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.

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.

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.

Ionization of nitrogen by extreme ultraviolet (XUV) light from the Sun has recently been recognized as an important driver of chemical reactions in the atmosphere of Titan. XUV photons with energies of 24 eV and above convert inert nitrogen molecules into reactive neutral and ionic fragments that initiate chemical reactions. Understanding the XUV-induced fragmentation poses significant challenges to modern theory owing to its ultrafast time scales, complex electronic rearrangements, and strong dependence on the XUV photon energy. Here, we apply femtosecond time-resolved photoelectron and photoion spectroscopy to study dissociative ionization of nitrogen, the most abundant molecule in Titan's atmosphere, at selected XUV photon energies using a table-top XUV time-compensating monochromator. We probe the resulting dynamics using a time-delayed infrared (IR) ionization pulse. Coupled with ab initio calculations, the results allow us to assign the major dissociation channels resulting from production of an inner-valence hole, with important implications for models of Titan's XUV-driven atmospheric chemistry.