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More on the catalysis of internal conversion in chlorophyll a by an adjacent carotenoid in light-harvesting complex (Chla/b LHCII) of higher plants: Time-resolved triplet-minus-singlet spectra of detergent-perturbed complexes
Abstract
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.
... The quenching of 3 Chl by Cars strongly depends on the arrangement of the pigments in the complex, with an exponential dependence on the distance between them, stemming from the Dexter exchange transfer mechanism (Siefermann-Harms, 1987). Comparatively small structural alterations, for example induced by increasing the detergent concentration, can affect the energetic coupling between pigments, reducing k T −T , and in turn raising the 3 Chl yield (Naqvi et al., 1999). The CD spectra of LHCII, especially in the Cars region, indicate that such conformational changes occur upon aggregation and in the lipid environment (Akhtar et al., 2015). ...
... These results indicate that Chl PB is a late event in photoinhibition in native thylakoid membranes and a consequence of the disruption of T-T transfer from Chl to Cars. As we do not observe substantial bleaching of Cars in isolated LHCII, similar to other results (Siefermann-Harms, 1990), it could then be postulated that T-T transfer is disrupted in the isolated antenna complexes, making the Chls more susceptible to PB (Naqvi et al., 1999). According to the kinetic model, isolated LHCII should be capable of producing enough 1 O 2 to explain the observed Chl PB. ...
Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments – protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl a photobleaching in membranes and detergent was found to be 3.4 × 10–5 and 1.4 × 10–5, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10–5 and 2 × 10–5). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition in vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.
... The lifetimes of 3 Car absorption decay in this work are shorter than those reported previously (Mozzo et al., 2008;Naqvi et al., 1997;Peterman et al., 1995). This should be attributed to the 0.1% DDM concentration we used for the sample because the former research has proposed that the increasing detergent concentration might lower the triplet energy transfer efficiency from Chla to Car (Naqvi et al., 1999). Since the purpose of this work is to investigate the influence of site-directed mutagenesis on the triplet energy transfer in LHCII, we focus on comparing the differences between WT and the mutants in the same detergent medium. ...
... This architecture makes it possible for the effective transfer of the excitation to the reaction center and the quenching of over-excitation to heat under too strong light conditions. Perturbing LHCII complexes using detergents has been used in earlier investigations to show that increasing the detergent concentration lowers the triplet energy transfer efficiency from Chla to Car and raises the yields of 3 Chla and fluorescence (Naqvi et al., 1999), which suggests that comparatively small structural alterations can strongly affect the energetic coupling of different pigments and consequently, the distribution of the excitations in the complex. Similarly, in the LHCII mutants, the conformational changes of the complex might lead to changes in the mutual separations and orientations of the pigments as evidenced by the changed absorption and fluorescence excitation spectra (Fig. 1). ...
Under strong light conditions, long-lived chlorophyll triplets (3Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench 3Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of 3Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching 3Chl.
... Zeaxanthin might also be directly involved in quenching via a charge-transfer heterodimer complex with chlorophyll (Holt et al. 2005). In general, carotenoids and carotenoid:chlorophyll dyads or clusters appear to play a special role in the dissipation of the excess excitation energy (Naqvi et al. 1999; Barzda et al. 2001 ; van Amerongen and van Grondelle 2001). As quenching can be formed even in the absence of zeaxanthin, its direct function has been doubted. ...
... These models incorporate many elements of earlier ones, in particular regarding the role of aggregation of the complexes and the dependence of the energy transfer pathways on the de-epoxidation state of xanthophylls (Standfuss et al. 2005; Pascal et al. 2005). Other authors, based on transient spectroscopic data, suggest that carotenoid:chlorophyll dyads or clusters might be responsible for the modulation of the fluorescence yield in LHCII (Naqvi et al. 1999; van Amerongen and van Grondelle 2001), and a charge-transfer complex might play a key role in NPQ (Holt et al. 2005), or xanthophyll cycle pigments outside the complexes can have a regulatory role via affecting the aggregation state and electronic transition levels of LHCII (Gruszecki et al. 2006). Although it cannot be ruled out that more than one mechanism operate in vivo, it is clear that these models are at variance with each other. ...
The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures
between 5 and 25°C and during rapid jumps of the leaf temperature. At 5°C, NPQ relaxed very slowly in the dark and was sustained
for up to 30min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to
25°C, the quenched state relaxed within 1min, characteristic for qE, the energy-dependent component of NPQ. Measurements
of the membrane permeability (ΔA515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of
low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These
data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves
a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending
the model of Horton and co-workers (FEBS Lett 579:4201–4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.
... Plants can properly alter the content of photosynthetic pigments according to their environment [49]. Our research found that different ratios of ON/IN had a certain positive impact on the Chl content of T. grandis leaves, while at ratios of 3/7, the Chla and total chlorophyll contents were significantly higher in all N treatment groups (N2 > N3 > N1 > N4 > CK). ...
Atmospheric nitrogen (N) deposition is coupled with organic nitrogen (ON) and inorganic nitrogen (IN); however, little is known about plant growth and the balance of elements in Torreya grandis growing under different ON/IN ratios. Here, we investigated the effects of ON/IN ratios (1/9, 3/7, 7/3, and 9/1) on leaf stoichiometry (LF), chlorophyll content, and chlorophyll fluorescence of T. grandis. We used ammonium nitrate as the IN source and an equal proportion of urea and glycine as the ON source. The different ON/IN ratios altered the stoichiometry and photochemical efficiency in T. grandis. Although the leaf P content increased significantly after treatment, leaf N and N:P maintained a certain homeostasis. Torreya grandis plants performed best at an ON/IN ratio of 3/7, with the highest values of chlorophyll-a, total chlorophyll, maximum photochemical efficiency, and photosynthetic performance index. Thus, both ON and IN types should be considered when assessing the responses of plant growth to increasing N deposition in the future. Our results also indicated that the leaf P concentration was positively correlated with Chl, Fv/Fm, and PIabs. This result further indicates the importance of the P element for plant growth against the background of nitrogen deposition. Overall, these results indicate that T. grandis might cope with changes in the environment by maintaining the homeostasis of element stoichiometry and the plasticity of PSII activity.
... In general, photosynthetic pigments mainly contain Chla, Chlb, and Car (Fargasova et al. 2006). Chla can absorb and transform light, whereas Chlb and Car can transmit the light absorbed to Chla, promoting the photochemical process (Naqvi et al. 1998). Plants can properly adjust the composition and content of photosynthetic pigments according to the balance between the absorbed and used energy (Foyer and Harbinson 1997;Fernández-Marín et al. 2018). ...
Warming and N (nitrogen) deposition are the two main driving factors of global change. We examined the effects of increased N deposition (8 kg ha⁻¹ year⁻¹) and warming, as well as their combined effect on the leaf photosynthetic pigments of Leymus secalinus, which is one of the key alpine plants growing in different grassland habitats on Qinghai-Tibetan plateau. In 2014, the experiments were established in 12 plots (2×5m) of three types of habitats including alpine meadow (AM), alpine steppe (AS), and cultivated grassland (CG) with the following treatments: CK (control treatment), N (only N deposition), W (only warming), and W&N (warming combined with N deposition). Results showed that the effects of warming and N deposition on photosynthetic pigments of Leymus secalinus varied with different grassland habitat types. In three grassland types, warming led to no significant effects on the total chlorophyll content of L. secalinus, while N deposition alone only significantly enhanced total chlorophyll content in alpine meadow and cultivated grassland. N deposition combined with warming only significantly enhanced total chlorophyll content of L. secalinus in alpine steppe and cultivated grassland. Chla content plays an important role in determining the variation of total chlorophyll content. Chla/Chlb ratio of L. secalinus was more stable in alpine meadow compared with that of L. secalinus in the other two grassland types. Car/Chl ratio of L. secalinus was not prone to be affected by warming and N deposition in all grassland types. Leaf N content was obviously positively correlated with photosynthetic pigments, especially Chla content. Warming and N deposition all affected photosynthetic pigment dynamics and tended to increase Chla by enhancing its weight. Our results highlighted that both warming and N deposition as well as their combination can alter the trade-off of photosynthetic pigments through enhancing the Chla ratio in L. secalinus. In addition, growing habitats should be within consideration when studying alpine plants adaptation mechanism to global change in the future.
... 33 Further, the incomplete occupancy or the vacancy of the neoxanthin site leads to the drop of 3 Chl a* quenching efficiency from 95 to 70%, 9 and the monomerization or the detergent treatment result in a substantial decrease in the Chl-to-Car TTET efficiency. 12,34 These observations prove that in addition to the TEET efficiency of quenching 3 Chl a* that depends tightly on the donor−acceptor distance, the conformational modulation of 1 Chl a* distribution over the LHCII complex can readily alter the 3 Chl a* photoproduction. ...
... 33 Further, the incomplete occupancy or the vacancy of the neoxanthin site leads to the drop of 3 Chl a* quenching efficiency from 95 to 70%, 9 and the monomerization or the detergent treatment result in a substantial decrease in the Chl-to-Car TTET efficiency. 12,34 These observations prove that in addition to the TEET efficiency of quenching 3 Chl a* that depends tightly on the donor−acceptor distance, the conformational modulation of 1 Chl a* distribution over the LHCII complex can readily alter the 3 Chl a* photoproduction. ...
... Also anisotropic CD spectra, which are in general more sensitive to isolate exciton interactions, failed to detect significant changes in the FR region upon aggregation (Akhtar et al. 2019a). Quite interestingly, and in direct contrast to all the proposed Chl-Car exciton quenching models, actually an increase, rather than a decrease, of Chl-Lut exciton coupling has been found when going from the non-solubilized aggregated LHCII to the unquenched detergent-solubilized LHCII trimers (Naqvi et al. 1999) along with the expected changes in the CD spectra (Lambrev et al. 2007;Akhtar et al. 2015). ...
Light-harvesting complex II (LHCII) is the major antenna complex in higher plants and green algae. It has been suggested that a major part of the excited state energy dissipation in the so-called "non-photochemical quenching" (NPQ) is located in this antenna complex. We have performed an ultrafast kinetics study of the low-energy fluorescent states related to quenching in LHCII in both aggregated and the crystalline form. In both sample types the chlorophyll (Chl) excited states of LHCII are strongly quenched in a similar fashion. Quenching is accompanied by the appearance of new far-red (FR) fluorescence bands from energetically low-lying Chl excited states. The kinetics of quenching, its temperature dependence down to 4 K, and the properties of the FR-emitting states are very similar both in LHCII aggregates and in the crystal. No such FR-emitting states are found in unquenched trimeric LHCII. We conclude that these states represent weakly emitting Chl-Chl charge-transfer (CT) states, whose formation is part of the quenching process. Quantum chemical calculations of the lowest energy exciton and CT states, explicitly including the coupling to the specific protein environment, provide detailed insight into the chemical nature of the CT states and the mechanism of CT quenching. The experimental data combined with the results of the calculations strongly suggest that the quenching mechanism consists of a sequence of two proton-coupled electron transfer steps involving the three quenching center Chls 610/611/612. The FR-emitting CT states are reaction intermediates in this sequence. The polarity-controlled internal reprotonation of the E175/K179 aa pair is suggested as the switch controlling quenching. A unified model is proposed that is able to explain all known conditions of quenching or non-quenching of LHCII, depending on the environment without invoking any major conformational changes of the protein.
... In fact, since they exhibit dramatically different fluorescence lifetimes (Van Oort et al. 2011) and excitation energy pathways (Enriquez et al. 2015), they must differ-albeit probably only subtly-also in their molecular architecture. On the other hand, as revealed by CD spectroscopy and fluorescence lifetimes of LHCII, there are significant differences between the detergent-solubilized states and the aggregated states in vitro, and between the in vitro and in vivo states (Naqvi et al. 1999;Lambrev et al. 2007, Miloslavina et al. 2008-a collaboration with Alfred Holzwarth, a 49er). As a model system, liposome-embedded trimers mimic reasonably well the in vivo state . ...
This short review, with a bit of historical aspect and a strong personal bias and emphases on open questions, is focusing on the (macro-)organization and structural-functional flexibilities of the photosynthetic apparatus of oxygenic photosynthetic organisms at different levels of the structural complexity-selected problems that have attracted most my attention in the past years and decades. These include (i) the anisotropic organization of the pigment-protein complexes and photosynthetic membranes-a basic organizing principle of living matter, which can, and probably should be adopted to intelligent materials; (ii) the organization of protein complexes into chiral macrodomains, large self-assembling highly organized but structurally flexible entities with unique spectroscopic fingerprints-structures, where, important, high-level regulatory functions appear to 'reside'; (iii) a novel, dissipation-assisted mechanism of structural changes, based on a thermo-optic effect: ultrafast thermal transients in the close vicinity of dissipation of unused excitation energy, which is capable of inducing elementary structural changes; it makes plants capable of responding to excess excitation with reaction rates proportional to the overexcitation above the light-saturation of photosynthesis; (iv) the 3D ultrastructure of the granum-stroma thylakoid membrane assembly and other multilamellar membrane systems, and their remodelings-associated with regulatory mechanisms; (v) the molecular organization and structural-functional plasticity of the main light-harvesting complex of plants, in relation to their crystal structure and different in vivo and in vitro states; and (vi) the enigmatic role of non-bilayer lipids and lipid phases in the bilayer thylakoid membrane-warranting its high protein content and contributing to its structural flexibility.
... Based on the above data we can conclude that, while lamellar aggregates of LHCII retain the native organization of complexes in the thylakoid membrane, detergent-solubilized trimers do not, and instead undergo perturbations. Detergent solubilization affects the orientation of carotenoids with respect to the membrane plane and perturbs a few excitonic CD bands probably arising from carotenoid: Chl interactions (Lambrev et al. 2007b); it also leads to a diminution in the efficiency of triplet-triplet energy transfer from Chl a to lutein and a rise in both the triplet formation yield and the fluorescence yield of Chl a (Naqvi et al. 1999). Upon the removal of detergent from gel-trapped isolated trimers, Ilioaia et al. (2008) observed similar alterations, but with the opposite sign -both in CD spectra and in fluorescence lifetimes. ...
This chapter focuses on the nature, physical mechanism and physiological significance of structural changes at different levels of structural complexity of the photosynthetic apparatus of oxygenic photosynthetic organisms – with special emphasis on non-photochemical quenching (NPQ) of the singlet-excited state of chlorophyll a. The dual role of the antenna system, light harvesting under low light and thermal dissipation under high light, requires substantial structural flexibility. Indeed, reversible structural changes induced by excess-light excitation have been observed in different organisms via several techniques, including electron microscopy, light scattering measurements, circular dichroism spectroscopy, and small-angle neutron scattering. These investigations have revealed reorganizations at the level of (i) the ultrastructure of thylakoid membranes, affecting repeat distances, (ii) macro-organization of light-harvesting antenna complexes within the membrane, perturbing the ordered arrays of the complexes, as well as (iii) isolated light-harvesting antenna systems. In some cases, correlations between the observed reorganizations and NPQ have been well established. In many cases, however, the relationship between changes in the macro-organization of the light-harvesting antenna or thylakoid membranes and NPQ remains to be explored. Nevertheless, the currently available data strongly suggest that, while an overall reorganization is a necessary condition for adjusting the functional activities to different growth light intensities and for photoprotection, in particular, reorganization per se is not sufficient for NPQ (that also requires the presence of effector molecules, such as, e.g., PsbS or zeaxanthin). This chapter also deals with the effect of dissipation of excess excitation energy on the photosynthetic apparatus: light-induced reversible structural changes in different antenna systems in vivo and in vitro, with rates linearly proportional to the intensity of excess light (that is not used for photosynthesis). These structural changes have been proposed to be driven by what is termed a thermo-optic mechanism: elementary structural changes elicited by ultrafast local heat transients due to the dissipation of photon energy, a photophysical feedback mechanism that appears to modulate NPQ and regulate enzymatic functions in light-harvesting antenna complexes.
... The molecular mechanism behind NPQ has so far not been unequivocally established 19 and there may, in fact, be several mechanisms acting in parallel, such as decay via the S 1 state of lutein 3,20 and via a Chl-Chl charge transfer state; 17,18,21 other mechanisms have been proposed as well. [22][23][24] While a great amount of effort has been centered on explaining the precise mechanism of quenching the excess excitation energy of Chls, in-depth analyses on examining the Chl energy transfer process in the quenched state of LHCII are scarce. Femtosecond transient absorption spectroscopy studies on LHCII aggregates previously using Chl a excitation have led to proposals of models and dynamics of energy flow to the intermediate quencher state, be it either a carotenoid S 1 state 20 or a Chl-Chl charge transfer state. ...
The pathways and dynamics of excitation energy transfer between the chlorophyll (Chl) domains in solubilized trimeric and aggregated light-harvesting complex II (LHCII) are examined using two-dimensional electronic spectroscopy (2DES). The LHCII trimers and aggregates exhibit the unquenched and quenched excitonic states of Chl a, respectively. 2DES allows direct correlation of excitation and emission energies of coupled states over population time delays, hence enabling mapping of the energy flow between Chls. By the excitation of the entire Chl b Qy band, energy transfer from Chl b to Chl a states is monitored in the LHCII trimers and aggregates. Global analysis of the two-dimensional (2D) spectra reveals that energy transfer from Chl b to Chl a occurs on fast and slow time scales of 240-270 fs and 2.8 ps for both forms of LHCII. 2D decay-associated spectra resulting from the global analysis identify the correlation between Chl states involved in the energy transfer and decay at a given lifetime. The contribution of singlet-singlet annihilation on the kinetics of Chl energy transfer and decay is also modelled and discussed. The results show a marked change in the energy transfer kinetics in the time range of a few picoseconds. Owing to slow energy equilibration processes, long-lived intermediate Chl a states are present in solubilized trimers, while in aggregates, the population decay of these excited states is significantly accelerated, suggesting that, overall, the energy transfer within the LHCII complexes is faster in the aggregated state.
... Significant aggregation-induced changes have been reported to occur also in the LD and CD spectra [31]. However, at least part of the differences between the detergent solubilized trimer and the LHCII aggregate might originate from a perturbation of the molecular architecture of the complexes by the detergent, as indicated first by triplet-minus-singlet transient spectroscopy [32], and also by a more recent CD and LD spectroscopic study, when comparison was made to the native TMs and small aggregates [33]. Detergents appear to affect mainly the orientation of carotenoids, and a few excitonic CD bands arising from carotenoid:chlorophyll interactions. ...
Chloroplast thylakoid membranes accommodate densely packed protein complexes in ordered, often semi-crystalline arrays and are assembled into highly organized multilamellar systems, an organization warranting a substantial degree of stability. At the same time, they exhibit remarkable structural flexibility, which appears to play important - yet not fully understood - roles in different short-term adaptation mechanisms in response to rapidly changing environmental conditions. In this review I will focus on dynamic features of the hierarchically organized photosynthetic machineries at different levels of structural complexity: (i) isolated light harvesting complexes, (ii) molecular macroassemblies and supercomplexes, (iii) thylakoid membranes and (iv) their multilamellar membrane systems. Special attention will be paid to the most abundant systems, the major light harvesting antenna complex, LHCII, and to grana. Two physical mechanisms, which are less frequently treated in the literature, will receive special attention: (i) thermo-optic mechanism - elementary structural changes elicited by ultrafast local heat transients due to the dissipation of photon energy, which operates both in isolated antenna assemblies and the native thylakoid membranes, regulates important enzymatic functions and appears to play role in light adaptatation and photoprotection mechanisms; and (ii) the mechanism by which non-bilayer lipids and lipid phases play key role in the functioning of xanthophyll cycle de-epoxidases and are proposed to regulate the protein-to-lipid ratio in thylakoid membranes and contribute to membrane dynamics.
... In order to address the idea of the link between the environment and indeed aggregation of LHCII and pH sensitivity of the fluorescence quenching we have used an in vitro quenching approach [27,34,40,53] to determine the pH response of LHCII trimers incubated at different concentrations of detergent and in different aggregation states. Although a number of papers dealt with the effect of the detergent on the spectroscopic properties of LHCII such as changes in time resolved triplet-minus-singlet signal [46] or variable linear dichroism and circular dichroism signals [47], no studies so far have been focused on measuring the pK for quenching of LHCII in different detergent environments and upon variations of the aggregation state. Previously, pH titrations of quenching in isolated LHCII trimers showed an increase in pK in the complexes that contained zeaxanthin in comparison to those that were enriched in violaxanthin [48], both xanthophylls bound to the extrinsic V1 site [49]. ...
Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6μM n-dodecyl β-D-maltoside) have been tested. It was found that at the detergent concentration of 6μM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
... Non-photochemical quenching contains a short-term (1-2 min) component, qE, which depends on the transmembrane ∆pH (3) as well as the presence of the PsbS protein (4) and zeaxanthin (5), resulting in a feedback de-excitation via a charge transfer state of the heterodimer zeaxanthin-Chl a complex (6). In general, carotenoids appear to play a special role in regulation of dissipation of the excess excitation energy in the lightharvesting antenna of PSII (7)(8)(9). ...
Evidence of nonequilibrium local heating in transient spectra of LHCII, the main light-harvesting complex of plants, was studied by using various excitation intensities over a wide temperature range, from 10 K to room temperature. No obvious manifestation of local heating was found at room temperature, whereas at 10 K, the local heating effect is discernible when more than 10 excitons per LHCII trimer per pulse are generated. Under these conditions, a major part of the excitation energy is converted into heat as a result of exciton-exciton annihilation. Initially, the heat energy is allocated on chlorophyll a molecules, reaching hundreds of degrees at the highest excitation intensities, which correspond to almost 100 excitons per trimer generated by a single excitation pulse. The decay of the nonequilibrium temperature is characterized well by two exponentials. The initial phase of cooling, which is most likely caused by the spreading of heat over the protein, corresponds to a characteristic time constant of approximately 20 ps. Later, the cooling rate decelerates to approximately 200 ps and is related to heat transfer to the solvent.
... 6. Difference spectra of Lhcb6 obtained from the spectra in Figs. 2 and 4 (WT– mutant). (A) Absorption; (B) CD. in LHCII might lead to a shortening of the Chl fluorescence lifetime [11,33] and it was proposed that excitonic interactions between lutein and Chl a would lead to mixing of their excited states [11]. Since the excited state of lutein is much shorter-lived (~ 10 ps) this will shorten the Chl excited-state lifetime. ...
In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575-578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.
The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light-harvesting complexes (LHC). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana-stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the FCP complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X-100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macro-aggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid-induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana-stroma differentiation.
Photoprotective thermal energy dissipation (as assessed via non-photochemical quenching of singlet-excited chlorophyll a, NPQ) in plants is driven by various mechanisms occurring over different time scales. The rapid and reversible part of NPQ, also called qE (for energy-dependent quenching), was demonstrated to correlate with the twisting of a neoxanthin molecule in the light-harvesting antenna as observed by resonance Raman spectroscopy (Nature 450: 575–578, 2007). Interestingly, the extent of fluorescence quenching correlates with the change in Raman signal in different situations: during NPQ in vivo, during fluorescence quenching upon aggregation of LHCII (the major light-harvesting complex in plants), and in crystals of LHCII. In the same study, it was proposed that the quenching is caused by excitation energy transfer from chlorophyll a to lutein in LHCII after a structural change that correlates with the twisting of the neoxanthin. However, this view has been challenged by others for different reasons. Here we discuss the arguments in favor and against this mechanism. A short overview is given of the spectroscopic data on chlorophyll-carotenoid interactions in plant light-harvesting systems, the changes in interactions upon aggregation or crystallization, and the possible relationship to the mechanism of NPQ.
Photosystem II (PS II) of green plants represents one of the most complex systems in plant biology. It consists of at least 29 different types of proteins, binds a huge number of pigments (chlorophylls and carotenoids), produces the oxygen that we breathe and plays essential roles in many regulation mechanisms of the photosynthesis. In this chapter, we discuss the mechanisms of light-harvesting and trapping of excitation energy in green plant PS II. We describe structural models of the pigment-binding proteins of PS II and their supramolecular organization, and consider the energy transfer processes between pigments in individual complexes, with an emphasis on the major trimeric light-harvesting complex II (LHCII) of PS II, which is by far the best studied protein of the PS II antenna. Energy transfer between different complexes also is addressed, as well as studies on complete PS II complexes. In contrast to many previous studies, we conclude that the relatively long excited-state lifetime in PS II should be explained by a combination of three different factors: 1) the relatively slow energy transfer in the outer antenna, 2) the small number of chlorophylls that connect the antenna to the reaction center, and 3) the relatively slow charge separation in the reaction center. The process of excitation trapping can therefore not be classified as being purely diffusion-, transfer-to-the-trap-, or trap-limited.
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.
Excitation energy transfer in the isolated light-harvesting chlorophyll (Chl)-a/b protein complex of photosystem II (LHC II) was studied by the one-colour pump-probe technique with femtosecond time resolution. After exciting Chl-b by 638 nm beam, the dynamic behaviour shows that the ultrafast energy transfer from Chl-b at positions of B2, B3, and B5 to the corresponding Chl-a molecules in monomeric subunit of LHC II is in the time scale of 230 fs. While with the excitation of Chl-a at 678 nm, the energy transfer between excitons of Chl-a molecules has the lifetime of about 370 fs, and two other slow decay components are due to the energy transfer between different Chl-a molecules in a monomeric subunit of LHC II or in different subunits, or due to change of molecular conformation.
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.
The absorption spectrum of the main antenna complex of photosystem II, LHCII, has been modeled using, as starting points, the chlorophyll (chl) atomic coordinates as obtained by the LHCII crystal analysis [Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X., and Chang, W. (2004) Nature 428, 287-292] of three different trimers. The chl site Q(y) transition energies have been obtained in terms of the chl macrocycle deformations influencing the energy level of the chl frontier orbitals. Using these chl site transition energy values and the entire set of interaction energies, calculated in the ideal dipole approximation, the complete Hamiltonians for the three LHCII trimers have been written and the full set of 42 eigenstates of each LHCII trimer have been calculated. With the 42 transition energies and transition dipole strengths, either unperturbed or associated to the eigenstates, the LHCII Q(y) absorption spectrum has been calculated using a chl absorption band shape. These calculations have been performed both in vacuo and in the presence of a medium. Despite the number of approximations, a good correlation with the measured absorption spectrum of a LHCII preparation is obtained. This analysis shows that, although a substantial C3 symmetry of the LHCII trimer in terms of both chl-chl distances and interaction energies is present, a marked variation among monomer subsets of site transition energies is estimated. This leads to a C3 symmetry breaking in the unperturbed chl site transition energies set and, consequently, in the trimer eigenstates. It is also concluded that interactions among chlorophylls do not significantly modify the light absorption role of LHCII in plant leaves.
Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 micromol m(-2) s(-1)) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV-Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600-1700 cm(-1)). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm(-1), corresponding to a protein in the alpha-helical structure at the expense of the band centered at 1621 cm(-1), characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.
Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.
Light-induced photooxidation of chlorophyll (Chl) a, b and xanthophylls was investigated in LHCIIb, the antenna pigment-protein complex of photosystem II. Absorption difference spectra at normal and low temperatures show initially (at less than 25% Chl a decay) a selective bleaching of a red-shifted Chl b with absorption bands at 487 and 655 nm, Chl b (460/650 nm) and Chl a (433/670 nm), which changes to a less selective photooxidation pattern at deeper bleaching stages. Difference absorption spectra and HPLC analyses indicate different photooxidation rates of pigments in the order neoxanthin>Chl a>lutein approximately Chl b. Despite significant pigment loss as monitored with absorption spectra, CD spectra indicate an essentially complete persistence of the protein secondary structure. Fluorescence excitation spectra suggest the conversion of a small fraction of Chl a into pheophytin a which acts as a fluorescence quencher, possibly through temporary charge separation process. The strong features in the electroabsorption (Stark effect) spectra due to chlorophyll b at 655 nm and a xanthophyll at 510 nm, and the spectral changes mentioned above are assigned to Chl molecules located at several binding sites in LHCIIb protein and are discussed in the context of spatial configuration and interactions of pigment molecules.
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.
Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.
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.
The structure of the light-harvesting chlorophyll a/b-protein complex, an integral membrane protein, has been determined at 3.4 A resolution by electron crystallography of two-dimensional crystals. Two of the three membrane-spanning alpha-helices are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophyll a is in close contact with chlorophyll b for rapid energy transfer, and with two carotenoids that prevent the formation of toxic singlet oxygen.
Electron cryo-microscopy of two-dimensional crystals of the major light-harvesting complex from pea chloroplasts has revealed the structure at 3A Å resolution. The atomic model provides insights into the molecular architecture, function and assembly of this key component of the photosynthetic membrane and will enable us to understand how energy is transferred to the photosynthetic reaction centres.
Excitation energy transfer and exciton-exciton annihilation in the isolated light-harvesting chlorophyll a/b protein complex of spinach photosystem II (LHC II) has been studied by two-color absorption difference spectroscopy with femtosecond time resolution. After selectively exciting Chl b at 645 nm, the transient absorption changes were monitored at wavelengths where either Chl b (655 nm) or Chl a (680 nm) dominates the absorption of LHC II. From the good correspondence of the lifetimes obtained from a numerical analysis of the very fast relaxation in the Chl b absorption band (160 [+-] 20 fs) and the rise kinetics in the Chl a absorption band (145 [+-] 20 fs), it is suggested that the Chl b [yields] Chl a excitation energy transfer occurs on a time scale of about 150 fs. In addition, at both probe wavelengths (655 and 680 nm) lifetimes of 3-7 ps were observed which likely arise from excitation energy transfer processes connected with spectral shifting. The kinetic curves of the transient absorption changes at 680 nm show a remarkable intensity dependence which is ascribed to exciton-exciton annihilation. Since at a probe wavelength of 655 nm no intensity effect on the kinetics was observed, it is concluded that annihilation processes preferably occur among excited singlet states of Chl a molecules. 28 refs., 6 figs.
Monomeric chlorophyll a (Chl a) was obtained from the isolated core antenna complex CP47 of photo-system II after incubation with the detergent triton X-100 and was studied by low-temperature polarized light spectroscopy with the aim to obtain model spectra for Chi a in intact photosynthetic complexes. Evidence is presented by circular dichroism and anisotropy measurements that the isolated chlorophyll is monomeric. The absorption bandwidths are relatively large compared to those found in photosynthetic complexes due to inhomogeneous broadening introduced by the detergent. By selective laser excitation at low temperature, considerable narrowing can be achieved. A number of vibrational bands are resolved in the site-selected, polarized absorption and fluorescence emission spectra. The emission spectrum of Chi a in detergent-damaged CP47 is compared with that of Chi a in the intact light-harvesting complex of photosystem II (LHC-II) from green plants. The spectra are remarkably similar indicating that the low-temperature thermal emitter in LHC-II has spectral properties that are very similar to those of monomeric Chl a.
Carotenoid triplets in isolated light harvesting complex (LHC) II of spinach at different concentrations were studied by absorbance-detected magnetic resonance (ADMR). Going from high to low LHC II concentrations, a change was observed in the intensity of the ADMR spectra of the |D|+|E| transition recorded at 507 nm relative to that recorded at 525 nm, from approx. 0.5 to approx. 1.0. The relative intensity of the 2|E| transition did not change. The change in relative intensity of the ADMR singla is due to a change of the ADMR signal intensity of the |D|+|E| transition that is detected at 525 nm. The effect is ascribed to an aggregation of trimeric LHC II into an oligomeric form of LHC II. Taking into account the narrowing of the zero-field resonance bands with oligomerisation, and the absence of bandshifts, the relative increase of the signal intensity of the |D|+|E| transition detected at 525 nm can be explained by assuming that the oligomer consists of a multiple of trimers, between which ‘inter-trimer’ energy transfer occurs. This yields an increase in the number of triplets that is transferred to the carotenoid having its triplet absorption maximum at 525 nm. Our new results indicate that the carotenoids are bound to Chl a monomers, and not dimers as proposed earlier (Van der Vos, R., Carbonera, D. and Hoff, A.J. (1991) Appl. Magn. Res. 2, 179–202).
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.
We present a compilation of spectral parameters associated with triplet–triplet absorption of organic molecules in condensed media. The wavelengths of maximum absorbance and the corresponding extinction coefficients, where known, have been critically evaluated. Other data, for example, lifetimes, energies and energy transfer rates, relevant to the triplet states of these molecules are included by way of comments but have not been subjected to a similar scrutiny. Work in the gas phase has been omitted, as have theoretical studies. We provide an introduction to triplet state processes in solution and solids, developing the conceptual background and offering an historical perspective on the detection and measurement of triplet state absorption. Techniques employed to populate the triplet state are reviewed and the various approaches to the estimation of the extinction coefficient of triplet–triplet absorption are critically discussed. A statistical analysis of the available data is presented and recommendations for a hierarchical choice of extinction coefficients are made. Data collection is expected to be complete through the end of 1984. Compound name, molecular formula and author indexes are appended.
A new strategy for ascertaining the pigment composition of photosynthetic specimens is proposed, and its advantages pointed out; it entails recording, on the same machine, the absorption spectrum of the pigment extract as well as the spectra of the cognate chromophores, and in using the latter for piecemeal reconstruction of the former.
It is argued that the double-beam method for the automatic correction of fluorescence excitation spectra, which proved to be a great boon in the days of analog instrumentation, has become, after the advent of digital techniques for acquiring and manipulating spectral data, more of a hindrance than a help; the benefits of reverting to a single beam device and making in situ measurement of the excitation intensity are pointed out.
Carotenoid pigments, ubiquitous in photosynthetic membranes, are essential for the survival of green plants1. Two facets of carotenoid function are recognized in photosynthetic membranes. First, carotenoids prevent the chlorophyll-photosensitized formation of highly destructive singlet oxygen by intercepting the chlorophyll triplet states2–10 and may also scavenge additional singlet oxygen present11,12. Second, carotenoids perform an antenna function by transferring the energy of absorbed light at the singlet excited state level to the chlorophyll system for the execution of photosynthetic work13–16. Nevertheless, the mechanisms by which carotenoids perform these functions are poorly understood. We now report that a unique synthetic carotenoporphyrin I consisting of a carotenoid part covalently linked to a synthetic tetraarylporphyrin successfully mimics both the photophysical functions of carotenoids in photosynthesis. The explanation for this seems to be the close interaction of the carotenoid and porphyrin π-electron systems.
Time-resolved femtosecond transient absorption measurements have been carried out at room temperature on light-harvesting chlorophyll a/b protein complex of photosystem II (LHC II) trimers prepared from spinach. Exciting in the chlorophyll (Chl) b region at 650 nm with very low intensity, virtually annihilation-free two-color transient absorption measurement of the kinetics over 100 ps, between 645 and 690 nm, yield global lifetimes of 175 fs, 625 fs, and 5 ps and a long component (≥790 ps) where the three fastest lifetimes reflect Chl b to Chl a energy transfer. Using a camera detection system, kinetics over 400 ps at still low annihilation levels and with much higher spectral resolution have been obtained. Short lifetime components of 180 fs, 480 fs, and 6 ps are comparable with the two-color data, but in addition, 34 and 85 ps components with small amplitudes are resolved and a long component (3.6 ns) is fixed at the longest lifetime value determined by fluorescence. Annihilation statistics have been calculated to compare these and earlier results. On the basis of these results and recent electron diffraction structural data, a preliminary three-pool Chl a, three-pool Chl b kinetic model is proposed. The possible influence of variable xanthophyll composition on quenching in LHC II preparations isolated from light- and dark-adapted leaves has been investigated using time-resolved picosecond fluorescence at room temperature. Global lifetimes of 5 ps, 170 ps and 3.6 ns, the lifetimes of the terminal LHC II excited state, were obtained. No discernable quenching effect due to the presence of zeaxanthin was observed.
A series of carotenoporphyrin dyad molecules in which the carotenoid is covalently linked to a tetraarylporphyrin at the ortho, meta, or para position of a meso aromatic ring has been prepared, and the molecules have been studied using steady-state and transient fluorescence emission, transient absorption, and H-1 NMR methods. Triplet-triplet energy transfer from the porphyrin moiety to the carotenoid has been observed, as has singlet-singlet energy transfer from the carotenoid polyene to the porphyrin. In addition, the carotenoid quenches the fluorescence of the attached porphyrin by a mechanism which increases internal conversion. The rates of all three of these processes are slower for the meta isomer than for the corresponding ortho and para molecules. Analysis of the data suggests that the triplet-triplet energy transfer is mediated by a through-bond (superexchange) mechanism involving the pi-electrons of the linkage bonds, rather than a direct, through-space coupling of the chromophores. The same appears to be true for the process leading to enhanced internal conversion. The results are consistent with a role for the through-bond mechanism in the singlet-singlet energy transfer as well. Simple Huckel molecular orbital calculations are in accord with the proposed through-bond process.
We show that isosbestic points generated by changes in temperature or solvent composition may be formed under a wider variety of conditions than previously recognized, so that the rules previously proposed for interpreting these points in terms of the number of absorbing species or the number of independent reaction parameters are unreliable. Examples of experimental cases in which these rules break down are cited. We also point out that in most applications it is more useful to show that spectra are linearly related than to show that they have isosbestic points.
A laser photolysis study, on the nanosecond time scale, has been carried out on the major light-harvesting chlorophyll-protein complex of photosystem II (LHC II..beta..). The transient triplet absorption of the isolated LHC II..beta.. has been compared to those of its constituent chromophores in dilute micellar aqueous solutions, namely, chlorophyll a, chlorophyll b, chlorophyll a + chlorophyll b, and chlorophyll a + ..beta..-carotene. The results indicate that the carotenoid in the LHC II..beta.. is photosensitized by triplet chlorophyll a and that the decay of the transient triplet absorption exhibits two transients, one in the approx. 50-ns range and the other in the approx. 10..mu..s time scales. The former transient is light-intensity dependent and is attributed to an annihilation process between two triplets of chlorophyll a, while the latter is due to the decay of the triplet carotenoid to its ground state.
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.
The dependence of the spectroscopic properties and aggregation state of a highly purified light-harvesting chlorophyll a/b protein complex (LHCII) population on detergent (octyl-β-d-glucopyranoside (OGP)) concentration in the solvent was investigated. The results, including circular dichroism, time-resolved fluorescence, sucrose gradient centrifugation and isoelectrofocusing, indicate that, depending on the detergent concentration, LHCII can be found in two main quaternary states whose minimal order is three. Thus, at high OGP concentrations (3% or greater), a trimer, which is identical with the trimeric form observed in two-dimensional crystals, is stable. At OGP concentrations lower than approximately 0.65%, macroscopic aggregates are formed, accompanied by the appearance of strong pigment—pigment interactions and fluorescence quenching. At intermediate OGP concentrations (0.650%–2%) an oligomeric form of LHCII, possibly a multiple of the trimer, is stable and is similar to the so-called CPII′ oligomeric form resolved by non-denaturing sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).
Steady-state and picosecond time-resolved fluorescence techniques in conjunction with circular dichroism have been used to study the light-harvesting chlorophyll-a/b protein complex (LHC) isolated from pea chloroplasts. In particular, the effect of changing the detergent / chlorophyll ratio on the state of the LHC has been investigated. Our results have been interpreted in light of the known protein geometry of the LHC in 2-dimensional crystals (Kühlbrandt, W. (1984) Nature 307, 478–479). The fluorescence lifetime data reveals 1 / e-lifetimes of 3.53 (±0.04) ns and 1.10 (±0.01) ns for a stable, efficiently energy-transferring state of the LHC. Subnanosecond lifetimes are observed under conditions leading to aggregation, while a long component of 5.50 (±0.16) ns corresponding to free Chl a is found when the detergent / chlorophyll ratio is high. The circular dichroism shows a major Chl-b exciton, a Chl-a / b exciton and a further ‘quenching’ Chl-b exciton. These have been attributed to: a C3 symmetric Chl-b interaction for which the intact C3 protein trimer geometry is a prerequisite; a dimeric Chl-a / b interaction, the presence of which is critically dependent on the detergent type; and a further Chl-b interaction which arises from the presence of aggregated trimers, respectively. We have found that the degree of heterogeneity with respect to the oligomeric state of the pigment-protein trimers is dependent upon the detergent / chlorophyll ratio used. Low detergent / chlorophyll ratios result in extensive aggregation of the trimers with a geometry similar to that found in 2-dimensional crystals of the LHC. Moderate detergent conditions yield predominantly non-aggregated trimers. Excess detergent conditions result in considerable chromophore heterogeneity and loss of the main Chl-b exciton consistent with protein denaturation through an initial break up of the trimer geometry. From these results we believe that in vitro the minimum stable functional unit corresponds to a C3 symmetric protein trimer.
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.
A model for the spectral characteristics, the transition dipole
moment orientations, and the energy transfer properties of chlorophyll
(Chl) a and b molecules in the light-harvesting complex (LHC) II is
proposed on the basis of the results from femtosecond transient
measurements and other spectroscopic data. The model uses the structural
data (Kuhlbrandt; et al. Nature 1994, 367, 614) and is obtained using a
genetic algorithm search of the large parameter space. Forster resonance
transfer has been assumed as the mechanism of energy transfer. The
spectral and orientational assignments of all twelve Chl molecules of a
LHC II monomer are proposed. In the best fit model two of the seven Chl
molecules that are proximal to the central luteins are Chl b. In contrast
to prior assumptions, the basic feature of the model consists of an
intermediately strong coupling (V < 100 cm(-1)) between the Chl a and b
molecules in close pairs and the absence of substantial excitonic coupling
between Chls a, thus indicating an overall limited influence of excitonic
effects on spectra and kinetics. A theoretical estimation of exciton
effects supports these model assumptions. Over most of the difference
absorption spectrum good agreement between experimental and theoretical
kinetics has been obtained. Energy transfer times in the symmetric LHC II
trimer range from 90 fs to 5.1 ps. For the monomeric complexes only the
longest lifetime is significantly affected and predicted to be just
slightly longer (6.6 ps). The predicted transition dipole moment
orientations result in weak coupling between the LHC II monomers. Several
possible routes to improve both the data fitting and the reliability of
the predictions in the future are discussed.
Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.
A detailed comparison has been made between dichroic steady-state spectroscopic properties at 77 K of several trimeric and monomeric forms of the major chlorophyll a/b binding protein (LHC-II) from pea. Monomeric forms were obtained by applying high concentrations of nonionic detergents, by a lipase treatment, or by a chymotrypsin/trypsin treatment. The latter treatments removed phosphatidyl glycerol essential for trimer formation. The absorption and dichroism spectra indicate that for trimeric LHC-II the chlorophyll b absorption region is centered around 649 nm and is composed of at least five subbands near 640, 647, 649, 652, and 656 nm. The chlorophyll a absorption region is centered around 670 nm and is composed of at least five bands near 661, 668, 671, 673, and 676 nm. The chlorophyll b band near 647 and 652 nm and the chlorophyll a bands near 668 and 673 nm are absent in the circular dichroism spectrum after monomerization. A configuration in which pigments of the same nature located on different monomers become excitonically coupled in the trimer could explain these results. In monomers obtained in high concentrations of nonionic detergents, no additional bands have disappeared, but the absorption spectra of the other two types of monomers lack the bands at 640 and 661 nm. These monomers have lost some chlorophyll a and b according to the fluorescence emission spectra, which show contributions from free chlorophyll a and b. The results suggest that phosphatidyl glycerol not only is involved in trimer formation but also has a structural role within the monomers.
Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.
Under many environmental conditions, plants are exposed to levels of sunlight in excess of those required for photosynthesis. Then, a regulated increase in the rate of nonradiative dissipation of excess excitation energy in the thylakoid membrane correlates with the conversion of the carotenoid violaxanthin into zeaxanthin and provides protection from the damaging effects of excessive irradiation. The hypothesis that these carotenoids specifically control the oligomerization of the light harvesting complexes of photosystem II was tested by investigating the effects of violaxanthin and zeaxanthin on the behavior of the major complex, LHCIIb, on sucrose gradients; it was found that zeaxanthin stimulated the formation of LHCIIb aggregates with reduced chlorophyll fluorescence yield whereas violaxanthin caused the inhibition of such aggregation and an elevation of fluorescence. Measurements of 77 K fluorescence indicated that zeaxanthin was not exerting an additional direct quenching of chlorophyll fluorescence. These effects can explain the physiological control of the light harvesting system by the xanthophyll cycle.
Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupa et al. (1987, Plant Physiol. 84, 19-24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, < or = 0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (deltaCD). (ii) At the optimal concentration, around 0.7 +/- 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0-1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (deltaCD). (iii) At detergent concentration above the optimum, around 0.8-1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor deltaCD. (iv) High concentrations (> or = 1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no deltaCD.
A spectral and functional assignment of the xanthophylls in monomeric and trimeric light-harvesting complex II of green plants has been obtained using HPLC analysis of the pigment composition, laser-flash induced triplet-minus-singlet, fluorescence excitation, and absorption spectra. It is shown that violaxanthin is not present in monomeric preparations, that it has most likely a red-most absorption maximum at 510 nm in the trimeric complex, and that it is involved in both light-harvesting and Chl-triplet quenching. Two xanthophylls (per monomer) have an absorption maximum at 494 nm. These play a major role in both singlet and triplet transfer. These two are most probably the two xanthophylls resolved in the crystal structure, tentatively assigned to lutein, that are close to several chlorophyll molecules [Kühlbrandt, W., Wang, N. D., & Fujiyoshi, Y. (1994) Nature 367, 614-621]. A last xanthophyll contribution, with an absorption maximum at 486 nm, does not seem to play a significant role in light-harvesting or in Chl-triplet quenching. On the basis of the assumption that the two structurally resolved xanthophylls are lutein, this 486 nm absorbing xanthophyll should be neoxanthin. The measurements demonstrate that violaxanthin is connected to at least one chlorophyll a with an absorption maximum near 670 nm, whereas the xanthophylls absorbing at 494 nm are connected to at least one chlorophyll a with a peak near 675 nm.
Energy transfer from chlorophyll b (Chl b) to chlorophyll a (Chl a) in monomeric preparations of light-harvesting complex II (LHCII) from spinach was studied at 77 K using pump-probe experiments. Sub-picosecond excitation pulses centered at 650 nm were used to excite preferentially Chl b and difference absorption spectra were detected from 630 to 700 nm. Two distinct Chl b to Chl a transfer times, approximately 200 fs and 3 ps, were found. A clearly distinguishable energy transfer process between Chl a molecules occurred with a time constant of 18 ps. The LHCII monomer data are compared to previously obtained LHCII trimer data, and both data sets are fitted simultaneously using a global analysis fitting routine. Both sets could be described with the following time constants: 140 fs, 600 fs, 8 ps, 20 ps, and 2.9 ns. In both monomers and trimers 50% of the Chl b to Chl a transfer is ultrafast (<200 fs). However, for monomers this transfer occurs to Chl a molecules that absorb significantly more toward shorter wavelengths than for trimers. Part of the transfer from Chl b to Chl a that occurs with a time constant of 600 fs in trimers is slowed down to several picoseconds in monomers. However, it is argued that observed differences between monomers and trimers should be ascribed to the loss of some Chl a upon monomerization or a shift of the absorption maximum of one or several Chl a molecules. It is concluded that Chl b to Chl a transfer occurs only within monomeric subunits of the trimers and not between different subunits.
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.
The energy transfer process in the minor light-harvesting antenna complex CP29 of green plants was probed in multicolor transient absorption experiments at 77 K using selective subpicosecond excitation pulses at 640 and 650 nm. Energy flow from each of the chlorophyll (Chl) b molecules of the complex could thus be studied separately. The analysis of our data showed that the "blue" Chl b (absorption around 640 nm) transfers excitation to a "red" Chl a with a time constant of 350 +/- 100 fs, while the 'red' Chl b (absorption at 650 nm) transfers on a picosecond time scale (2.2 +/- 0.5 ps) toward a "blue" Chl a. Furthermore, both fast (280 +/- 50 fs) and slow (10-13 ps) equilibration processes among the Chl a molecules were observed, with rates and associated spectra very similar to those of the major antenna complex, LHC-II. Based on the protein sequence homology between CP29 and LHC-II, a basic modelling of the observed kinetics was performed using the LHC-II structure and the Förster theory of energy transfer. Thus, an assignment for the spectral properties and orientation of the two Chl's b, as well as for their closest Chl a neighbors, is put forward, and a comparison is made with the previous assignments and models for LHC-II and CP29.
The molecular mass of an oligomeric integral membrane protein, the light-harvesting chlorophyll a/b-protein complex from the photosynthetic membranes of chloroplasts, has been determined in detergent solution by analytical ultracentrifugation and measurement of the density increment at constant chemical potential of all diffusible solutes. The technique used eliminates any problems resulting from detergent binding to the protein, is independent of the particular detergent used (in this case the nonionic n-octyl beta-D-glucopyranoside), and gives the apparent weight-average molecular mass at different protein concentrations, allowing extrapolation to zero concentration. It means that the solutions of the complex must be brought to dialysis equilibrium with the solvent detergent solution and also requires a reliable method for measuring the protein concentration, for which amino acid analysis was used. The detergent-solubilized complex was a trimer that dissociated into monomers and dimers at low protein concentration. The accurate concentration determinations also allowed the molar chlorophyll-to-protein ratio to be measured as 15, corresponding to 8 chlorophyll a and 7 chlorophyll b molecules.
A new carotenoporphyrin has been prepared in which a synthetic carotenoid is joined to a tetraarylporphyrin through a flexible
trimethylene linkage. This molecule exists primarily in an extended conformation with the carotenoid chromophore far from
the porphyrin π-electron system. In benzene solution, where large-amplitude molecular motions are rapid, the molecule can momentarily assume
less stable conformations which favor triplet energy transfer, and quenching of the porphyrin triplet by the carotenoid is
fast. In a polystyrene matrix or frozen glass such motions are slow, and energy transfer cannot compete with other pathways
for depopulating the triplet state. These observations help establish the requirements for biological photoprotection.
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