Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe³⁺-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H⁻ from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) P ^G –O ^3B bond cleavage; 3) four concomitant events: W1–PO ₃ ⁻ formation, OH ⁻ and proton cleavage, nucleophilic attack by the OH ⁻ against P ^G , and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Electrocatalytic carbon dioxide (CO ₂ ) reduction reaction (CO ₂ RR) has emerged as a promising strategy for sustainable energy conversion and carbon utilization. Despite intensive research efforts, the understanding of intermediates and pathways leading from CO ₂ RR to multicarbon (C ₂₊ ) chemicals remains incomplete. The challenge is to gain insight into the activation of adsorbed CO and the subsequent pathways. Here, we design a specially tailored Cu nanowire array facing a hydrophobic interface as an electrode to highly enhance Raman signals in the in situ environment, allowing sensitive observation of the sequential change of various elusive intermediates during CO ₂ RR, such as CO, CH ₂ , CO coexisting with CH ₂ , CH ₂ CO, and CH ₃ . Density functional theory calculations reveal that the C─C coupling during CO ₂ RR originates from an asymmetric coupling between CH ₂ and CO to form CH ₂ CO, identified as the rate‐determining step in the formation of C ₂₊ products. These findings deepen the understanding of the C─C coupling processes, which are crucial for advancing catalyst development in electrochemical CO ₂ upgrading.

κ-H₃(Cat-EDT-TTF)₂ (H-TTF) is a hydrogen-bonded π-electron system. Only its isotopologue, D-TTF, shows the phase transition. We obtained a symmetric single-well effective-PEC for H-TTF and low-barrier effective-PEC for D-TTF.

Although hydrogen plays a crucial role in ammonia synthesis, very little is known about its poisoning of Ru catalysts. In this study, density functional theory calculations of H₂ and N₂ dissociations, and H atom binding on Ru₁₅₃ were performed to provide a fundamental understanding of hydrogen poisoning. Because of the kinetic dominance of the H₂ dissociation over N₂ (vertically or horizontally adsorbed) splitting, the dissociated H atoms block the active sites required for horizontal (less energetically demanding dissociation) N₂ adsorption to occur either from the gas phase or after its geometrical transformation from being adsorbed vertically. Additionally, the dissociated H atoms withdraw electrons from the surface, which reduces the ability of the neighboring Ru atoms to donate electrons for N₂ activation, hindering its dissociation and suppressing ammonia synthesis.