Claudio Cirelli

Ultrafast pump–probe serial femtosecond crystallography (SFX) experiments are typically performed with extremely high excitation laser fluence to maximize the occupancy of the light-induced state. This has been justified by assuming that a dominant fraction (up to 99%) of the pump laser light is scattered by the jetting medium, strongly reducing the pump laser fluence in the crystals which, therefore, do not absorb multiple photons and thus undergo the biologically relevant single-photon photoreaction. However, this notion is strongly contested in the field. To address the issue of pump laser scattering losses in high viscosity jets, we performed time-resolved SFX experiments on fatty acid photodecarboxylase crystals using high-viscosity extrusion (HVE) and both patterned and non-patterned sheet-on-sheet (SOS) fixed-target sample delivery systems on the Cristallina-MX instrument at SwissFEL. Tightly focused pump laser beams were used to mitigate light contamination (the undesired accidental illumination of neighboring crystals) in SOS chips, paving the way for their use in pump–probe investigations of lipidic-cubic-phase-grown membrane protein crystals. Determination of the light-induced reaction intermediate occupancies enabled a comparison of the influence of the delivery method on the pump laser fluence reaching the crystals. We show that optically transparent high-viscosity jets and chip crystal delivery methods result in photoproduct yields that approach 70–80% and 60–70%, respectively, of computationally derived yields that take into account excitation probabilities. These results demonstrate that clear viscous jets scatter pump laser light far less than has been claimed. Consequently, the use of excessive photoexcitation energy densities is neither needed nor justified in optically pumped time-resolved SFX experiments when using appropriately sized crystals.

The Alvra experimental station at the Swiss X-ray free-electron laser, SwissFEL, investigates ultrafast dynamics in chemical and biological systems using X-ray scattering and spectroscopy techniques. A key feature of Alvra is its unique capability to perform time-resolved X-ray emission and resonant inelastic X-ray scattering in the tender X-ray regime, currently not available at other XFEL endstations, combined with simultaneous access to X-ray absorption spectroscopy and X-ray solution scattering. Together with sub-35 fs time resolution enabled by advanced timing diagnostics, this positions Alvra as a versatile instrument for ultrafast chemical dynamics in the liquid phase. Here we cover the various technical aspects of the beamline and experimental station, and present some examples of experimental results measured during the first years of SwissFEL commissioning and user operation.

Cyanobacteria have produced Earth’s oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity– dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCP ^O ) generates a metastable intermediate (OCP ^1hν ) that either relaxes thermally or, upon absorption of a second photon within ∼1 s, converts to the active photoprotective state (OCP ^R ). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP ^1hν , establish its functional relevance and capture structural snapshots along the OCP ^O →OCP ^1hν and OCP ^1hν →OCP ^R photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

Cytochrome c oxidase (CcO) utilizes the energy released as dioxygen is reduced to two water molecules to pump protons across an energy transducing biological membrane. X-ray structures of CcO have been known for three decades1-3 and all electron transfer and proton uptake and release steps have been characterized.4,5 However, it remains unclear how the reduction of dioxygen at the enzyme’s active-site achieves proton pumping. Here we apply time-resolved serial femtosecond X-ray crystallography to observe structural changes in the ba3-type CcO from Thermus thermophilus following the release of photocaged oxygen using a UV laser flash. In the resting oxidised state, chloride ions are observed to ligate CuB of the active site and the farnesyl oxygen of haem a3. Both ions are displaced when the enzyme is fully reduced. Water molecules are observed to order and disorder within the active-site 3.3 ms after photocaged oxygen is released, creating and disrupting water mediated pathways for proton transport through the enzyme. Requiring that the active-site charges sum to zero for every step of the catalytic cycle yields a step-by-step atomistic model for proton pumping by ba3-type CcOs.

Solvation plays a key role in many photochemical and biological processes. Yet, direct atomic-scale observation of solvent reorganization around photoexcited molecules containing only light atoms has so far remained elusive. Here, we use time-resolved X-ray scattering at an X-ray free-electron laser to track with a ~ 120 fs time resolution the photoinduced ultrafast rearrangement of polar acetonitrile solvent molecules around an organic hemithioindigo chromophore. The experiments reveal that the solvation shell reorganizes with a ~ 0.3 ps time constant driven by photoinduced charge transfer in the chromophore, and then reequilibrates over ~ 3 ps as the excited state population returns to the ground state. These results demonstrate the viability of using ultrashort X-ray pulses to directly visualize the motion of light atoms in solution, paving the way for atomic-scale understanding and control of photoinduced processes in biological and synthetic photoactive organic molecules.

The possible mechanistic roles of bridging ligands in chromophore-catalyst assemblies are examined to assess if the bridge can play an active role in light-driven electron storage towards multi-redox photocatalysis, as opposed to its traditional role facilitating electron transfer (ET) from chromophore-to-catalyst. In a dyad linking a Ru polypyridyl chromophore and Rh proton reduction catalyst, [Ru-Rh]3+, the central azaacene motif in the tetrapyridophenazine (tpphz) bridging ligand is shown here to accumulate electrons, rather than supporting ET to the catalyst. Ultrafast time-resolved X-ray absorption spectroscopies probing the chromophore (Ru L3-edge) and catalyst (Rh L3- and Cl K-edges) atoms demonstrate that photoexcitation induces ET away from the chromophore, but not to the catalyst motif. Ab initio calculations show that the spectral features are consistent with electron accumulation on tpphz. An alternative photocatalytic mechanism is identified, in which tpphz stores electrons and the multi-electron Rh catalyst becomes activated by a disproportionation mechanism. The results indicate a broader role for bridging ligands in photocatalytic assemblies to be considered in their future design.

Photochemical C–H activation reactions mediated by transition metal complexes often proceed via formation of σ-complexes, in which a C–H σ-bond coordinates to the metal and is ultimately broken by specific charge-transfer interactions. Since metal-alkane σ-complexes are short-lived transient intermediates and because their reactivity is determined by electronic-structure effects, direct experimental characterization is challenging and understanding of their reactivity is limited. Here, we demonstrate how optical pump and metal-specific X-ray probe spectroscopy reveals the decisive nature of charge-transfer interactions in σ-complexes photochemically prepared with three different Rh-carbonyl complexes. We use femtosecond-resolution X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray scattering (RIXS) at the Rh L3-edge to probe with orbital-level accuracy how the charge-transfer interactions between the Rh centers, the ancillary ligands and the alkane C–H σ-bonds vary with varying reactivity towards C–H activation. Supported by theoretical calculations, we reveal the importance of specific occupied molecular orbitals with specific symmetry in modulating the reactivity of the σ-complexes by providing, channeling and directing charge density via the metal to and from the C–H bond. Elucidating the electronic factors that facilitate C–H bond activation provides a basis for future ligand substitutions aimed at enhancing the reactivity of metal-alkane σ-complexes with enhanced efficiency of the activation step.

Solvation plays a key role in photoinduced charge transfer, a fundamental process in many photochemical and biological systems. However, direct experimental observation of the atomic-scale solvent reorganization around an organic molecule following electronic excitation has so far remained elusive. We report time-resolved X-ray scattering measurements of a photoexcited organic hemithioindigo chromophore in the polar solvent acetonitrile, a system containing only light atoms. Exploiting an ~120 fs time resolution and the high X-ray photon flux offered by an X-ray free-electron laser, the experiments capture the ultrafast dipolar rearrangement of the solvent molecules surrounding the solute driven by photoinduced charge transfer. The transient solvation shell structure is found to be formed with a ~0.5 ps time constant and to reequilibrate over ~3 ps as the excited state population returns to the ground state. Transient absorption spectroscopy measurements and molecular dynamics simulations support the interpretation of the structural dynamics. These results demonstrate the viability of using ultrashort X-ray pulses to directly visualize the motion of light atoms in solution, paving the way for atomic-scale understanding and control of photoinduced processes in biological and synthetic photoactive organic molecules.

Transient X-ray absorption spectroscopy at the sulfur K edge probes the ultrafast internal conversion and intersystem crossing of thiopyridone isomers in solution.