Capturing the Quenching Mechanism of Light-Harvesting Complexes of Plants by Zooming in on the Ensemble
The light-harvesting complexes (LHCs) of plants can regulate the energy flux to the reaction centers in response to fluctuating light by virtue of their vast conformational landscape. They do so by switching from a light-harvesting state to a quenched state, dissipating the excess absorbed energy as heat. However, isolated LHCs are prevalently in their light-harvesting state, which makes the identification of their photoprotective mechanism extremely challenging. Here, ensemble time-resolved fluorescence experiments show that monomeric CP29, a minor LHC of plants, exists in various emissive states with identical spectra but different lifetimes. The photoprotective mechanism active in a subpopulation of strongly quenched complexes is further investigated via ultrafast transient absorption spectroscopy, kinetic modeling, and mutational analysis. We demonstrate that the observed quenching is due to excitation energy transfer from chlorophylls to a dark state of the centrally bound lutein.
INTRODUCTION<!>RESULTS<!>Heterogeneous Target Modeling Highlights the Involvement of a Carotenoid Dark State in the Quenching<!>DISCUSSION<!>EXPERIMENTAL PROCEDURES Sample Preparation<!>Time-Resolved Fluorescence
<p>In plants and green algae, photosynthetic reactions start with light absorption by pigment-protein complexes embedded in the thylakoid membrane. Such antenna proteins efficiently deliver the absorbed energy to the reaction centers, where it is used to promote charge separation, the initial step of converting light to energy. Effective regulation of this energy flux in response to changing light conditions is necessary for plants to avoid photodamage and optimize growth. 1,2 Antenna proteins play a crucial role in these regulatory mechanisms, being able to switch conformation and, consequently, their functional state: from highly fluorescent states, whose long excited-state lifetime is imperative for light harvesting, to short lifetime (quenched) states, carrying out photoprotection by safely dissipating excitation energy as heat. [3][4][5][6][7] But what is the quenching mechanism? Although many studies have addressed this question, the answer is still debated because of the technical challenges of the measurements. In principle, the most suitable way to study the mechanism of quenching would be to perform time-resolved spectroscopic measurements on isolated monomeric light-harvesting complexes (LHC). This permits the control of the experimental conditions, and the relatively low number of pigments per protein helps to avoid power-dependent artifacts such as annihilation. 8,9 Moreover, the above-mentioned conformational variety has been disclosed by single-molecule techniques for a wide range of isolated LHCs. 10,11 However, such a variegated landscape usually falls through the net of bulk experiments due to ensemble</p><p>The Bigger Picture</p><p>The light-harvesting complexes of plants have a dual and seemingly opposite role: in low light they act as antennae, collecting energy for photochemistry, whereas in high light they switch to a quenched state, dissipating the excess absorbed energy as heat to avoid photodamage. Quenching is necessary for plants to survive in high light but also causes a drop in photosynthetic efficiency. Unveiling the photoprotective mechanism is therefore important for improving crop productivity. The identification of the quenching mechanism is hindered by the fact that, in isolated solubilized complexes, the lightharvesting conformation largely dominates. Here, we have taken advantage of the presence of a subpopulation of quenched complexes in the ensemble of isolated proteins to simultaneously analyze the lightharvesting and quenched states. This allowed us to understand the nature of the photoprotective mechanism and to identify the quenching site.</p><p>averaging. When isolated, the vast majority of LHCs are found in a highly fluorescent light-harvesting state, and ensemble measurements generally fail to capture any detailed information about the quenched state. 7,12,13 Promoting the photoprotective state of photosynthetic complexes in vitro is possible but requires protein aggregation, 14,15 crystallization, 3 or embedding into gel matrices in the absence of detergent. 16 These conditions, however, pose more experimental challenges 9,17 and might be more difficult to relate to physiological conditions. As a result, the investigation of antenna quenching in vitro remains a complicated task, and the exact nature of the photoprotective mechanism is still controversial. [17][18][19][20] Only two cases have been reported so far of solubilized monomeric antennae that are constitutively in a quenched state. 21,22 Both studies evidenced the involvement of a carotenoid excited state as a quencher of chlorophyll excitation. Staleva et al. 21 showed that a carotenoid S 1 state could efficiently quench chlorophyll in one of the cyanobacterial light-inducible proteins (Hlips), which are generally regarded as the ancestors of modern LHCs. 23 More recently, our group demonstrated that in a LHC from a mutant tobacco plant containing astaxanthin, chlorophyll excitation is constitutively quenched by the astaxanthin S* state, which becomes accessible via a twist of the carotenoid end-ring. 22 However, the experimental proof that the same quenching mechanism is active in LHCs with their natural pigment content is still missing.</p><p>In this work, we were able to identify different emissive states in a wildtype monomeric LHC from plants in a bulk experiment. The antenna investigated is CP29, one of the minor LHCs of plants and algae, which is part of the photosystem II supercomplex. 24,25 It is an integral membrane protein binding 13-14 chlorophylls (mostly chlorophylls a) and 3 carotenoids (Figure 1A). [25][26][27] CP29 occupies a central position in the energy transfer routes of photosystem II, receiving excitations from peripheral LHCII and delivering them to the core protein CP47 (Figure 1B), and it was also shown to play a role in photoprotection. 28,29 All our measurements were performed on the solubilized monomeric protein. Most of the complexes were in their unquenched light-harvesting state, but a significant subset was found in a strongly quenched state.</p><p>The nature of the observed photoprotective mechanism and of the pigments involved was investigated by a combination of time-resolved spectroscopy, compartmental target modeling, and mutational analysis.</p><!><p>Time-Resolved Fluorescence and Transient Absorption Reveal Lifetime Heterogeneity Time-resolved fluorescence traces of CP29 measured at room temperature at 680 nm upon 468 nm excitation required three lifetime components for proper fitting (first row of Table 1; Figure S1). The larger contribution (70%) stems from a 4.1 ns component. The remaining contributions are from a 1.6 ns (about 14% of total signal) and a $100 ps (16% of total amplitude) component. The resulting average lifetime is around 3.1 ns. Time-resolved fluorescence traces obtained after 468 nm excitation were measured at different emission wavelengths (Figure S2) and globally analyzed to obtain the decay-associated spectrum (DAS) of each component (Figure 2A). The average lifetime is essentially wavelength independent, and all the three DAS have a profile identical to that of the steady-state spectrum of the complex (Figures 2  and S3).</p><p>The lifetime heterogeneity, which is observed in many antenna proteins, 12,13,30,31 can be explained by the presence of different emissive states of CP29, with the majority of the complexes exhibiting a long lifetime (about 4 ns). A smaller but significant fraction of antennae is found in slightly (1.6 ns) and strongly ($0.1 ns) quenched states. The high similarity of the fluorescence spectra of these states implies that the terminal emitter domain of all is identical. This suggests that redemitting (e.g., chlorophyll-chlorophyll charge transfer 32,33 ) states do not play a role in the quenching mechanism and supports the idea that the quencher in CP29 is a dark (non-emissive) state populated from chlorophyll excited states. It should be noted that previous studies have shown that the lifetime heterogeneity observed here in CP29 is an intrinsic property of all LHCs not only in detergent 11,12,31,[34][35][36] but also in liposomes, 12,37,38 lipid nanodisks, 39 and the thylakoid membrane, 6,40 suggesting that the conformations observed here are the same occurring in vivo.</p><p>To shed light on the quenching mechanism, we performed transient absorption (TA) measurements on CP29 in its monomeric state. This technique allows the observation of dark states (such as carotenoid excited states), which cannot be detected with fluorescence measurements. 41 The monomers were excited in the red edge of the absorption spectrum (682 nm; see Figure S4) and a large part of the visible was probed (from 480 to 720 nm) to be able to identify carotenoid excited states possibly involved in quenching. Two additional experiments were performed with 489 and 509 nm excitation wavelengths (direct carotenoid excitation) in order to extract the spectra of the carotenoid excited states. Global analysis was performed on all measured data (Figures 3A and 3B; see Experimental Procedures for details on data analysis). In first approximation, a sequential scheme can be used to describe the data (Figure 3C), with each species (evolution-associated difference spectrum [EADS]; Figure 3E) decaying into the following one with a specific lifetime. Since the resolved lifetimes are well separated, the resulting DADS (decayassociated difference spectra, which originate instead from a parallel kinetic scheme; Figures 3D, 3F, and S5) are essentially the spectral difference between consecutive EADS.</p><p>After excitation in the Qy band, the signal is dominated by a bleach in the Qy region due to ground-state bleaching and stimulated emission of chlorophyll excited states (blue signal in Figure 3B; black, red, and blue EADS in Figure 3E). A small positive signal at wavelengths < 600 nm is due to chlorophyll excited-state absorption (ESA; the weak red signal in Figures 3A and 3B). Global analysis of the TA data for the 682 nm excitation reveals four main components (see EADS in Figure 3E and DADS in Figure S5). Energy equilibration between different pools of chlorophylls (mostly involving uphill energy transfer; see black to red EADS in Figure 3E and blue DADS in Figure S5) takes place in 2.4 ps, in agreement with previous reports, 42,43 followed by a loss of 16% of the total bleach in 54 ps (red to blue EADS). The remaining bleach (blue EADS) decays in 2.4 ns, populating a long-lived species (green EADS), whose lifetime is far longer than the experimental time window. Such a component can be assigned to a mix of carotenoid and chlorophyll triplet states. 44 The fractions and lifetimes of the 54 ps and of the 2.4 ns components (represented by the DADS of Figure 3F) are consistent with what was observed in time-resolved fluorescence measurements (with the difference that the two longlifetime components of 1.6 and 4 ns are merged into one single nanosecond component due to the limited experimental time window). The lifetime of 54 ps for the quenched component is also a more precise estimate as compared to the lifetime obtained via TCSPC owing to the higher time resolution of TA (100 fs versus 100 ps). We can therefore relate the fast component (red DADS in Figure 3F) to the fraction of strongly quenched monomers and the slow component (blue DADS in Figure 3F) to the unquenched complexes. Notably, the quenched and unquenched components have very similar shapes and both peak at 681 nm (inset of Figure 3F). The excellent overlap in the emissive region is consistent with our time-resolved fluorescence data (Figure 2B) and shows that the quenching in CP29 is not related to red-emitting states. Compared to the chlorophyll ESA profile shown by EADS 1, 2, and 3 in Figures 3C-3E, the DADS of the 54 ps component displays larger amplitude at shorter wavelengths, suggesting the involvement of a quencher which does not have a chlorophyll excited-state character (and whose spectrum peaks instead in the 500-520 nm region; see Figure S5).</p><!><p>We have so far demonstrated that detergent-solubilized CP29 exhibits a heterogeneous distribution of lifetimes, ranging from 4 ns to less than 100 ps. Both time-resolved fluorescence and TA data exclude the contribution of chlorophyll-chlorophyll charge-transfer states in the spectra of the quenched complexes, indicating that the terminal emitter domain in both quenched and unquenched CP29 is a purely excitonic state. In light of this finding, the strong lifetime reduction in quenched complexes implies the existence of a short-lived dark state able to quench chlorophyll excitations. To resolve the spectra of the pigments active in energy transfer, we therefore applied a compartment target model to the 682 nm excitation dataset. Each compartment represents a species with a difference spectrum (SADS [species-associated difference spectrum]). 45 Compartments can communicate via energy transfer processes whose rates (and connectivity) are determined to reproduce the measured kinetics (Figure 4A). To account for the heterogeneity of CP29 emission lifetimes, the model adopts two parallel schemes referring to unquenched and quenched complexes (Figure 4A), each containing three compartments. In the scheme representing unquenched complexes (violet box), energy equilibration between two pools of chlorophylls (red and cyan compartments) is followed by the decay of their excited state in a few nanoseconds, which populates long-lived triplets (triplet formation is approximated as a one-step process from the cyan to the green compartment). In the scheme of the quenched complexes (yellow box), an identical energy equilibration between chlorophylls (red and black compartments) precedes energy transfer with a rate of 20/ns to a quencher (blue compartment). The quencher finally decays with a rate of 160/ns. Because of these inverted kinetics, the concentration of the quencher will remain small (see Figure S6). At time zero, only the red chlorophyll pools are excited, and the amount of excitation going into the quenched and unquenched compartment groups represents the fraction of quenched/unquenched complexes. More details about the compartmental model are given in Text S1.</p><p>Because of the weaker signal at wavelengths below 630 nm, the fitting in this region is very sensitive and required additional constraints on the shape of the ESA of different chlorophyll compartments. We therefore tested different sets of equality constraints (see Text S1 and Figures S7-S10 for details); the obtained quencher SADS, despite some spectral changes, always exhibited typical features of carotenoid excited states. The model leading to the best fitting is discussed below.  The first two compartments (red and black-cyan) in both quenched and unquenched complexes represent chlorophyll pools that equilibrate in a few picoseconds. The spectral bleach of bulk unquenched chlorophylls (cyan SADS in Figure 4B) has more amplitude in the region between 660 and 675 nm than that of the red chlorophyll pool (red SADS in Figure 4B), compatible with an uphill energy equilibration taking place after selective excitation at 682 nm. The triplet spectrum (green SADS; Figure 4B) has a peak around 505 nm and a shallow bleach below 500 nm, which is typical for lutein triplets in LHCs. 44 Note that by equating in the model the minima of the Qy bleach for unquenched and quenched bulk chlorophylls (implying that the two emissive states have similar oscillator strength), we obtained that the amount of initial excitation sitting on quenched (unquenched) complexes is 17% (83%) of the total (Figure 4A), meaning that the number of quenched monomers is around 1/6 of the total. This agrees with the fraction of strongly quenched complexes estimated from time-resolved fluorescence traces at 680 nm (16%; see Table 1).</p><p>The shape of the quencher spectrum (blue SADS in Figures 4B and 4C) does not have the characteristics of a xanthophyll S 1 state, since it peaks at shorter wavelengths than expected. 46,47 This can be appreciated by comparing the quencher spectrum to the S 1 spectrum obtained upon direct carotenoid excitation (see Figure S11 for details), which has a maximum at 532 nm and decays in around 13 ps (orange SADS in Figure 4C). Strikingly, the spectrum of the quencher resembles that of the carotenoid triplet (green SADS in Figure 4C), but it has the lifetime (around 6 ps) of a singlet carotenoid excited state 48 (carotenoid triplets have instead lifetimes of several microseconds). Based on these characteristics, we assign the quencher species to a carotenoid S* state, [49][50][51][52] since similar properties for carotenoid S* states have been observed for many other carotenoids in solution 47,51,53,54 and in LHCs 22,49,50 (as an example, Figure S12 shows a comparison between the S 1 , S*, and triplet spectra of the carotenoid spheroidene as obtained in the work of Papagiannakis et al. 49 from TA experiments on the LH2 antenna of Rb. Sphaeroides). The quencher spectrum also shows a wavy signal in the Qy region (dotted blue line in Figure 4B), consistent with what was previously observed. 22,55 However, because of the very low concentration of the quencher (see Figure S6 for concentration profiles of all species) and the high absolute intensity of the Qy bleach, the contribution of this feature to the dynamics in the Qy region is practically negligible (see Text S2 for further discussion).</p><p>To further confirm the involvement of a carotenoid excited state in CP29 quenching, an alternative heterogeneous scheme without the quencher compartment, implying that quenched chlorophyll quickly decays without any additional quencher (see Figure S13A), was tested. Despite yielding a similar fitting quality, such a model results in an unusual ESA profile for the quenched chlorophyll compartment (Figure S13B). This SADS, which is markedly different from that of all other chlorophyll species (Figure S13C), exhibits a peak in the carotenoid triplet region (i.e., where the quencher SADS would peak according to the above discussed heterogeneous model; compare Figures S13 and 4). Such an unrealistic ESA profile can be interpreted as a mixture of the chlorophyll and the quencher spectra, thus reinforcing our conclusion that a quencher with the features of a carotenoid excited state is needed to explain the observed quenching.</p><p>Locating the Quenching Site: The Role of Lutein Our target model suggests that the quenching mechanism in monomeric CP29 relies on energy transfer from chlorophyll to a carotenoid dark excited state, which we assign to S*. The available structures for CP29 [24][25][26] show three distinct carotenoid binding sites: L1, L2, and N1, which bind lutein, violaxanthin, and neoxanthin, respectively (Figure 1). Which of these xanthophylls is responsible for the quenching?</p><p>An obvious candidate is a carotenoid located in the proximity of the lowest-energy chlorophylls. It was recently shown that both chlorophylls a612 and a603 (nomenclature from Pan et al. 26 ) are associated with low energy states, implying that CP29 has two terminal emitter domains localized on two different chlorophyll clusters (Figure 1B). 27 Chlorophyll a612 is the nearest neighbor of the lutein in site L1, whereas chlorophyll a603 is into contact with the violaxanthin in L2 (Figure 1B). 25,26 To investigate the role of these two chlorophyll-carotenoid pairs in the observed quenching mechanism, time-resolved fluorescence was recorded for two CP29 chlorophyll-deficient mutants, namely KO612 and KO603, lacking chlorophyll a612 and a603, respectively. 27 Time-resolved fluorescence traces measured at room temperature at 680 nm upon 468 nm excitation were satisfactorily fitted with three lifetime components for both mutants (Table 1, second and third rows; Figure S14). The largest contribution (more than 70%) comes from a lifetime component longer than 4 ns. The remaining contributions are from shorter lifetimes, as in the WT (wild type), accounting for fractions of slightly (1.5-2 ns) and strongly quenched (100-200 ps) antennae. As for the shortest lifetime component, KO603 has a very similar lifetime ($100 ps) and amplitude as the WT. On the other hand, the fraction of quenched KO612 complexes has a significantly longer lifetime (around 200 ps) and a smaller amplitude (<10%). In both mutants, global analysis of fluorescence time traces measured at different wavelengths yields very similar DAS for quenched and unquenched components, as already found in CP29 WT (Figures S15-S17). This trend is further confirmed by TA measurements. After 672 nm excitation, the TA traces of CP29 WT and KO603 show a similar fast decaying component (accounting for around 15% of total bleach, black, and blue lines in Figure 5; see Table S1  These results indicate that the quenching observed in monomeric CP29 WT is conserved in KO603, but it is altered in KO612. We conclude that the carotenoid quenching site active in the monomer of CP29 WT is L1 and thus that the observed carotenoid S* state belongs to lutein, which receives excitation energy mainly from chlorophyll a612.</p><!><p>Our results show that a heterogeneous model can adequately account for the observed fraction of fluorescence quenching in monomeric CP29 in both  S1).</p><p>time-resolved fluorescence and TA data. The heterogeneity of the emissive states of LHCs was proposed before on the basis of time-resolved fluorescence measurements 12,31 and demonstrated by single-molecule spectroscopy. 10,11 Single-molecule spectroscopy provided evidence that LHCs can exist in and reversibly switch between quenched and unquenched states, which reflect the vast conformational landscape of these proteins. 5,10 Experiments performed on single LHCs at room temperature have shown that quenched complexes can exhibit emission profiles very similar to those of unquenched complexes, i.e., a major fluorescence band peaking at 680 nm. 4 This is consistent with our observation that quenched and unquenched CP29 monomers show nearly identical fluorescence profiles, suggesting that the quencher is a dark state (such as a carotenoid excited state). By applying a heterogeneous model to our TA data, we were then able to assign the quencher SADS to a carotenoid S* state. Such a mechanism was recently proposed by our group to explain the constitutive quenching observed in LHCII containing astaxanthin instead of the native xanthophylls. 22 In the same work, molecular dynamics simulations suggested that the switch from the light-harvesting to the photoprotective state of a complex might be ascribed to a twist of a carotenoid end-ring, which makes its S* state accessible as energy acceptor from chlorophyll excited states. We speculate that a similar molecular switch might also explain the quenching observed in our sample, with the difference that native xanthophylls inside the isolated CP29 seem to be more prone to remain in a conformation that makes their S* inaccessible, therefore favoring the light-harvesting state of the complex. The tested kinetic scheme is such that the quencher is populated on a slower timescale (60 ps) than its overall lifetime (6 ps), which makes its concentration very small in time (see concentration profiles in Figure S6). This type of kinetics is usually referred as inverted kinetics 41,56 and comes from the fact that the observed quenching rates of LHCs (normally in the order of [100 ps] À1 ) 7,17,19,22 are much slower than the typical carotenoid lowest-excited-state relaxation rates (in the order of [10 ps] À1 ). 48 It should be noted that due to the very low concentration of the quencher species, the experimental observation of the described inverted kinetics is extremely challenging, especially at the low excitation powers which are necessary to avoid singlet-singlet annihilation. 17 Chlorophyll to carotenoid energy transfer via an inverted kinetic scheme was observed for the first time in TA experiments performed on LHCII aggregates. 19 Aggregation is indeed known to foster the photoprotective function of antenna complexes. 14,15 However, the excitation power used in those measurements was not low enough to avoid singlet-singlet annihilation, which was later found to possibly produce carotenoid excited states as an artifact. 9 Measuring in annihilation-free conditions is therefore essential to validate any evidence of carotenoid excited-state formation in the observed kinetics. As a result, in this work, much care was devoted to measure below the annihilation threshold, as shown in Text S3 and Figure S20. In this respect, the use of the monomeric protein rather than the aggregate form sets the annihilation threshold to much higher values and represents a valuable ally in experimental terms.</p><p>The observed quenching mechanism is very effective, leading to a fluorescence yield that is only 2% of what it is in unquenched complexes (lifetimes drop from 3-4 ns to 60 ps only). The observed lifetime is consequently short enough to efficiently quench photosystem II, helped by the position occupied by CP29 within the supercomplex, i.e., on the energy transfer path from the peripheral antenna to the core complex. Furthermore, a recent work by Valkunas and collaborators 7 has shown that quenching in LHCII aggregates can be explained by a (relatively) small number of quenched monomeric units (with 50-100 ps lifetimes) connected to a larger amount of unquenched complexes. Finally, a lifetime of 150 ps was measured in CP29 crystals. 57 Quantum mechanical calculations on the structural data suggested that quenching in CP29 crystals is due to chlorophyll to carotenoid energy transfer. It was also shown that, even though all bound xanthophylls can lead to a significant reduction of chlorophyll lifetime, the major effect on quenching can be ascribed to lutein bound to L1, which is in agreement with our results. However, the fact that many chlorophyllcarotenoid couples can potentially contribute to quenching might also explain why removing a specific chlorophyll can reduce the extent of quenching but not suppress it completely. Interestingly, a previous work 58 demonstrated that the spectroscopic properties of the binding site containing chlorophylls a611 and a612 and lutein 1 are preserved in all LHCs of PSII, which on the basis of the results presented here suggests that a similar quenching mechanism might be active in other antennae.</p><p>In conclusion, our data resulting from bulk experiments show that, even in its monomeric form, CP29 can be found in different emissive states, marked by different lifetimes but similar fluorescence spectra. The majority of the complexes is found in a state with a long fluorescence lifetime, which favors the light-harvesting function. However, a small but functionally important fraction of complexes (around 16% of the total) is efficiently quenched by a dark state, which we assign to a carotenoid S* state. Comparison with two chlorophyll-deficient mutants of CP29 suggests that quenching in the WT monomer is due to an energy transfer from chlorophylls to lutein located in the L1 binding site. The observed quenching mechanism is very efficient (lifetime below 100 ps) and is therefore a suitable candidate for non-photochemical quenching in physiological conditions. Finally, the high structural homology between CP29 and other antenna complexes such as LHCII, suggests that this photoprotective strategy can be generalized to other members of the LHC family.</p><!><p>Monomeric CP29 and its chlorophyll-deficient mutants were isolated from Arabidopsis thaliana as described in Xu et al. 27 For all the measurements, the sample was diluted to the desired OD in a buffer of HEPES 10 mM (pH $ 7.5) and 0.03% alpha-DM.</p><!><p>Time-resolved fluorescence was measured via TCSPC on a FluoTime 200 from PicoQuant at 10 C. The excitation wavelength was 468 nm for all measurements, with a power of 100 mW and a repetition rate of 10 MHz. The OD of all samples was %0.05. Measurements were performed in a 1x1 cm cuvette and samples were gently stirred. Under these conditions, no power dependency was observed. Signal was acquired at different wavelengths until a number of 20,000 counts at peak maximum, using a time window of 20 ns and a time bin of 4 ps. The instrument response function (IRF), estimated via the measured decay of pinacyanol iodide in methanol (lifetime of around 6 ps 59 ), had a full width at half maximum of 88 ps. Fluorescence time traces were globally analyzed using a number of parallel, non-interacting kinetic components, so that the total dataset can be described with the fitting function Fðt; lÞ as follows:</p><p>where each DAS (DAS k ) is the amplitude factor associated with a decay component k having a decay lifetime t k . Fitting quality was assessed by the c 2 value and by residual inspection.</p><p>Ultrafast Transient Absorption Spectroscopy TA experiments were performed at room temperature on a setup described in more detail elsewhere. 60 In summary, mode-locked pulses centered at 800 nm from a Coherent MIRA seed were amplified by means of regenerative amplification in a Coherent-Rega 9050, where their repetition rate was set to 40 kHz. The Rega output was then split into pump and probe pathways. Optical parametric amplification (Coherent OPA 9400) was employed to tune the wavelength of the pump pulses, which were then narrowed down to a FWHM of 10 nm around their central wavelength via interference filters (THORLABS). Probe pulses were focused into a sapphire crystal to produce a dispersed white-light continuum, which was detected via a 76-channel photodiode array, with a spectral window of around 130 nm. The recorded spectral windows were in the Qx/Qy region (590-720 nm) and in the 480-610 nm region. By changing the pump-pathway length, a delay line allowed recording of TA difference spectra (pumped minus un-pumped) up to 3.5 ns after excitation. Samples were measured at an OD of around 0.8 mm À1 at the Qy absorption maximum (678 nm; Figure S4) in a 1 mm Quartz cuvette, and a shaker was used to refresh the sample during the scans. An oxygen scavenging mixture consisting of glucose oxidase 0.1 mg/mL, catalase 0.05 mg/mL, and glucose 10 mM was added to prevent sample degradation (with a HEPES buffer concentration of 50 mM). The pump energy per pulse was set to 5 nJ for 489 nm excitation, 3 nJ for 672 and 682 nm excitation, and 10 nJ for 509 excitation. The amount of annihilation at these pump energies is negligible (see Text S3 and Figure S20, where exhaustive information about the power dependency of the measured signal can be found). The excitation density (calculated as N chl 3 DOD max /2OD max , where the maxima are restricted to the Qy region and N chl is the number of chlorophylls per monomer) was below 6% of all monomers in all measurements.</p><p>Global and target analysis were performed on the TA data matrices as described in van Stokkum et al. 45 Briefly, TA traces recorded simultaneously at different wavelengths were globally analyzed according to Equation 1 to obtain a set of kinetic components, named DADS, representing parallel processes taking place at the excited state with a specific lifetime (as sketched in Figure 3D). The IRF Gaussian profile (FWHM of around 100 fs) was directly estimated from the fitting. A sequential kinetic scheme was also applied to yield so-called EADS, corresponding to the difference spectra of species evolving into the following one (as sketched in Figure 3C) on the same timescales as obtained for DADS. Target analysis was used to extract the difference spectra (species-associated difference spectra [SADS]) of the actual species involved in the measured excited-state kinetics (see van Stokkum et al. 45 for details). Details about the use of spectral equality constraints within the target modeling are given in Text S1. In all global and target analyses, the chirp was explicitly taken into account (by a parametric description of the wavelength-dependent location of the maximum of the IRF).</p>