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.

Abstract:The valence excited states of ferric and ferrous hexacyanide ions in aqueous solution were mapped with resonant inelastic X-ray scattering (RIXS) at the Fe L2,3-and N K-edges. Probing of both the central Fe and the ligand N atoms enabled identification of the metal-and ligand-centered excited states, as well as ligandto-metal and metal-to-ligand charge transfer excited states. Ab initio calculations utilizing the RASPT2 method was used to simulate the Fe L2,3-edge RIXS spectra and enabled quantification of the covalency of both occupied and empty orbitals of π and σ symmetry. We find that π back-donation in the ferric complex is smaller compared to the ferrous complex. This is evidenced by the relative amount of Fe 3d character in the nominally 2π CN -molecular orbital of 7% and 9% in ferric and ferrous hexacyanide, respectively. Utilizing the direct sensitivity of Fe L3-edge RIXS to the Fe 3d character in the occupied molecular orbitals we also find that the donation interactions are dominated by σ-bonding. The latter is found to be stronger in the ferric complex with a Fe 3d contribution to the nominally 5σ CN -molecular orbitals of 29% compared to 20% in the ferrous complex. These results are consistent with the notion that a higher charge at the central metal atom increases donation and decreases back-donation.

We present a liquid flatjet system for solution phase soft-x-ray spectroscopy. The flatjet set-up utilises the phenomenon of formation of stable liquid sheets upon collision of two identical laminar jets. Colliding the two single water jets, coming out of the nozzles with 50 μm orifices, under an impact angle of 48° leads to double sheet formation, of which the first sheet is 4.6 mm long and 1.0 mm wide. The liquid flatjet operates fully functional under vacuum conditions (<10−3 mbar), allowing soft-x-ray spectroscopy of aqueous solutions in transmission mode. We analyse the liquid water flatjet thickness under atmospheric pressure using interferomeric or mid-infrared transmission measurements and under vacuum conditions by measuring the absorbance of the O K-edge of water in transmission, and comparing our results with previously published data obtained with standing cells with Si3N4 membrane windows. The thickness of the first liquid sheet is found to vary between 1.4–3 μm, depending on the transverse and longitudinal position in the liquid sheet. We observe that the derived thickness is of similar magnitude under 1 bar and under vacuum conditions. A catcher unit facilitates the recycling of the solutions, allowing measurements on small sample volumes (∼10 ml). We demonstrate the applicability of this approach by presenting measurements on the N K-edge of aqueous NH4+. Our results suggest the high potential of using liquid flatjets in steady-state and time-resolved studies in the soft-x-ray regime.

A combined experimental and theoretical approach reveals correlations of metal L-edge X-ray absorption energies to local charge and spin densities.

The 3d transition metals play a pivotal role in many charge transfer processes in catalysis and biology. X-ray absorption spectroscopy at the L-edge of metal sites probes metal 2p-3d excitations, providing key access to their valence electronic structure, which is crucial for understanding these processes. We report L-edge absorption spectra of Mn(acac) and Mn(acac) complexes in solution, utilizing a liquid flatjet for X-ray absorption spectroscopy in transmission mode. With this, we derive absolute absorption cross-sections for the L-edge transitions with peak magnitudes as large as 12 and 9 Mb for Mn(acac) and Mn(acac), respectively. We provide insight into the electronic structure with ab initio restricted active space calculations of these L-edge transitions, reproducing the experimental spectra with excellent agreement in terms of shapes, relative energies, and relative intensities for the two complexes. Crystal field multiplet theory is used to assign spectral features in terms of the electronic structure. Comparison to charge transfer multiplet calculations reveals the importance of charge transfer in the core-excited final states. On the basis of our experimental observations, we extrapolate the feasibility of 3d transition metal L-edge absorption spectroscopy using the liquid flatjet approach in probing highly dilute biological solution samples and possible extensions to table-top soft X-ray sources.