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.

Photosynthesis powers life on our planet. The basic photosynthetic architecture consists of antenna complexes that harvest solar energy and reaction centres that convert the energy into stable separated charge. In oxygenic photosynthesis, the initial charge separation occurs in the photosystem II reaction centre, the only known natural enzyme that uses solar energy to split water. Both energy transfer and charge separation in photosynthesis are rapid events with high quantum efficiencies. In recent nonlinear spectroscopic experiments, long-lived coherences have been observed in photosynthetic antenna complexes, and theoretical work suggests that they reflect underlying electronic-vibrational resonances, which may play a functional role in enhancing energy transfer. Here, we report the observation of coherent dynamics persisting on a picosecond timescale at 77 K in the photosystem II reaction centre using two-dimensional electronic spectroscopy. Supporting simulations suggest that the coherences are of a mixed electronic-vibrational (vibronic) nature and may enhance the rate of charge separation in oxygenic photosynthesis.

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 center with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, where S1 is the dark stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution2,3. A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site4-6, is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) 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 structure of PS II7 at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, RT measurements are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10-13. Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

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→ S3transition, 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 O2formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2→ S3transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QAand QB, are observed. At the donor site, tyrosine YZand 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→ S3transition mirrors the appearance of OXelectron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Two-dimensional electronic spectroscopy (2DES) reveals connections between an optical excitation at a given frequency and the signals it creates over a wide range of frequencies. These connections, manifested as cross-peak locations and their lineshapes, reflect the underlying electronic and vibrational structure of the system under study. How these spectroscopic signatures evolve in time reveals the system dynamics and provides a detailed picture of coherent and incoherent processes. 2DES is rapidly maturing and has already found numerous applications, including studies of photosynthetic energy transfer and photochemical reactions and many-body interactions in nanostructured materials. Many systems of interest contain electronic transitions spanning the ultraviolet to the near infrared and beyond. Most 2DES measurements to date have explored a relatively small frequency range. We discuss the challenges of implementing 2DES and compare and contrast different approaches in terms of their information content, ease of implementation, and potential for broadband measurements.

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.

Two-dimensional decay associated spectra 2C2DES Two-color two-dimensional electronic spectroscopy 2DES Two-dimensional electronic spectroscopy 2DIR Two-dimensional infrared spectroscopy 2PE Two-pulse photon echo BBO Beta-barium borate BChl Bacteriochlorophyll BRC Bacterial reaction center CCD Charge-collecting device CGS Common ground state Chl Chlorophyll CP Cross-peak CS Charge separation DAS Decay associated spectra DMSO Dimethyl sulfoxide DO Diffractive optic DSP Digital signal processor EET Excitation energy transfer ESM Exponential series method ESA Excited state absorption xiv ESE Excited state emission ET Energy transfer FRET Fluorescence resonance energy transfer GSB Ground state bleach LHCII Light-harvesting complex II LN Liquid nitrogen LO Local oscillator NMR Nuclear magnetic resonance NOPA Non-collinear optical parametric amplifier OD Optical density PC Prism compressor PERY N,N'-bis (2,6-dimethylphenyl) perylene-3,4,9,10-tetracarboxylicdiimide Pheo Pheophytin PSI Photosystem I PSII Photosystem II RC D1D2-cyt.b559 reaction center complex SFG Sum frequency generation SHB Spectral hole burning SNR Signal-to-noise ratio SPM Self-phase modulation SVD Singular value decomposition TA Transient absorption TRF Time-resolved fluorescence ZAP-SPIDER Zero additional phase spectral interferometry for direct electric field reconstruction xv ABSTRACT Two-Dimensional Electronic Spectroscopy of the Photosystem II D1D2-cyt.b559

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. Implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, 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.