 (LHC II) has been studied by femtosecond spectroscopy. In the main absorption band of Chl b the ground state recovery consists of two components of 0.5 ps and 2.0 ps, respectively. Also in the Chl a absorption band, at 665 nm, the ground state recovery is essentially bi-exponential. In this case is, however, the fastest relaxation lifetime is a 2.0 ps component followed by a slower component with a lifetime in the order of 10-20 ps. In the Chl b absorption band a more or less constant anisotropy of r = 0.2 was observed during the 3 ps the system was monitored. In the Chl a absorption band there was, however, a relaxation of the anisotropy from r = 0.3 to a quasi steady state level of r = 0.18 in about 1 ps. Since the 0.5 ps component is only seen upon selective excitation of Chl b we assign this component to the energy transfer between Chl b and Chl a. The other components most likely represents redistribution processes of energy among spectrally different forms of Chl a. The energy transfer process between Chl b and Chl a can well be explained by the Förster mechanism which also gives a calculated distance of 13 A between interacting chromophores. The organisation of chlorophylls in LHC II is discussed in view of the recent crystal structure data (1991) Nature 350, 130].
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