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Global photosynthesis consumes ten times more CO 2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption 1 . The high efficiency of algal photosynthesis relies on a mechanism concentrating CO 2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO 2 fixation 2 . Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified 3 , 4 , how microalgae supply energy to concentrate CO 2 against a thermodynamic gradient remains unknown 4 – 6 . Here we show that in the green alga Chlamydomonas reinhardtii , the combined action of cyclic electron flow and O 2 photoreduction—which depend on PGRL1 and flavodiiron proteins, respectively—generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO 2 . We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity. The CO 2 -concentrating mechanism of the green alga Chlamydomonas reinhardtii is dependent on pH gradients generated by both cyclic electron flow and O 2 photoreduction.

Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S 2 to S 3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons. The oxygen-evolving complex in Photosystem II (PSII) catalyzes the light-driven oxidation of water to oxygen and it is still under debate how the water reaches the active site. Here, the authors analyse time-resolved XFEL-based crystal structures of PSII that were determined at room temperature and report the structures of the waters in the putative channels surrounding the active site at various time-points during the reaction cycle and conclude that the O1 channel is the likely water intake pathway and the Cl1 channel the likely proton release pathway.

Diatom is an important group of marine algae and contributes to around 20% of the global photosynthetic carbon fixation. Photosystem I (PSI) of diatoms is associated with a large number of fucoxanthin-chlorophyll a / c proteins (FCPIs). We report the structure of PSI-FCPI from a diatom Chaetoceros gracili s at 2.38 Å resolution by single-particle cryo-electron microscopy. PSI-FCPI is a monomeric supercomplex consisting of 12 core and 24 antenna subunits (FCPIs), and 326 chlorophylls a , 34 chlorophylls c , 102 fucoxanthins, 35 diadinoxanthins, 18 β -carotenes and some electron transfer cofactors. Two subunits designated PsaR and PsaS were found in the core, whereas several subunits were lost. The large number of pigments constitute a unique and huge network ensuring efficient energy harvesting, transfer and dissipation. These results provide a firm structural basis for unraveling the mechanisms of light-energy harvesting, transfer and quenching in the diatom PSI-FCPI, and also important clues to evolutionary changes of PSI-LHCI. Diatoms are marine algae with an important role in global photosynthetic carbon fixation. Here, the authors present the 2.38 Å cryo-EM structure of photosystem I (PSI) in complex with its 24 fucoxanthin chlorophyll a/c -binding (FCPI) antenna proteins from the diatom Chaetoceros gracilis , which provides mechanistic insights into light-energy harvesting, transfer and quenching of the PSI-FCPI supercomplex.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn 4 CaO 5 cluster first stores four oxidizing equivalents, the S 0 to S 4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry 1 – 3 . Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S 3 →[S 4 ]→S 0 transition where O 2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn 4 CaO 5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O x , which was introduced as a bridging ligand between Ca and Mn1 during the S 2 →S 3 transition 4 – 6 , disappears or relocates in parallel with Y z reduction starting at approximately 700 μs after the third flash. The onset of O 2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide. Using serial femtosecond X-ray cystallography, we provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, specifically the S 3 →[S 4 ]→S 0 transition where O 2 is formed.

Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria 1 . Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane 2 , but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I ( Ec CI) in different redox states, including catalytic turnover. Ec CI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues 3 , Ec CI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a ‘domino effect’ series of proton transfers and electrostatic interactions: the forward wave (‘dominoes stacking’) primes the pump, and the reverse wave (‘dominoes falling’) results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes. Cryo-electron microscopy studies of Escherichia coli complex I suggest a conserved mechanism of coupled proton transfers and electrostatic interactions that result in proton ejection from the complex exclusively at the distal NuoL subunit.

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.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of S i states ( i  = 0–4) at the Mn 4 CaO 5 cluster 1 – 3 , during which an extra oxygen (O6) is incorporated at the S 3 state to form a possible dioxygen 4 – 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). Y Z , a tyrosine residue that connects the reaction centre P680 and the Mn 4 CaO 5 cluster, 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 Ca 2+ 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. Serial femtosecond crystallography reveals the structural dynamics of photosystem II during the S-state transitions that produce dioxygen, providing insight into electron transfer, water insertion, proton release and O–O bond formation on sub-microsecond timescales.

The structure of the V O subcomplex of yeast V-ATPase, solved by electron cryomicroscopy, reveals a new subunit and suggests a mechanism for the translocation of protons across membranes. Structure of a membrane rotary ATPase The cell powers many cellular processes by pumping protons through membrane-embedded rotary ATPases. It has been difficult to determine the structure of a physiological ATPase complex, given the membrane association. John Rubinstein and colleagues have now solved this problem, and determine the structure of the V O complex of the yeast vacuolar ATPase within a membrane. At approximately 3.9 Å, the resolution of the structure is sufficient to permit the authors to propose a previously unidentified subunit. Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V 1 region drives proton translocation through the membrane-embedded V O region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase 1 , 2 , 3 , 4 , 5 . We induced dissociation and auto-inhibition of the V 1 and V O regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae 6 , 7 , allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the V O complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac 8 c′c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H + transport. The AAC-mediated H + current requires free fatty acids and resembles the H + leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H + leak, but does not completely inhibit it. This suggests that the H + leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H + leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria. The mitochondrial ADP/ATP carrier mediates the proton leak in mitochondria from all tissues that lack UCP1, thereby linking coupled (ATP production) and uncoupled (thermogenesis) energy conversion.

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 tepidum at 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 Q B with 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.