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 consider the molecular dynamics along a reaction coordinate using a calculated or suggested potential energy surface^1,2,3,4,5. But fully mapping such dynamics experimentally, by following all nuclear motions in a time-resolved manner—that is, the motions of wavepackets—is challenging and has not yet been realized even for the simple stereotypical bimolecular reaction^6,7,8: A–B + C → A + B–C. Here we track the trajectories of these vibrational wavepackets during photoinduced bond formation of the gold trimer complex [Au(CN)₂⁻]₃ in an aqueous monomer solution, using femtosecond X-ray liquidography^9,10,11,12 with X-ray free-electron lasers^13,14. In the complex, which forms when three monomers A, B and C cluster together through non-covalent interactions^15,16, the distance between A and B is shorter than that between B and C. Tracking the wavepacket in three-dimensional nuclear coordinates reveals that within the first 60 femtoseconds 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 femtoseconds to give a linear and covalently bonded trimer complex A–B–C. The trimer exhibits harmonic vibrations that we map and unambiguously assign to specific normal modes using only the experimental data. In principle, more intense X-rays could visualize the motion not only of highly scattering atoms such as gold but also of lighter atoms such as carbon and nitrogen, which will open the door to the direct tracking of the atomic motions involved in many chemical reactions.

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.

Ionic species often play important roles in chemical reactions occurring in water and other solvents, but it has been elusive to determine the solvent-dependent molecular structure with atomic resolution. The triiodide ion has a molecular structure that sensitively changes depending on the type of solvent and its symmetry can be broken by strong solute-solvent interaction. Here, by applying pump-probe x-ray solution scattering, we characterize the exact molecular structure of $\mathrm{I}_3{}^{-}$ ion in water, methanol, and acetonitrile with subangstrom accuracy. The data reveal that $\mathrm{I}_3{}^{-}$ ion has an asymmetric and bent structure in water. In contrast, the ion keeps its symmetry in acetonitrile, while the symmetry breaking occurs to a lesser extent in methanol than in water. The symmetry breaking of $\mathrm{I}_3{}^{-}$ ion is reproduced by density functional theory calculations using 34 explicit water molecules, confirming that the origin of the symmetry breaking is the hydrogen-bonding interaction between the solute and solvent molecules.