The making and breaking of atomic bonds are essential processes in chemical reactions. Although the ultrafast dynamics of bond breaking have been studied intensively using time-resolved techniques, it is very difficult to study the structural dynamics of bond making, mainly because of its bimolecular nature. It is especially difficult to initiate and follow diffusion-limited bond formation in solution with ultrahigh time resolution. Here we use femtosecond time-resolved X-ray solution scattering to visualize the formation of a gold trimer complex, [Au(CN)2(-)]3 in real time without the limitation imposed by slow diffusion. This photoexcited gold trimer, which has weakly bound gold atoms in the ground state, undergoes a sequence of structural changes, and our experiments probe the dynamics of individual reaction steps, including covalent bond formation, the bent-to-linear transition, bond contraction and tetramer formation with a time resolution of ∼500 femtoseconds. We also determined the three-dimensional structures of reaction intermediates with sub-ångström spatial resolution. This work demonstrates that it is possible to track in detail and in real time the structural changes that occur during a chemical reaction in solution using X-ray free-electron lasers and advanced analysis of time-resolved solution scattering data.

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I1, I2, and I3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I1 intermediate is generated within 100 ps and transforms to the R-like I2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I3 intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.

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

Here we report structural dynamics of equine myoglobin (Mb) in response to the CO photodissociation visualized by picosecond time-resolved X-ray solution scattering. The data clearly reveals new structural dynamics that occurs in the timescale of ~360 picoseconds (ps) and 9 nanoseconds (ns), which have not been clearly detected in previous studies.Myoglobin (Mb) is a heme protein that carries small-molecule ligands such as O 2 , CO and NO in muscles, and can be considered as a subunit of hemoglobin, a paradigm protein for the study of allostery. Due to its small size, availability and photosensitivity of the hemeligand bond, Mb has served as a prototypical model system for studying protein structural dynamics. Accordingly, structural dynamics of Mb have been intensively studied with various spectroscopic 1-8 and structural 9-16 tools. The ligands form covalent bonds with Fe 2+ of the heme group and can be photolyzed by visible light on sub-picosecond time scale. [2][3] Upon the CO photolysis of MbCO, a small portion of the dissociated CO ligands geminately rebind to the heme, while the remainder travels to various pockets that can accommodate the ligand and eventually escapes the protein matrix to the solvent. On a longer time scale, the vacant heme recovers the ligand via non-geminate recombination.To directly track the structural changes associated with the ligand migration and rebinding and capture structurally distinct intermediates, we used pump-probe time-resolved X-ray solution scattering technique, where the time-dependent scattering of short X-ray pulses from a synchrotron are used to interrogate the structural dynamics of a liquid solution sample that is pumped with optical laser pulses in a pump-probe manner. Time-resolved Xray solution scattering [17][18][19] together with time-resolved X-ray crystallography 20 , X-ray absorption spectroscopy 21 and electron diffraction 21 can provide direct structural information, and thus complements time-resolved optical spectroscopy in the analysis of solution-phase reaction mechanisms. Recently time-resolved solution scattering technique has been applied to follow conformational changes in proteins with nanosecond 22-24 and picosecond 25 time resolution. Here, we show its application to another type of protein, Mb from equine heart, with picosecond time resolution.Time-resolved X-ray solution scattering data were measured at 14IDB beamline of Advanced Photon Source. The usual experimental protocol 22, 25 was followed. Specifically,

Intramolecular charge transfer (ICT) in a multibranched push–pull chromophore is a key photophysical process which is attracting attention due to its relevance to the development of highly efficient organic light-emitting diodes, but the excited-state dynamics of multibranched push–pull chromophores is still unclear. Here, using femtosecond transient absorption spectroscopy and singular value decomposition analysis, we studied the excited state dynamics of 2,6-bis­(diphenylamino)­anthraquinone (DPA-AQ-DPA), which contains two diphenylamino (DPA) groups as electron-donors (D) and anthraquinone (AQ) as an electron-acceptor (A) and is a candidate for an efficient red TADF (thermally activated delayed fluorescence) emitter. The emission of DPA-AQ-DPA exhibits large Stokes shifts with increasing solvent polarity, indicating that the emission can be attributed to an ICT process. The charge separated (CS) state formed by ICT undergoes torsional dynamics, involving twisting between D and A, resulting in the formation of a twisted charge separated state (CStwisting). This twisting reaction between D and A is accelerated in high-polarity solvents compared with that in low-polarity solvents. Such faster CT-induced torsional dynamics in high-polarity solvents is explained in terms of the localization of ICT on one of two ICT branches, suggesting that DPA-AQ-DPA in localized CStwisting formed in high-polarity solvents has two different dihedral angles between a single A group and two D groups. On the other hand, with increasing solvent polarity, the CS and CStwisting states of DPA-AQ-DPA become stabilized, making their energy levels considerably lower than that of 3(π,π*), consequently blocking the formation of the triplet excited state and TADF in a high-polarity solvent such as acetonitrile. By contrast, the energy levels of CS and CStwisting states in a low-polarity solvent, such as diethyl ether, are higher than that of 3(π,π*), allowing for deactivation into 3DPA-AQ-DPA* through intersystem crossing. This result indicates that the energy levels of CS and CStwisting states can be adjusted by controlling aspects of the local environment, such as solvents, so that intersystem crossing can be either inhibited or promoted. In other words, the energy gap (ΔE ST) between the lowest singlet and triplet excited states for DPA-AQ-DPA can be regulated by changing the solvent polarity.

Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.

Time-resolved X-ray diffraction provides direct information on three-dimensional structures of reacting molecules and thus can be used to elucidate structural dynamics of chemical and biological reactions. In this review, we discuss time-resolved X-ray diffraction on small molecules and proteins with particular emphasis on its application to crystalline (crystallography) and liquid-solution (liquidography) samples. Time-resolved X-ray diffraction has been used to study picosecond and slower dynamics at synchrotrons and can now access even femtosecond dynamics with the recent arrival of X-ray free-electron lasers.

The [Au(CN)2−]3 trimer in water experiences a strong van der Waals interaction between the d10 gold atoms due to large relativistic effect and can serve as an excellent model system to study the bond formation process in real time. The trimer in the ground state (S0) exists as a bent structure without the covalent bond between the gold atoms, and upon the laser excitation, one electron in the antibonding orbital goes to the bonding orbital, thereby inducing the formation of a covalent bond between gold atoms. This process has been studied by various time-resolved techniques, and most of the interpretation on the structure and dynamics converge except that the structure of the first intermediate (S1) has been debated due to different interpretations between femtosecond optical spectroscopy and femtosecond X-ray solution scattering. Recently, the excitation wavelength of 267 nm employed in our previous scattering experiment was suggested as the culprit for misinterpretation. Here, we revisited this issue by performing femtosecond X-ray solution scattering with 310 nm excitation and compared the results with our previous study employing 267 nm excitation. The data show that a linear S1 structure is formed within 500 fs regardless of excitation wavelength and the structural dynamics observed at both excitation wavelengths are identical to each other within experimental errors.