Fundamental studies of chemical reactions often draw molecular dynamics along a reaction coordinate in a calculated or suggested potential energy surface (PES) 1-5 . But fully mapping such dynamics experimentally, by following all nuclear motions in a timeresolved manner, that is the motions of wavepackets, is challenging and has not even been realized for the simple stereotypical bimolecular reaction 6-8 of A-B + C → A + B-C. Here we report such tracking of vibrational wavepacket trajectories during photo-induced bond formation in the gold trimer complex [Au(CN)2 -]3 in an aqueous solution, using femtosecond x-ray solution scattering (liquidography 9-12 ) at x-ray free electron lasers 13,14 . We find that the complex forms from an assembly of three monomers A, B and C clustered together through non-covalent interactions 15,16 and with the distance between A and B shorter than between B and C. Tracking of the wavepacket in three-dimensional nuclear coordinates (RAB, RBC, and RAC) reveals that within the first 60 fs after photoexcitation, a covalent bond forms between A and B to give A-B + C. The second covalent bond, between B and C, subsequently forms within 360 fs to give a linear and covalently-bonded trimer complex A-B-C. The trimer exhibits harmonic vibrations that we are also able to map, and unambiguously assign to specific normal modes using only the experimental data. More intense x-rays can in principle visualize the motion of not only highly-scattering atoms such as gold but also of lighter atoms such as carbon and nitrogen, which will open the door for the direct tracking of the atomic motions involved in many chemical reactions.The [Au(CN)2 -]3 complex has served as a valuable model system for studying photoinitiated processes in solution. Irradiation with ultraviolet light excites it from the ground state (S0) to the singlet state (S1), which within 20 fs undergoes intersystem crossing to reach a triplet excited state (T1') 18 . A further transition from T1' to another triplet excited state (T1) then occurs with a time constant of 1~2 ps, completing formation of covalent bonds and transformation of the complex from a bent to a linear structure 9,17,18 (see the Supplementary Information (SI) for details of the notations of electronic states).Formation of the bonds could involve any of the three possible candidate trajectories sketched in Fig. 1b. The equilibrium structure in the ground state determines the position of the

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.

The structural dynamics of in-cage isomerization of CH2I2 and the unusual structure of the loosely-bound isomer precursor were unveiled with femtosecond X-ray liquidography (solution scattering).

The quaternary transition between the relaxed (R) and tense (T) states of heme-binding proteins is a textbook example for the allosteric structural transition. Homodimeric hemoglobin (HbI) from Scapharca inaequivalvis is a useful model system for investigating the allosteric behavior because of the relatively simple quaternary structure. To understand the cooperative transition of HbI, wild-type and mutants of HbI have been studied by using time-resolved X-ray solution scattering (TRXSS), which is sensitive to the conformational changes. Herein, we review the structural dynamics of HbI investigated by TRXSS and compare the results of TRXSS with those of other techniques.

Small molecules such as molecular oxygen, nitric oxide, and carbon monoxide play important roles in life, and many proteins require the transport of small molecules to and from the bulk solvent for their function. Ligand migration within a protein molecule is expected to be closely related to the overall structural changes of the protein, but the detailed and quantitative connection remains elusive. For example, despite numerous studies, how occluded ligand migration affects the kinetics and structural dynamics of the R−T transition remains unclear. To shed light on this issue, we chose homodimeric hemoglobin (HbI) with the I114F mutation (I114F), which is known to interfere with ligand migration between the primary and secondary docking sites, and studied its kinetics and structural dynamics using time-resolved X-ray solution scattering. The kinetic analysis shows that I114F has three structurally distinct intermediates (I 1 , I 2 , and I 3 ) as in the wild type (WT), but its geminate CO recombination occurs directly from I 1 without the path via I 2 observed in WT. Moreover, the structural transitions, which involve ligand migration (the transitions from I 1 to I 2 and from I 3 to the initial state), are decelerated compared to WT. The structural analysis revealed that I114F involves generally smaller structural changes in all three intermediates compared to WT.

Salt bridge, one of the representative structural factors established by non-covalent interactions, plays a crucial role in stabilizing the structure and regulating the protein function, but its role in dynamic...

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