Architectures of photosynthetic RC-LH1 supercomplexes from Rhodobacter blasticus.

The reaction center-light-harvesting complex 1 (RC-LH1) plays an essential role in the primary reactions of bacterial photosynthesis. Here, we present high-resolution structures of native monomeric and dimeric RC-LH1 supercomplexes from Rhodobacter (Rba.) blasticus using cryo-electron microscopy. The RC-LH1 monomer is composed of an RC encircled by an open LH1 ring comprising 15 αß heterodimers and a PufX transmembrane polypeptide. In the RC-LH1 dimer, two crossing PufX polypeptides mediate dimerization. Unlike Rhodabacter sphaeroides counterpart, Rba. blasticus RC-LH1 dimer has a less bent conformation, lacks the PufY subunit near the LH1 opening, and includes two extra LH1 αß subunits, forming a more enclosed S-shaped LH1 ring. Spectroscopic assays reveal that these unique structural features are accompanied by changes in the kinetics of quinone/quinol trafficking between RC-LH1 and cytochrome bc1. Our findings reveal the assembly principles and structural variability of photosynthetic RC-LH1 supercomplexes, highlighting diverse strategies used by phototrophic bacteria to optimize light-harvesting and electron transfer in competitive environments.

Design principles for energy transfer in the photosystem II supercomplex from kinetic transition networks.

Photosystem II (PSII) has the unique ability to perform water-splitting. With light-harvesting complexes, it forms the PSII supercomplex (PSII-SC) which is a functional unit that can perform efficient energy conversion, as well as photoprotection, allowing photosynthetic organisms to adapt to the naturally fluctuating sunlight intensity. Achieving these functions requires a collaborative energy transfer network between all subunits of the PSII-SC. In this work, we perform kinetic analyses and characterise the energy landscape of the PSII-SC with a structure-based energy transfer model. With first passage time analyses and kinetic Monte Carlo simulations, we are able to map out the overall energy transfer network. We also investigate how energy transfer pathways are affected when individual protein complexes are removed from the network, revealing the functional roles of the subunits of the PSII-SC. In addition, we provide a quantitative description of the flat energy landscape of the PSII-SC. We show that it is a unique landscape that produces multiple kinetically relevant pathways, corresponding to a high pathway entropy. These design principles are crucial for balancing efficient energy conversion and photoprotection.

Optimizing photosynthetic light-harvesting under stars: simple and general antenna models.

In the next 10-20 years, several observatories will aim to detect the signatures of oxygenic photosynthesis on exoplanets, though targets must be carefully selected. Most known potentially habitable exo-planets orbit cool M-dwarf stars, which have limited emission in the photosynthetically active region of the spectrum (PAR, 400 < λ < 700 nm) used by Earth's oxygenic photoautotrophs. Still, recent experiments have shown that model cyanobacteria, algae, and non-vascular plants grow comfortably under simulated M-dwarf light, though vascular plants struggle. Here, we hypothesize that this is partly due to the different ways they harvest light, reflecting some general rule that determines how photosynthetic antenna structures may evolve under different stars. We construct a simple thermodynamic model of an oxygenic antenna-reaction centre supercomplex and determine the optimum structure, size and absorption spectrum under light from several star types. For the hotter G (e.g. the Sun) and K-stars, a small modular antenna is optimal and qualitatively resembles the PSII-LHCII supercomplex of higher plants. For the cooler M-dwarfs, a very large antenna with a steep 'energy funnel' is required, resembling the cyanobacterial phycobilisome. For the coolest M-dwarfs an upper limit is reached, where increasing antenna size further is subject to steep diminishing returns in photosynthetic output. We conclude that G- and K-stars could support a range of niches for oxygenic photo-autotrophs, including high-light adapted canopy vegetation that may generate detectable bio-signatures. M-dwarfs may only be able to support low light-adapted organisms that have to invest considerable resources in maintaining a large antenna. This may negatively impact global coverage and therefore detectability.

Development of artificial photosystems based on designed proteins for mechanistic insights into photosynthesis.

This review aims to provide an overview of the progress in protein-based artificial photosystem design and their potential to uncover the underlying principles governing light-harvesting in photosynthesis. While significant advances have been made in this area, a gap persists in reviewing these advances. This review provides a perspective of the field, pinpointing knowledge gaps and unresolved challenges that warrant further inquiry. In particular, it delves into the key considerations when designing photosystems based on the chromophore and protein scaffold characteristics, presents the established strategies for artificial photosystems engineering with their advantages and disadvantages, and underscores the recent breakthroughs in understanding the molecular mechanisms governing light-harvesting, charge separation, and the role of the protein motions in the chromophore's excited state relaxation. By disseminating this knowledge, this article provides a foundational resource for defining the field of bio-hybrid photosystems and aims to inspire the continued exploration of artificial photosystems using protein design.

Tuning by Hydrogen Bonding in Photosynthesis.

Hydrogen bonding plays a crucial role in stabilizing proteins throughout their folding process. In photosynthetic light-harvesting chromoproteins, enriched with pigment chromophores, hydrogen bonds also fine-tune optical absorption to align with the solar irradiation spectrum. Despite its significance for photosynthesis, the precise mechanism of spectral tuning through hydrogen bonding remains inadequately understood. This study investigates wild-type and genetically engineered LH2 and LH1 light-harvesting complexes from Rhodobacter sphaeroides using a unique set of advanced spectroscopic techniques combined with simple exciton modeling. Our findings reveal an intricate interplay between exciton and site energy shift mechanisms, challenging the prevailing belief that spectral changes observed in these complexes upon the modification of tertiary structure hydrogen bonds almost directly follow shifting site energies. These deeper insights into natural adaptation processes hold great promise for advancing sustainable solar energy conversion technologies.

Discovery of Multiple Light-Harvesting States of the Photosynthetic Protein PE545.

Cryptophytes are photosynthetic microalga that flourish in a remarkable diversity of natural environments by using pigment-containing proteins with absorption maxima tuned to each ecological niche. While this diversity in the absorption has been well established, the subsequent photophysics is highly sensitive to the local protein environment and so may exhibit similar variation. Thermal fluctuations of the protein conformation are expected to introduce photophysical heterogeneity of the pigments that may have evolved important functional properties in a manner similar to that of the absorption. However, such heterogeneity is averaged out in ensemble measurements and, therefore, has not yet been probed. Here, we report single-molecule measurements of phycoerythrin 545 (PE545), the prototypical cryptophyte antenna protein, in its native dimeric form. A conformational ensemble was resolved consisting of distinct photophysical states with different light-harvesting properties. Proteins that did not quench, partially quenched, or fully quenched absorbed light were observed. Light intensity increased the quenched-state population of the dimer, potentially as a mechanism to deal with the extreme light intensities found in aqueous environments. Cross-linking, which mimics local interactions, introduces this light-dependent functionality while also suppressing other conformational dynamics. The cellular organization can, therefore, actively modulate the protein conformation and dynamics, selecting for distinct levels of light harvesting. Thus, the complex conformational equilibrium provides an additional mechanism for cryptophytes and likely other photosynthetic organisms to optimize solar energy capture and conversion.

Controlling Vibronic Coupling in Chlorophyll Proteins: The Effects of Excitonic Delocalization and Vibrational Localization.

Vibrational-electronic (vibronic) coupling plays a critical role in excitation energy transfer in molecular aggregates and pigment-protein complexes (PPCs). But the interplay between excitonic delocalization and vibronic interactions is complex, often leaving even qualitative questions as to what conceptual framework (e.g., Redfield versus Förster theory) should be used to interpret experimental results. To shed light on this issue, we report here on the interplay between excitonic delocalization and vibronic coupling in site-directed mutants of the water-soluble chlorophyll protein (WSCP), as reflected in 77 K fluorescence spectra. Experimentally, we find that in PPCs where excitonic delocalization is disrupted (either by mutagenesis or heterodimer formation), the relative intensity of the vibrational sideband (VSB) in fluorescence spectra is suppressed by up to 37% compared to that of the native protein. Numerical simulations reveal that this effect results from the localization of high-frequency vibrations in the coupled system; while excitonic delocalization suppresses the purely electronic transition due to H-aggregate-like dipole-dipole interference, high-frequency vibrations are unaffected, leading to a relative enhancement of the VSB. By comparing VSB intensities of PPCs both in the presence and absence of excitonic delocalization, we extract a set of "local" Huang-Rhys (HR) factors for Chl a in WSCP. More generally, our results suggest a significant role for geometric effects in controlling energy-transfer rates (which depend sensitively on absorption/fluorescence line shapes) in molecular aggregates and PPCs.

Expression pattern of Stlhcb gene family in potato and effects of overexpression of Stcp24 gene on potato photosynthesis.

Potato is one of the four staple food crops in the world. It has a wide range of cultivation, high yield, and high nutritional value. Enhancing the photosynthesis of potato is particularly important as it leads to an increase in the potato yield. The light-harvesting pigment-binding protein complex is very important for plant photosynthesis. We identified 12 Stlhcb gene family members from the potato variety "Atlantic" using transcriptome sequencing and bioinformatics. The proteins encoded by the Stlhcb gene family have between 3358 and 4852 atomic number, a relative molecular weight between 24060.16 and 34624.54 Da, and an isoelectric point between 4.99 and 8.65. The RT-qPCR results showed that the 12 Stlhcb genes were expressed in a tissue-specific and time-dependent fashion under low light. The relative expression of the Stlhcb genes in the leaves was significantly higher than that in the stems and roots, and the relative expression of these genes first increased and then decreased with the prolongation of light exposure time. The Stcp24 gene with the highest expression was cloned, and an expression vector was constructed. A subcellular localization analysis was performed in tobacco and an overexpression experiment was performed in potato using an Agrobacterium-mediated method. The subcellular localization analysis showed that the protein encoded by Stcp24 was located in chloroplasts as expected. Overexpression of Stcp24 in transgenic potato increased the yield of potatoes and the content of chlorophyll a and b; increased the net photosynthetic rate, transpiration rate, stomatal conductance, electron transport efficiency, and semi-saturated light intensity; and promoted photosynthesis and plant growth. This study provides a reference for the study of the function of the potato light-harvesting pigment-binding protein gene family. It lays a foundation for further study of the mechanism of the photosynthesis of potato, improvement of the light energy utilization of potato, and molecular breeding of potato.

Spectral modulation of B850 bacteriochlorophyll a in light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum by detergents and calcium ions.

Spectral variations of light-harvesting (LH) proteins of purple photosynthetic bacteria provide insight into the molecular mechanisms underlying spectral tuning of circular bacteriochlorophyll (BChl) arrays, which play crucial roles in photoenergy conversion in these organisms. Here we investigate spectral changes of the Qy band of B850 BChl a in LH2 protein from purple sulfur bacterium Thermochromatium tepidum (tepidum-LH2) by detergents and Ca2+. The tepidum-LH2 solubilized with lauryl dimethylamine N-oxide and n-octyl-ß-D-glucoside (LH2LDAO and LH2OG, respectively) exhibited blue-shift of the B850 Qy band with hypochromism compared with the tepidum-LH2 solubilized with n-dodecyl-ß-D-maltoside (LH2DDM), resulting in the LH3-like spectral features. Resonance Raman spectroscopy indicated that this blue-shift was ascribable to the loss of hydrogen-bonding between the C3-acetyl group in B850 BChl a and the LH2 polypeptides. Ca2+ produced red-shift of the B850 Qy band in LH2LDAO by forming hydrogen-bond for the C3-acetyl group in B850 BChl a, probably due to a change in the microenvironmental structure around B850. Ca2+-induced red-shift was also observed in LH2OG although the B850 acetyl group is still free from hydrogen-bonding. Therefore, the Ca2+-induced B850 red-shift in LH2OG would originate from an electrostatic effect of Ca2+. The current results suggest that the B850 Qy band in tepidum-LH2 is primarily tuned by two mechanisms, namely the hydrogen-bonding of the B850 acetyl group and the electrostatic effect.

Structural basis for the distinct core-antenna assembly of cryptophyte photosystem II.

Photosystem II (PSII) catalyzes the light-driven charge separation and water oxidation reactions of photosynthesis. Eukaryotic PSII core is usually associated with membrane-embedded light-harvesting antennae, which greatly increase the absorbance cross-section of the core. The peripheral antennae in different phototrophs vary considerably in protein composition and arrangement. Photosynthetic cryptophytes possess chlorophyll a/c binding proteins (CACs) that serve as their antennae. How these CACs assemble with the PSII core remains unclear. Here, we report the 2.57-Å resolution structure of cryptophyte PSII-CAC purified from cells at nitrogen-limited stationary growth phase. We show that each monomer of the PSII homodimer contains a core complex, six chlorophyll a/c binding proteins (CACs) and a previously unseen chlorophyll-binding protein (termed CAL-II). Six CACs are arranged as a double-layered arc-shaped non-parallel belt, and two such belts attach to the dimeric core from opposite sides. The CAL-II simultaneously interacts with a number of core subunits and five CACs. The distinct organization of CACs and the presence of CAL-II may play a critical role in stabilizing the dimeric PSII-CAC complex under stress conditions. Our study provides mechanistic insights into the assembly and function of the PSII-CAC complex as well as the possible adaptation of cryptophytes in response to environmental stresses.

Molecular events accompanying aggregation-induced energy quenching in fucoxanthin-chlorophyll proteins.

In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL,HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to.

The stromal side of the cytochrome b6f complex regulates state transitions.

In oxygenic photosynthesis, state transitions distribute light energy between PSI and PSII. This regulation involves reduction of the plastoquinone pool, activation of the state transitions 7 (STT7) protein kinase by the cytochrome (cyt) b6f complex, and phosphorylation and migration of light harvesting complexes II (LHCII). In this study, we show that in Chlamydomonas reinhardtii, the C-terminus of the cyt b6 subunit PetB acts on phosphorylation of STT7 and state transitions. We used site-directed mutagenesis of the chloroplast petB gene to truncate (remove L215b6) or elongate (add G216b6) the cyt b6 subunit. Modified complexes are devoid of heme ci and degraded by FTSH protease, revealing that salt bridge formation between cyt b6 (PetB) and Subunit IV (PetD) is essential to the assembly of the complex. In double mutants where FTSH is inactivated, modified cyt b6f accumulated but the phosphorylation cascade was blocked. We also replaced the arginine interacting with heme ci propionate (R207Kb6). In this modified complex, heme ci is present but the kinetics of phosphorylation are slower. We show that highly phosphorylated forms of STT7 accumulated transiently after reduction of the PQ pool and represent the active forms of the protein kinase. The phosphorylation of the LHCII targets is favored at the expense of the protein kinase, and the migration of LHCII toward PSI is the limiting step for state transitions.

Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center.

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an "encounter" complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a "productive" ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c2 (cyt c2). Importantly, SMFS resolves the distribution of interaction forces into low (∼150 pN) and high (∼330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c2 and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c2. The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

Simulation of the Pump-Probe Spectra and Excitation Energy Relaxation of the B850 Band of the LH2 Complex in Purple Bacteria.

Ultrafast spectroscopic techniques have been vital in studying excitation energy transfer (EET) in photosynthetic light harvesting complexes. In this paper, we simulate the pump-probe spectra of the B850 band of the light harvesting complex 2 (LH2) of purple bacteria, by using the hierarchical equation of motion method and the optical response function approach. The ground state bleach, stimulated emission, and excited state absorption components of the pump-probe spectra are analyzed in detail. The laser pulse-induced population dynamics are also simulated to help understand the main features of the pump-probe spectra and the EET process. It is shown that the excitation energy relaxation is an ultrafast process with multiple time scales. The first 40 fs of the pump-probe spectra is dominated by the relaxation of the k = ±1 states to both the k = 0 and higher energy states. Dynamics on a longer time scale around 200 fs reflects the relaxation of higher energy states to the k = 0 state.

Structure of the red-shifted Fittonia albivenis photosystem I.

Photosystem I (PSI) from Fittonia albivenis, an Acanthaceae ornamental plant, is notable among green plants for its red-shifted emission spectrum. Here, we solved the structure of a PSI-light harvesting complex I (LHCI) supercomplex from F. albivenis at 2.46-Å resolution using cryo-electron microscopy. The supercomplex contains a core complex of 14 subunits and an LHCI belt with four antenna subunits (Lhca1-4) similar to previously reported angiosperm PSI-LHCI structures; however, Lhca3 differs in three regions surrounding a dimer of low-energy chlorophylls (Chls) termed red Chls, which absorb far-red beyond visible light. The unique amino acid sequences within these regions are exclusively shared by plants with strongly red-shifted fluorescence emission, suggesting candidate structural elements for regulating the energy state of red Chls. These results provide a structural basis for unraveling the mechanisms of light harvest and transfer in PSI-LHCI of under canopy plants and for designing Lhc to harness longer-wavelength light in the far-red spectral range.

Martini 3 Coarse-Grained Model for the Cofactors Involved in Photosynthesis.

As a critical step in advancing the simulation of photosynthetic complexes, we present the Martini 3 coarse-grained (CG) models of key cofactors associated with light harvesting (LHCII) proteins and the photosystem II (PSII) core complex. Our work focuses on the parametrization of beta-carotene, plastoquinone/quinol, violaxanthin, lutein, neoxanthin, chlorophyll A, chlorophyll B, and heme. We derived the CG parameters to match the all-atom reference simulations, while structural and thermodynamic properties of the cofactors were compared to experimental values when available. To further assess the reliability of the parameterization, we tested the behavior of these cofactors within their physiological environments, specifically in a lipid bilayer and bound to photosynthetic complexes. The results demonstrate that our CG models maintain the essential features required for realistic simulations. This work lays the groundwork for detailed simulations of the PSII-LHCII super-complex, providing a robust parameter set for future studies.

Rhodopsin-based light-harvesting system for sustainable synthetic biology.

Rhodopsins, a diverse class of light-sensitive proteins found in various life domains, have attracted considerable interest for their potential applications in sustainable synthetic biology. These proteins exhibit remarkable photochemical properties, undergoing conformational changes upon light absorption that drive a variety of biological processes. Exploiting rhodopsin's natural properties could pave the way for creating sustainable and energy-efficient technologies. Rhodopsin-based light-harvesting systems offer innovative solutions to a few key challenges in sustainable engineering, from bioproduction to renewable energy conversion. In this opinion article, we explore the recent advancements and future possibilities of employing rhodopsins for sustainable engineering, underscoring the transformative potential of these biomolecules.

Light harvesting in purple bacteria does not rely on resonance fine-tuning in peripheral antenna complexes.

The ring-like peripheral light-harvesting complex 2 (LH2) expressed by many phototrophic purple bacteria is a popular model system in biological light-harvesting research due to its robustness, small size, and known crystal structure. Furthermore, the availability of structural variants with distinct electronic structures and optical properties has made this group of light harvesters an attractive testing ground for studies of structure-function relationships in biological systems. LH2 is one of several pigment-protein complexes for which a link between functionality and effects such as excitonic coherence and vibronic coupling has been proposed. While a direct connection has not yet been demonstrated, many such interactions are highly sensitive to resonance conditions, and a dependence of intra-complex dynamics on detailed electronic structure might be expected. To gauge the sensitivity of energy-level structure and relaxation dynamics to naturally occurring structural changes, we compare the photo-induced dynamics in two structurally distinct LH2 variants. Using polarization-controlled 2D electronic spectroscopy at cryogenic temperatures, we directly access information on dynamic and static disorder in the complexes. The simultaneous optimal spectral and temporal resolution of these experiments further allows us to characterize the ultrafast energy relaxation, including exciton transport within the complexes. Despite the variations in PPC molecular structure manifesting as clear differences in electronic structure and disorder, the energy-transport and-relaxation dynamics remain remarkably similar. This indicates that the light-harvesting functionality of purple bacteria within a single LH2 complex is highly robust to structural perturbations and likely does not rely on finely tuned electronic- or electron-vibrational resonance conditions.

Light-independent pathway of STN7 kinase activation under low temperature stress in runner bean (Phaseolus coccineus L.).

BACKGROUND: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet. RESULTS: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation. CONCLUSIONS: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.

Structural basis for an early stage of the photosystem II repair cycle in Chlamydomonas reinhardtii.

Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.