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Plants are designed to utilize visible light for photosynthesis. Expanding this light absorption toward the far-red could boost growth in low-light conditions and potentially increase crop productivity in dense canopies. A promising strategy is broadening the absorption of antenna complexes to the far-red. In this study, we investigated the capacity of the photosystem I antenna protein Lhca4 to incorporate far-red absorbing chlorophylls dand fand optimize their spectra. We demonstrate that these pigments can successfully bind to Lhca4, with the protein environment further red-shifting the chlorophyll dabsorption, markedly extending the absorption range of this complex above 750 nm. Notably, chlorophyll dsubstitutes the canonical chlorophyll ared-forms, resulting in the most red-shifted emission observed in a plant light-harvesting complex. Using ultrafast spectroscopy, we show that the introduction of these novel chlorophylls does not interfere with the excited state decay or the energy equilibration processes within the complex. The results demonstrate the feasibility of engineering plant antennae to absorb deeper into the far-red region while preserving their functional and structural integrity, paving the way for innovative strategies to enhance photosynthesis.

Photosynthetic reaction centers catalyze the majority of solar energy conversion on the Earth. Under low-intensity illumination, this is achieved with a near-unity quantum efficiency, almost every absorbed photon producing a photochemical charge separation. Biohybrid technologies seek to capture the high efficiency of natural photoproteins by combining them with man-made electrodes. However, the transfer of photoproteins from their membrane environment into an abiotic architecture invariably results in efficiency losses. Here, we combined spectroscopy and analytical electrochemistry to identify the loss processes in a reaction-center-based biophotoelectrode. While over 90% efficient under low-intensity illumination, the biophotoelectrode efficiency dropped to ∼11% under high-intensity illumination. This loss stemmed from bottlenecks in electron transfer that rendered 60% of reaction centers inactive, as well as a short-circuiting of 73% of the separated charge from active reaction centers. The quantitative insights into loss processes presented in this work will be instrumental in shaping future rational design of biophotoelectrode devices.

Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6fcomplex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRedto PSI can be regulated externally by Mg2+ions in solution.

Increasing the absorption cross section of plants by introducing far-red absorbing chlorophylls (Chls) has been proposed as a strategy to boost crop yields. To make this strategy effective, these Chls should bind to the photosynthetic complexes without altering their functional architecture. To investigate if plant-specific antenna complexes can provide the protein scaffold to accommodate these Chls, we have reconstituted the main light-harvesting complex (LHC) of plants LHCII in vitroand in silico, with Chl d. The results demonstrate that LHCII can bind Chl din a number of binding sites, shifting the maximum absorption ∼25 nm toward the red with respect to the wild-type complex (LHCII with Chl aand b) while maintaining the native LHC architecture. Ultrafast spectroscopic measurements show that the complex is functional in light harvesting and excitation energy transfer. Overall, we here demonstrate that it is possible to obtain plant LHCs with enhanced far-red absorption and intact functional properties.

Light-harvesting complex II (LHCII) is a pigment–protein complex present in higher plants and green algae. LHCII represents the main site of light absorption, and its role is to transfer the excitation energy toward the photosynthetic reaction centers, where primary energy conversion reactions take place. The optical properties of LHCII are known to depend on protein conformation. However, the relation between the structural and spectroscopic properties of the pigments is not fully understood yet. In this respect, previous classical molecular dynamics simulations of LHCII in a model membrane [Sci. Rep.2015, 5, 1–10] have shown that the configuration and excitonic coupling of a chlorophyll (Chl) dimer functioning as the main terminal emitter of the complex are particularly sensitive to conformational changes. Here, we use quantum chemistry calculations to investigate in greater detail the effect of pigment–pigment interactions on the excited-state landscape. While most previous studies have used a local picture in which electrons are localized on single pigments, here we achieve a more accurate description of the Chl dimer by adopting a supramolecular picture where time-dependent density functional theory is applied to the whole system at once. Our results show that specific dimer configurations characterized by shorter inter-pigment distances can result in a sizable intensity decrease (up to 36%) of the Chl absorption bands in the visible spectral region. Such a decrease can be predicted only when accounting for Chl–Chl charge-transfer excitations, which is possible using the above-mentioned supramolecular approach. The charge-transfer character of the excitations is quantified by two types of analyses: one focusing on the composition of the excitations and the other directly on the observable total absorption intensities.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes–the phycobilisomes–via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

We present a nonlinear spectroelectrochemical technique to investigate photosynthetic protein complexes. The PEC2DES setup combines photoelectrochemical detection (PEC) that selectively probes the protein photogenerated charges output with two-dimensional electronic spectroscopy (2DES) excitation that spreads the nonlinear optical response of the system in an excitation–detection map. PEC allows us to distinguish the contribution of charge separation (CS) from other de-excitation pathways, whereas 2DES allows us to disentangle congested spectral bands and evaluate the exciton dynamics (decays and coherences) of the photosystem complex. We have developed in operandophase-modulated 2DES by measuring the photoelectrochemical reaction rate in a biohybrid electrode functionalized with a plant photosystem complex I–light harvesting complex I (PSI-LHCI) layer. Optimizing the photoelectrochemical current signal yields reliable linear spectra unequivocally associated with PSI-LHCI. The 2DES signal is validated by nonlinear features like the characteristic vibrational coherence at 750 cm–1. However, no energy transfer dynamics is observed within the 450 fs experimental window. These intriguing results are discussed in the context of incoherent mixingresulting in reduced nonlinear contrast for multichromophoric complexes, such as the 160 chlorophyll PSI. The presented PEC2DES method identifies generated charges unlike purely optical 2DES and opens the way to probe the CS channel in multichromophoric complexes.

In nature, plants experience large fluctuations in light intensity and they need to balance the absorption and utilization of this energy appropriately. Non-photochemical quenching (NPQ) is a rapidly switchable mechanism that protects plants from photodamage caused by high light exposure by dissipating the excess absorbed energy as heat. It is triggered by the pH gradient across the thylakoid membrane and requires the protein PsbS and the xanthophyll zeaxanthin. However, the site and mechanism of the quencher(s) remain unknown. Here, we constructed a mutant of Arabidopsis thalianathat lacks light-harvesting complex II (LHCII), the main antenna complex of plants, to verify its contribution to NPQ. The mutant plant has normally stacked thylakoid membranes, displays no upregulation of other LHCs but shows a relative decrease in Photosystem I (PSI), which compensates for the decrease of the PSII antenna. The mutant plant exhibits a reduction in NPQ of about 60% and the remaining NPQ resembles that of mutant plants lacking chlorophyll (Chl) b, which lack all PSII peripheral antenna complexes. We thus report that PsbS-dependent NPQ occurs mainly in LHCII, but there is an additional quenching site in the PSII core.

In the major peripheral plant light-harvesting complex LHCII, excitation energy is transferred between chlorophylls along an energetic cascade before it is transmitted further into the photosynthetic assembly to be converted into chemical energy. The efficiency of these energy transfer processes involves a complicated interplay of pigment-protein structural reorganization and protein dynamic disorder, and the system must stay robust within the fluctuating protein environment. The final, lowest energy site has been proposed to exist within a trimeric excitonically coupled chlorophyll (Chl) cluster, comprising Chls a610-a611-a612. We studied an LHCII monomer with a site-specific mutation resulting in the loss of Chls a611and a612, and find that this mutant exhibits two predominant overlapping fluorescence bands. From a combination of bulk measurements, single-molecule fluorescence characterization, and modeling, we propose the two fluorescence bands originate from differing conditions of exciton delocalization and localization realized in the mutant. Disruption of the excitonically coupled terminal emitter Chl trimer results in an increased sensitivity of the excited state energy landscape to the disorder induced by the protein conformations. Consequently, the mutant demonstrates a loss of energy transfer efficiency. On the contrary, in the wild-type complex, the strong resonance coupling and correspondingly high degree of excitation delocalization within the Chls a610-a611-a612 cluster dampens the influence of the environment and ensures optimal communication with neighboring pigments. These results indicate that the terminal emitter trimer is thus an essential design principle for maintaining the efficient light-harvesting function of LHCII in the presence of protein disorder.

In this work, we have investigated the role of the individual antenna complexes and of the low-energy forms in excitation energy transfer and trapping in Photosystem I of higher plants. To this aim, a series of Photosystem I (sub)complexes with different antenna size/composition/absorption have been studied by picosecond fluorescence spectroscopy. The data show that Lhca3 and Lhca4, which harbor the most red forms, have similar emission spectra (λmax= 715–720 nm) and transfer excitation energy to the core with a relative slow rate of ∼25/ns. Differently, the energy transfer from Lhca1 and Lhca2, the “blue” antenna complexes, occurs about four times faster. In contrast to what is often assumed, it is shown that energy transfer from the Lhca1/4 and the Lhca2/3 dimer to the core occurs on a faster timescale than energy equilibration within these dimers. Furthermore, it is shown that all four monomers contribute almost equally to the transfer to the core and that the red forms slow down the overall trapping rate by about two times. Combining all the data allows the construction of a comprehensive picture of the excitation-energy transfer routes and rates in Photosystem I.

Plant photosynthesis relies on the capacity of chlorophylls and carotenoids to absorb light. One of the roles of carotenoids is to harvest green-blue light and transfer the excitation energy to the chlorophylls. The corresponding dynamics were investigated here for the first time, to our knowledge, in the CP26 and CP24 minor antenna complexes. The results for the two complexes differ substantially. In CP26 fast transfer (80 fs) occurs from the carotenoid S2state to chlorophylls aabsorbing at 675 and 678 nm, whereas transfer from the hot S1state to the lowest energy chlorophylls is observed in <1 ps. In CP24, energy transfer from the S2state leads in 80 fs to the population of chlorophylls band high-energy chlorophylls aabsorbing at 670 nm, whereas the low-energy chlorophylls aare populated only in several picoseconds. The results suggest that CP26 has a structural and functional organization similar to that of LHCII, whereas CP24 differs substantially from the other Lhc complexes, especially regarding the lutein L1 binding domain. No energy transfer from the carotenoid S1state to chlorophylls was observed in either complex, suggesting that this state is energetically below the chlorophyll Qy state and therefore may play a role in the quenching of chlorophyll excitations.

We performed picosecond fluorescence measurements on well-defined Photosystem II (PSII) supercomplexes from Arabidopsiswith largely varying antenna sizes. The average excited-state lifetime ranged from 109 ps for PSII core to 158 ps for the largest C2S2M2complex in 0.01% α-DM. Excitation energy transfer and trapping were investigated by coarse-grained modeling of the fluorescence kinetics. The results reveal a large drop in free energy upon charge separation (700 cm−1) and a slow relaxation of the radical pair to an irreversible state (∼150 ps). Somewhat unexpectedly, we had to reduce the energy-transfer and charge-separation rates in complexes with decreasing size to obtain optimal fits. This strongly suggests that the antenna system is important for plant PSII integrity and functionality, which is supported by biochemical results. Furthermore, we used the coarse-grained model to investigate several aspects of PSII functioning. The excitation trapping time appears to be independent of the presence/absence of most of the individual contacts between light-harvesting complexes in PSII supercomplexes, demonstrating the robustness of the light-harvesting process. We conclude that the efficiency of the nonphotochemical quenching process is hardly dependent on the exact location of a quencher within the supercomplexes.

Photosystem I (PSI) plays a major role in the light reactions of photosynthesis. In higher plants, PSI is composed of a core complex and four outer antennas that are assembled as two dimers, Lhca1/4 and Lhca2/3. Time-resolved fluorescence measurements on the isolated dimers show very similar kinetics. The intermonomer transfer processes are resolved using target analysis. They occur at rates similar to those observed in transfer to the PSI core, suggesting competition between the two transfer pathways. It appears that each dimer is adopting various conformations that correspond to different lifetimes and emission spectra. A special feature of the Lhca complexes is the presence of an absorption band at low energy, originating from an excitonic state of a chlorophyll dimer, mixed with a charge-transfer state. These low-energy bands have high oscillator strengths and they are superradiant in both Lhca1/4 and Lhca2/3. This challenges the view that the low-energy charge-transfer state always functions as a quencher in plant Lhc's and it also challenges previous interpretations of PSI kinetics. The very similar properties of the low-energy states of both dimers indicate that the organization of the involved chlorophylls should also be similar, in disagreement with the available structural data.

Antenna complexes are key components of plant photosynthesis, the process that converts sunlight, CO2, and water into oxygen and sugars. We report the first (to our knowledge) femtosecond transient absorption study on the light-harvesting pigment-protein complexes CP26 (Lhcb5) and CP24 (Lhcb6) of Photosystem II. The complexes are excited at three different wavelengths in the chlorophyll (Chl) Qy region. Both complexes show a single subpicosecond Chl bto Chl atransfer process. In addition, a reduction in the population of the intermediate states (in the 660–670 nm range) as compared to light-harvesting complex II is correlated in CP26 to the absence of both Chls a604 and b605. However, Chl forms around 670 nm are still present in the Chl aQy range, which undergoes relaxation with slow rates (10–15 ps). This reduction in intermediate-state amplitude CP24 shows a distinctive narrow band at 670 nm connected with Chls band decaying to the low-energy Chl astates in 3–5 ps. This 670 nm band, which is fully populated in 0.6 ps together with the Chl alow-energy states, is proposed to originate from Chl 602 or 603. In this study, we monitored the energy flow within two minor complexes, and our results may help elucidate these structures in the future.

The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

The topology of the long N-terminal domain (∼100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.

Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5±2.5ps) and rates of excitation energy transfer from LHCI to core (kLC) and vice versa (kCL). The ratio between these rates, R=kCL/kLC, appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. kLCdepends slightly but nonsystematically on detection wavelength, averaging (9.4±4.9ps)−1. Rranges from 0.5 (<690nm) to ∼1.3 above 720nm.

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1–4 peak, respectively, at 704, 708, and 709nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to “normal” chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400–450cm−1), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705nm and originates from a broad absorption band peaking at 690nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.

With the availability of structural models for photosystem I (PSI) in cyanobacteria and plants it is possible to compare the excitation transfer networks in this ubiquitous photosystem from two domains of life separated by over one billion years of divergent evolution, thus providing an insight into the physical constraints that shape the networks’ evolution. Structure-based modeling methods are used to examine the excitation transfer kinetics of the plant PSI-LHCI supercomplex. For this purpose an effective Hamiltonian is constructed that combines an existing cyanobacterial model for structurally conserved chlorophylls with spectral information for chlorophylls in the Lhca subunits. The plant PSI excitation migration network thus characterized is compared to its cyanobacterial counterpart investigated earlier. In agreement with observations, an average excitation transfer lifetime of ∼49ps is computed for the plant PSI-LHCI supercomplex with a corresponding quantum yield of 95%. The sensitivity of the results to chlorophyll site energy assignments is discussed. Lhca subunits are efficiently coupled to the PSI core via gap chlorophylls. In contrast to the chlorophylls in the vicinity of the reaction center, previously shown to optimize the quantum yield of the excitation transfer process, the orientational ordering of peripheral chlorophylls does not show such optimality. The finding suggests that after close packing of chlorophylls was achieved, constraints other than efficiency of the overall excitation transfer process precluded further evolution of pigment ordering.

The energy transfer processes between carotenoids and Chls have been studied by femtosecond transient absorption in the CP29-WT complex, which contains only two carotenoids per polypeptide located in the L1 and L2 sites, and in the CP29-E166V mutant in which only the L1 site is occupied. The comparison of these two samples allowed us to discriminate between the energy transfer pathways from the two carotenoid binding sites and thus to obtain detailed information on the Chl organization in CP29 and to assign the acceptor chlorophylls. For both samples, the main transfer occurs from the S2 state of the carotenoid. In the case of the L1 site the energy acceptor is the Chl a 680nm (A2), whereas the Chl a 675nm (A4–A5) and the Chl b 652nm (B6) are the acceptors from the xanthophyll in the L2 site. These transfers occur with lifetimes of 80–130 fs. Two additional transfers are observed with 700-fs and 8- to 20-ps lifetimes. Both these transfers originate from the carotenoid S1 states. The faster lifetime is due to energy transfer from a vibrationally unrelaxed S1 state, whereas the 8- to 20-ps component is due to a transfer from the S1,0 state of violaxanthin and/or neoxanthin located in site L2. A comparison between the carotenoid to Chl energy transfer pathways in CP29 and LHCII is presented and differences in the structural organization in the two complexes are discussed.

The energy transfer processes between Chls b and Chls a have been studied in the minor antenna complex CP29 by femtosecond transient absorption spectroscopy. Two samples were analyzed: the native CP29, purified from higher plants, and the recombinant one, reconstituted in vitro with the full pigment complement. The measurements indicate that the transfer kinetics in the two samples are virtually identical, confirming that the reconstituted CP29 has the same spectroscopic properties as the native one. In particular, three lifetimes (150 fs, 1.2ps, and 5–6ps) were identified for Chl b-652nm to Chl a energy transfer and at least one for Chl b-640nm (600–800 fs). Considering that the complexes bind two Chls b per polypeptide, the observation of more than two lifetimes for the Chl b to Chl a energy transfer, in both samples, clearly indicates the presence of the so-called mixed Chl binding sites—sites which are not selective for Chl a or Chl b, but can accommodate either species. The kinetic components and spectra are assigned to specific Chl binding sites in the complex, which provides further information on the structural organization.

The light harvesting complex Lhca1, one of the four gene products comprising the photosystem I antenna system, has been analyzed by site-directed mutagenesis with the aim of determining the chromophore(s) responsible for its long wavelength chlorophyll spectral form, a specific characteristic of the LHCI antenna complex. A family of mutant proteins, each carrying a mutation at a single chlorophyll-binding residue, was obtained and characterized by biochemical and spectroscopic methods. A map of the chromophores bound to each of the 10 chlorophyll-binding sites was drawn, and the energy levels of the Qytransition were determined in most cases. When compared with Lhcb proteins previously analyzed, Lhca1 is characterized by stronger interactions between individual chromophores as detected by both biochemical and spectroscopic methods; most mutations, although targeted to a single residue, lead to the loss of more than one chromophore and of conservative CD signals typical of chlorophyll-chlorophyll interactions. The lower energy absorption form (686 nm at 100K, 688 nm at room temperature), which is responsible for the red-shifted emission components at 690 and 701 nm, typical of Lhca1, is associated with a chlorophyll a/chlorophyll a excitonic interaction originating from a pigment cluster localized in the protein domain situated between helix C and the helix A/helix B cross. This cluster includes chlorophylls bound to sites A5-B5-B6 and a xanthophyll bound to site L2.

We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-α-d-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.

The energy transfer kinetics from carotenoids to chlorophylls and among chlorophylls has been measured by femtosecond transient absorption kinetics in a monomeric unit of the major light-harvesting complex (LHCII) from higher plants. The samples were reconstituted complexes with different carotenoid contents. The kinetics was measured both in the carotenoid absorption region and in the chlorophyll Qy region using two different excitation wavelengths suitable for selective excitation of the carotenoids. Analysis of the data shows that the overwhelming part of the energy transfer from the carotenoids occurs directly from the initially excited S2 state of the carotenoids. Only a small part (<20%) may possibly take an S1 pathway. All the S2 energy transfer from carotenoids to chlorophylls occurs with time constants <100 fs. We have been able to differentiate among the three carotenoids, two luteins and neoxanthin, which have transfer times of ∼50 and 75 fs for the two luteins, and ∼90 fs for neoxanthin. About 50% of the energy absorbed by carotenoids is initially transferred directly to chlorophyll b (Chl b), while the rest is transferred to Chl a. Neoxanthin almost exclusively transfers to Chl b. Due to various complex effects discussed in the paper, such as a specific coupling of Chl b and Chl a excited states, the percentage of direct Chl b transfer thus is somewhat lower than estimated by us previously for LHCII from Arabidopsis thaliana. (Connelly, J. P., M. G. Müller, R. Bassi, R. Croce, and A. R. Holzwarth. 1997. Biochemistry. 36:281). We can distinguish three different Chls b receiving energy directly from carotenoids. We propose as a new mechanism that the carotenoid-to-Chl b transfer occurs to a large part via the Bx state of Chl b and to the Qx state, while the transfer to Chl a occurs only via the Qx state. We find no compelling evidence in favor of a substantial S1 transfer path of the carotenoids, although some transfer via the S1 state of neoxanthin can not be entirely excluded. The S1 lifetimes of the two luteins were determined to be 15ps and 3.9ps. A detailed quantitative analysis and kinetic model of the processes described here will be presented in a separate paper.

The energy transfer rates between chlorophylls in the light harvesting complex CP29 of higher plants at room temperature were calculated ab initio according to the Förster mechanism (Förster T. 1948, Ann. Physik. 2:55–67). Recently, the transition moment orientation of CP29 chlorophylls was determined by differential linear dichroism and absorption spectroscopy of wild-type versus mutant proteins in which single chromophores were missing (Simonetto R., Crimi M., Sandonà D., Croce R., Cinque G., Breton J., and Bassi R. 1999. Biochemistry. 38:12974–12983). In this way the Qy transition energy and chlorophyll a/b affinity of each binding site was obtained and their characteristics supported by reconstruction of steady-state linear dichroism and absorption spectra at room temperature. In this study, the spectral form of individual chlorophyll a and b ligands within the protein environment was experimentally determined, and their extinction coefficients were also used to evaluate the absolute overlap integral between donors and acceptors employing the Stepanov relation for both the emission spectrum and the Stokes shift. This information was used to calculate the time-dependent excitation redistribution among CP29 chlorophylls on solving numerically the Pauli master equation of the complex: transient absorption measurements in the (sub)picosecond time scale were simulated and compared to pump-and-probe experimental data in the Qy region on the native CP29 at room temperature upon selective excitation of chlorophylls b at 640 or 650nm. The kinetic model indicates a bidirectional excitation transfer over all CP29 chlorophylls a species, which is particularly rapid between the pure sites A1-A2 and A4-A5. Chlorophylls b in mixed sites act mostly as energy donors for chlorophylls a, whereas site B5 shows high and bidirectional coupling independent of the pigment hosted.

Abstract: The absorption spectra of two light harvesting complexes from higher plants, CP29 and LHC II, have been analysed in the Soret region in order to obtain a description in terms of the absorption spectra of the individual pigments. This information is of great practical use when applying spectroscopic techniques to the study of energy transfer in photosynthesis such as time-resolved spectroscopy thus allowing determination of the relative absorption cross-section for the different chromophores in the system as a function of wavelength. In this study, recombinant Lhc proteins carrying point mutations in pigment-binding residues have been used in order to obtain the spectral shape of individual chromophores by differential spectroscopy with respect to the WT protein. Combinations of spectra thus obtained were then used to fit the absorption spectra of WT and mutant pigment-proteins according to the constraints posed by stoichiometry of pigments as derived by biochemical analysis. This procedure allowed identification of each pigment in term of its wavelength position, spectral shape and extinction coefficient. The data obtained by this procedure have been successfully applied to the description of other higher plant Lhc proteins thus supporting the view that the Lhc superfamily members share specific pigment–protein interactions as suggested by sequence homology.

Abstract: The spectral forms of the two chlorophyll species in higher plant Photosystem II antenna proteins have been experimentally determined within their protein environment. Recombinant CP29 and LHC II antenna proteins missing individual chromophores were obtained by over-expression in bacteria without any changing of the primary protein sequence and in vitro reconstitution. Difference absorption spectroscopy with respect to the corresponding proteins binding the complete pigment complement yielded the spectral shape and extinction of single chlorophyll a and b. A functional relation of their absorption was given by Gaussian subband decomposition covering the entire Qx and Qy optical region together with the absolute value of the molar extinction coefficient. With respect to analogous determinations reported in the literature for organic solvents, this information is valuable for further understanding the in-protein chlorophyll excited states and excited state dynamics: in particular, for the calculation of Förster transfer rates by means of chlorophyll–chlorophyll overlap integral employing the Stepanov relation for emission and single chromophore transition energies according to the results of mutational analysis of chlorophyll binding sites [Bassi et al. (1999) Proc Natl Acad Sci USA 96: 10056–10061; Remelli et al. (1999) J Biol Chem 274: 33510–33521].

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site‐directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57±1.5° with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site-directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57±1.5° with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coliand then refoldedin vitrowith purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Qytransition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.