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Photosystem I (PSI) and II (PSII) balance their light energy distribution absorbed by their light-harvesting complexes (LHCs) through state transition to maintain the maximum photosynthetic performance and to avoid photodamage. In state 2, a part of LHCII moves to PSI, forming a PSI-LHCI-LHCII supercomplex. The green alga Chlamydomonas reinhardtii exhibits state transition to a far larger extent than higher plants. Here we report the cryo-electron microscopy structure of a PSI-LHCI-LHCII supercomplex in state 2 from C. reinhardtii at 3.42 Å resolution. The result reveals that the PSI-LHCI-LHCII of C. reinhardtii binds two LHCII trimers in addition to ten LHCI subunits. The PSI core subunits PsaO and PsaH, which were missed or not well-resolved in previous Cr-PSI-LHCI structures, are observed. The present results reveal the organization and assembly of PSI core subunits, LHCI and LHCII, pigment arrangement, and possible pathways of energy transfer from peripheral antennae to the PSI core. Photosystems (PS) I and II undergo state transitions to optimize photosynthesis and photoprotection. Here the authors report a cryo-electron microscopy structure of the state 2 PSI-LHCI-LHCII supercomplex from C. reinhardtii revealing subunit organization and possible pathways of energy transfer.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis 1 , 2 . The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs) 3 . Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS–PSII–PSI–LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis 4 , providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS–PSII–PSI–LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI. In situ structures of PBS–PSII–PSI–LHC megacomplexes from the alga P. purpureum at near-atomic resolution using cryogenic-electron tomography and in situ single-particle analysis are reported, providing interaction details between PBS, PSII and PSI.

A new approach, time-resolved serial femtosecond crystallography, is used to view the intermediate states of a photosystem complex following illumination, shedding light on proton transfer and O=O bond formation. Bond formation in photosystem II Technical developments, such as X-ray free electron lasers (XFEL), allow for a more detailed view of the structure of the photosystem complexes, making it possible to get a glimpse of the mechanisms of proton transfer and bond formation. Jian-Ren Shen and colleagues use a new approach, time-resolved serial femtosecond crystallography, with X-ray free electron lasers to view the intermediate states formed after two-flash illumination. Upon illumination, the authors see that the disappearance of one water molecule relocates another water molecule towards an oxygen atom, in a manner that may reflect proton transfer. They also gain evidence for the inclusion of a new oxygen atom that would be positioned to form an O=O bond that has been hypothesized but never previously detected. These insights increase our understanding of the mechanism of water oxidation in photosystem II. Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC) 1 , 2 , 3 . The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn 4 CaO 5 cluster organized in an asymmetric, ‘distorted-chair’ form 4 . This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ 5 structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the Q B /non-haem iron and the Mn 4 CaO 5 cluster. The changes around the Q B /non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn 4 CaO 5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ 4 -oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4 , 5 ). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously 6 , 7 .

Plants and cyanobacteria use chlorophyll-rich photosystem complexes to convert light energy into chemical energy. Some organisms have developed adaptations to take advantage of longer-wavelength photons. Nürnberg et al. studied photosystem complexes from cyanobacteria grown in the presence of far-red light. The authors identified the primary donor chlorophyll as one of a few chlorophyll molecules in the far-red light–adapted enzymes that were chemically altered to shift their absorption spectrum. Kinetic measurements demonstrated that far-red light is capable of directly driving water oxidation, despite having less energy than the red light used by most photosynthetic organisms. Science , this issue p. 1210 A chlorophyll variant with far-red absorption is involved in photosynthesis in cyanobacteria adapted to far red light. Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy “red limit” of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.

Photosynthetic water oxidation is catalyzed by the Mn₄CaO₅ cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S₀ to S₄). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S₁, S₂, and S₃ states by x-ray free-electron laser serial crystallography. No insertion of water was found in S₂, but flipping of D1 Glu189 upon transition to S₃ leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn₄CaO₆ cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.

In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b 6 f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.

We review the mechanism underlying nonphotochemical chlorophyll fluorescence quenching (NPQ) and its role in protecting plants against photoinhibition. This review includes an introduction to this phenomenon, a brief history of major milestones in our understanding of NPQ, definitions, and a discussion of quantitative measurements of NPQ. We discuss the current knowledge and unknown aspects in the NPQ scenario, including the following: ΔpH, the proton gradient (trigger); light-harvesting complex II (LHCII), PSII light harvesting antenna (site); and changes in the antenna induced by ΔpH (change), which lead to the creation of the quencher. We conclude that the minimum requirements for NPQ in vivo are ΔpH, LHCII complexes, and the PsbS protein. We highlight the most important unknown in the NPQ scenario, the mechanism by which PsbS acts upon the LHCII antenna. Finally, we describe a novel, emerging technology for assessing the photoprotective \"power\" of NPQ and the important findings obtained through this technology

The structures of three intermediate states of photosystem II, which is crucial for photosynthesis, have been solved at room temperature, shedding new light on this process. Room temperature structures of photosystem II During the conversion of light into energy in plants, photosystem II oxidizes water within a Mn 4 CaO 5 cluster in the oxygen evolving complex (OEC). This process involves five intermediate states that have eluded structural determination until now. Junko Yano and colleagues use a femtosecond X-ray free electron laser (XFEL) to capture three of these states at room temperature. As the structure was solved in the presence of ammonia, a water analogue, the authors are able to conclude that the ammonia-binding Mn site is not a substrate water site. Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn 4 CaO 5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S 0 to S 4 ) 1 , in which S 1 is the dark-stable state and S 3 is the last semi-stable state before O–O bond formation and O 2 evolution 2 , 3 . A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site 4 , 5 , 6 . Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S 1 ), two-flash illuminated (2F; S 3 -enriched), and ammonia-bound two-flash illuminated (2F-NH 3 ; S 3 -enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL 7 provided a damage-free view of the S 1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions 8 , 9 , and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn 4 CaO 5 cluster in the S 2 and S 3 states 10 . Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site 10 , 11 , 12 , 13 . This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

Photosynthetic organisms are frequently exposed to light intensities that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O₂, which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosynthetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii, the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipation of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii, we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light-harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1.