Water Networks in Photosystem II Using Crystalline Molecular Dynamics Simulations and Room-Temperature XFEL Serial Crystallography.

Structural dynamics of water and its hydrogen-bonding networks play an important role in enzyme function via the transport of protons, ions, and substrates. To gain insights into these mechanisms in the water oxidation reaction in Photosystem II (PS II), we have performed crystalline molecular dynamics (MD) simulations of the dark-stable S1 state. Our MD model consists of a full unit cell with 8 PS II monomers in explicit solvent (861 894 atoms), enabling us to compute the simulated crystalline electron density and to compare it directly with the experimental density from serial femtosecond X-ray crystallography under physiological temperature collected at X-ray free electron lasers (XFELs). The MD density reproduced the experimental density and water positions with high fidelity. The detailed dynamics in the simulations provided insights into the mobility of water molecules in the channels beyond what can be interpreted from experimental B-factors and electron densities alone. In particular, the simulations revealed fast, coordinated exchange of waters at sites where the density is strong, and water transport across the bottleneck region of the channels where the density is weak. By computing MD hydrogen and oxygen maps separately, we developed a novel Map-based Acceptor-Donor Identification (MADI) technique that yields information which helps to infer hydrogen-bond directionality and strength. The MADI analysis revealed a series of hydrogen-bond wires emanating from the Mn cluster through the Cl1 and O4 channels; such wires might provide pathways for proton transfer during the reaction cycle of PS II. Our simulations provide an atomistic picture of the dynamics of water and hydrogen-bonding networks in PS II, with implications for the specific role of each channel in the water oxidation reaction.

Room temperature XFEL crystallography reveals asymmetry in the vicinity of the two phylloquinones in photosystem I.

Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.

Structural dynamics in the water and proton channels of photosystem II during the S2 to S3 transition.

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 S2 to S3 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.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo-bridged Mn4 CaO5 cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn4 CaO5 cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S0  - S4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S2 state of PSII with structural data collected of the S1 , S2 and S3 states by serial crystallography at neutral pH (∼6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S2 state, the room temperature crystallography data can be well-described by just one S2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

Structures of the intermediates of Kok's photosynthetic water oxidation clock.

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

Crystallophore: a versatile lanthanide complex for protein crystallography combining nucleating effects, phasing properties, and luminescence.

Macromolecular crystallography suffers from two major issues: getting well-diffracting crystals and solving the phase problem inherent to large macromolecules. Here, we describe the first example of a lanthanide complex family named "crystallophore" (Xo4), which contributes to tackling both bottlenecks. This terbium complex, Tb-Xo4, is an appealing agent for biocrystallography, combining the exceptional phasing power of the Tb(iii) heavy atom with powerful nucleating properties, providing ready-to-use crystals for structure determination. Furthermore, protein/Tb-Xo4 co-crystals can be easily detected and discriminated from other crystalline by-products using luminescence. We demonstrate the potential of this additive for the crystallisation and structure determination of eight proteins, two of whose structures were unknown.

Structure of photosystem II and substrate binding at room temperature.

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 Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. 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. 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 (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, 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 Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases.

Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.