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Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Sistates (i= 0–4) at the Mn4CaO5cluster1–3, during which an extra oxygen (O6) is incorporated at the S3state to form a possible dioxygen4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.

In plants and green algae, the core of photosystem I (PSI) is surrounded by a peripheral antenna system consisting of light-harvesting complex I (LHCI). Here we report the cryo-electron microscopic structure of the PSI–LHCI supercomplex from the green alga Chlamydomonas reinhardtii. The structure reveals that eight Lhca proteins form two tetrameric LHCI belts attached to the PsaF side while the other two Lhca proteins form an additional Lhca2/Lhca9 heterodimer attached to the opposite side. The spatial arrangement of light-harvesting pigments reveals that Chlorophylls bare more abundant in the outer LHCI belt than in the inner LHCI belt and are absent from the core, thereby providing the downhill energy transfer pathways to the PSI core. PSI–LHCI is complexed with a plastocyanin on the patch of lysine residues of PsaF at the luminal side. The assembly provides a structural basis for understanding the mechanism of light-harvesting, excitation energy transfer of the PSI–LHCI supercomplex and electron transfer with plastocyanin. This cryo-electron microscopy structure of a photosystem I–light-harvesting complex I supercomplex from Chlamydomonasreveals eight Lhca proteins forming two tetrameric light-harvesting complex I belts on one side, and a Lhca heterodimer on the other.

Light-harvesting complex 1 (LH1) and the reaction centre (RC) form a membrane-protein supercomplex that performs the primary reactions of photosynthesis in purple photosynthetic bacteria. The structure of the LH1–RC complex can provide information on the arrangement of protein subunits and cofactors; however, so far it has been resolved only at a relatively low resolution. Here we report the crystal structure of the calcium-ion-bound LH1–RC supercomplex of Thermochromatium tepidumat a resolution of 1.9 Å. This atomic-resolution structure revealed several new features about the organization of protein subunits and cofactors. We describe the loop regions of RC in their intact states, the interaction of these loop regions with the LH1 subunits, the exchange route for the bound quinone QBwith free quinone molecules, the transport of free quinones between the inside and outside of the LH1 ring structure, and the detailed calcium-ion-binding environment. This structure provides a solid basis for the detailed examination of the light reactions that occur during bacterial photosynthesis. The structure of the Thermochromatium tepidum calcium-ion-bound light-harvesting–reaction centre (LH1–RC) supercomplex, which performs the primary reactions of photosynthesis in purple photosynthetic bacteria, is resolved to the atomic level.

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). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5cluster organized in an asymmetric, ‘distorted-chair’ form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ 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 QB/non-haem iron and the Mn4CaO5cluster. The changes around the QB/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 Mn4CaO5cluster 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 μ4-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.