One of the most fundamental questions in biology, chemistry, and physics is how the molecules interact, so called, reaction mechanisms. Our laboratory strives to understand the reaction mechanisms in molecular detail in gas phase, solution phase, and solid phase spanning amorphous phase, polycrystalline and single crystals. Traditionally, femtosecond spectroscopy has been used to achieve this goal, however, spectroscopic data, in most cases, fail to provide direct information on the structural changes such as bond lengths and bond angles. To remedy this, we combine the traditional femtoscience with direct structural tools such as diffraction (scattering and crystallography), EXAFS, and NMR. These techniques can be applied to a wide range of systems, encompassing small molecules, nano-scale complexes, and macromolecules such as polymers, proteins and DNA. A typical experiment is conducted in a pump-probe manner; an optical pulse such as femtosecond laser pulse is directed to the sample of interest to initiate a desired reaction, and after a well-defined time delay, a probing pulse such as an ultrashort x-ray pulse is sent to the sample undergoing a reaction. Then, the signal resulted from the interaction of the reacting system and the probing pulse captures the molecular actions in real time. Since the measured signal (in this case, diffraction signal) is a function of molecular structures, the time-dependant data at various time delays contains a clue to the molecular reaction mechanisms and a novel data analysis of the time-resolved signal finally reveals the mechanism. 

Molecular Dynamics of Small Molecules, Nano-Scale Molecules and Macromolecules in Solution: Time-Resolved X-ray Liquidography (Solution Scattering)

Third-generation synchrotron radiation facility such as European Synchrotron Radiation Facility (ESRF) and Advanced Photon Source (APS) can generate an X-ray pulse as short as 100 picoseconds. With the advent of X-ray free electron lasers (XFELs) such as PAL-XFEL, SACLA and LCLS, even femtosecond X-ray pulses are now available, opening up a new possibility of capturing the movement of molecules in real time. Our laboratory conducts time-resolved experiments by utilizing such ultrashort X-ray pulses as a probe. Of great interest is the molecular structure of short-lived intermediates and solute-solvent interactions in solution phase. X-ray has a much longer penetration depth than electron does, and this characteristic makes X-ray suited for solution studies. A series of successful time-resolved X-ray diffraction experiments have been conducted on reactions of various small molecules and macromolecules such as nano-scale complexes and proteins.

Time-Resolved Spectroscopy: Femtosecond Transient Absorption Spectroscopy, 2-Dimensional Electronic Coherent Spectroscopy, Nanosecond Transient Absorption Spectrosocpy and Nanosecond Transient Grating Spectroscopy

Along with time-resolved X-ray/electron diffraction, our laboratory conducts a variety of time-resolved optical spectroscopy experiments such as femtosecond transient absorption spectroscopy, 2-dimensional electronic coherent spectroscopy, nanosecond transient absorption spectroscopy and nanosecond transient grating spectroscopy. Porous materials such as COFs and MOFs as well as small molecules and proteins have been studied by utilizing these tools. 

Protein Structural Dynamics: Time-Resolved X-ray Crystallography

Our laboratory is interested in the reaction mechanisms of various proteins such as a blue light photoreceptor, bacteriorhodopsin, phytochrome, and LOV2 domain. These proteins play indispensible roles in the signal transduction pathway of a cellular system. The time-resolved X-ray Laue diffraction on a single crystal is currently the only method which can provide us detailed real-time structural information in sub-nanosecond time resolution at room temperature.The experiment is conducted at a third generation synchrotron facility such as ESRF and APS as the time-resolved X-ray liquid experiment. The difference is that the long-range order found in a single crystal enhances the diffraction signal and reduce the background significantly. Diffuse signal from disordered molecules becomes sharp peaks. Generally, a ns or fs laser pulse triggers the protein dynamics in a single crystal of proteins and a polychromatic, ultrashort X-ray pulse from a beamline of a synchrotron radiation facility sweeps through the single crystal. The resulting diffraction pattern is then recorded in a charge-coupled device (CCD) based detection system. Typically a better time resolution can be achieved when the single crystal is kept still and a polychromatic X-ray beam is used for diffraction unlike a traditional X-ray crystallographic experiment where a diffraction pattern of a monochromatic X-ray beam is measured on an oscillating crystal. However, our laboratory is also developing a new technique of achieving a better time resolution with the traditional monochromatic X-ray pulses so that a wider range of protein reactions can be studied.

Ab-Initio, DFT Quantum Chemical Calculation, and Molecular Dynamics Simulation

Modern scientific advances have confirmed that experiments alone cannot provide a full story and convincing explanations on complex reaction mechanisms. Therefore, all our experimental endeavors accompany theoretical calculations including both ab-initio and DFT quantum chemical calculations and molecular dynamics simulations. Selected examples are molecular structures of short-lived intermediates, the photo-reaction of solute molecules in solution, and reaction pathway of proteins. 

Time-Resolved Electron Diffraction

With the advance of fs laser technology and electron gun design, the time resolution of ultrafast electron diffraction has reached near 1 ps. This impressive time resolution has enabled us to capture the molecular structure of short-lived intermediates. However, the fundamental bond-making and bond-breaking processes occur in femtosecond regime, which is beyond the current technology. Our laboratory is developing a next-generation ultrafast electron diffraction technique, which is femtosecond electron diffraction (FED). This new technique is based on a light-driven RF electron gun producing a near-relativistic electron pulse. Upon realization of this new technique, a molecular vibration will be directly captured, fulfilling one of chemists' dreams. 

Research Highlights

Light-induced protein structural dynamics in bacterio- phytochrome revealed by time-resolved x-ray solution scattering

Sci. Adv., 8, eabm6278 (2022)

Professor Ihee and his research group elucidated the structural dynamics of bacteriophytochrome, a type of photosensitive protein, using time-resolved X-ray liquidography (TRXL). In the TRXL experiment, the photoresponse of the protein was initiated by light and sequent structural changes were monitored by time-resolved X-ray scattering signals. We extracted the structural information of intermediates occuring in the process of the photoreaction by utilizing a structural modeling technique aided by molecular dynamics simulations. In this study, we confirmed that the subunits of bacteriophytochrome are arranged in a linear and horizontal conformation in the ground state. Upon the exposure to infrared light, these subunits gradually undergo conformational changes, eventually forming an “O”-shaped structure. Based on the structural dynamics information of bacteriophytochrome, we could explain why the activity of bacteriophytochrome increases after receiving infrared light compared to its pre-exposure state.

Determining the charge distribution and the direction of bond cleavage with femtosecond anisotropic x-ray liquidography

Nat. Commun., 13, 522 (2022)

Professor Ihee and his research group revealed the charge distribution within molecules in solution using femtosecond time-resolved X-ray liquidography (fs-TRXL) with X-ray free electron lasers (XFELs). In this work, we analyzed the X-ray scattering signal arising from the correlation between solute and solvent molecules in order to identify the arrangement of the solvent molecules around the solute. Based on this information, we determined the charge distribution within the solute molecule with the aid of molecular dynamics simulations. Furthermore, we determined the correlation between the charge distribution and the dissociation properties of the molecules. We investigated the charge distribution and reaction dynamics of a triiodide ion (I3-). Using fs-TRXL, we determined the charge distribution of I3- in solution and elucidated the direction of bond cleavage upon the photoexcitation, thereby elucidating the relationship between the charge distribution and corresponding reaction pathway.

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering

Nat. Commun., 12, 3677 (2021)

The movement of molecules occurring in the ultrafast time domain is one of the significant factors determining the reaction pathways of molecules. However, studies on the ultrafast molecular motions have predominantly focused on small molecules, and research on macromolecules such as proteins remains rare due to experimental difficulties and challenges in analyzing the structure of these large molecules. Professor Ihee and his research group succeeded in revealing ultrafast structural change of homodimeric hemoglobin (HbI) protein using femtosecond time-resolved X-ray liquidography (fs-TRXL). In this study, we conducted research on the movement of proteins in the ultrafast time domain. As a result, we successfully elucidated the specific movements of Hb. We observed the coherent motion of HbI involved in the protein quake process as well as the rearrangement of helices. Furthermore, by utilizing the information in the small-angle X-ray scattering region, we successfully unveiled not only the protein but also the temporal variations in the hydration shell.

Optical Kerr Effect of Liquid Acetonitrile Probed by Femtosecond Time-Resolved X-ray Liquidography

J. Am. Chem. Soc., 143, 14261–14273 (2021)

Professor Ihee and his research group utilized femtosecond time-resolved X-ray liquidography (fs-TRXL) to study the ultrafast dynamics of molecular liquids, Optical Kerr effect (OKE). With fs-TRXL, we monitored the microscopic structural motions related to the OKE response. By applying fs-TRXL to acetonitrile and a dye solution in acetonitrile, different types of molecular motions around photoaligned molecules could be resolved selectively, based on the anisotropy of two-dimensional scattering patterns as well as the additional structural information manifested in the scattering data. As a result, different types of reorientational (libration and orientational diffusion) and translational (interaction-induced motion) motions are captured separately through anisotropic and isotropic scattering signals, respectively. Excellent agreement between the measured and simulated scattering signals obtained from molecular dynamics simulations, provides a detailed structural description of the OKE response in liquid acetonitrile, without relying on theoretical modeling.

Filming ultrafast roaming-mediated isomerization of bismuth triiodide in solution 

Nat. Commun., 12, 4732 (2021)

Isomerization and dissociation processes of molecules play a crucial role in chemical reactions. Despite numerous studies on the products and reaction kinetics of these chemical reactions, direct observation of the molecular motion involved in these reactions has been exceedingly rare. Professor Ihee and his research group investigated the photoisomerization and photodissociation reactions of bismuth triiodide (BiI3) using femtosecond X-ray liquidography (fs-TRXL) to directly observe the molecular motion associated with the recently proposed roaming reaction, which is one of the major mechanisms driving the photoisomerization. With the operation of PAL-XFEL in Pohang, the temporal resolution was significantly improved, enabling us to successfully obtain the experimental data arising from the ultrafast structural change during the roaming reaction. The analysis on the data revealed important chemical processes accompanied in the photoisomerization such as bond formation, dissociation, and motion of the dissociated iodine atom.

Mapping the emergence of molecular vibrations mediating bond formation

Nature 582 (2020) 520–524

Professor Ihee and his research group achieved the visualization of wavepacket motions, the fully mapped time-resolved all-nuclear motions, in a gold trimer complex, [Au(CN)2]3, with atomic resolution using femtosecond X-ray liquidography at an X-ray free electron laser (XFEL). This groundbreaking research surpassed prior achievements, the direct observation of chemical bond formation in chemical reactions in solution phase, and served as a remarkable demonstration of the direct tracking of the atomic motions via femtosecond X-ray liquidography.

Direct observation of bond formation in solution with femtosecond X-ray liquidography 

Nature 518 (2015) 385389; J. Phys. B: At. Mol. Opt. Phys. 48 (2015) 244005; Struct. Dyn. 3 (2016) 043209 

Professor Ihee and his research group visualized the real-time progress of bond formation in a gold trimer complex, [Au(CN)2]3, with atomic resolution using femtosecond X-ray liquidography at an X-ray free electron laser (XFEL). The making and breaking of chemical bonds are essential processes in chemical reactions, and the ultrafast dynamics of bond breaking processes have been well studied using time-resolved techniques. However, it is extremely difficult to study the dynamics of bond making because the bond making is a bimolecular process and thus is limited by slow diffusion in solution phase. This work was the first example of directly observing the structural dynamics of bond formation process in solution phase and demonstrated the full power of femtosecond X-ray liquidography equipped with femtosecond time resolution and atomic-level structural resolution. 

Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography  

Nat. Chem. 5 (2013) 212; Nat. Chem. 6 (2014) 259 

Professor Ihee and his research group revealed the atomic-level structural changes involved in the signaling process of photoactive yellow protein using time-resolved X-ray crystallography. From time-resolved X-ray crystallography measurement of the isomerization process in photoactive yellow protein, the Ihee group revealed that the early stage of the isomerization on the nanosecond time scale proceeds via bifurcated pathways with different chromophore structures. This result was highly valued because it provided the atomic-level structural change of the chromophore, which cannot be probed by time-resolved spectroscopy, with the progress of the protein structural transition.

Protein structural dynamics in solution revealed by pump-probe X-ray protein liquidography and time-resolved X-ray Laue crystallography; Hemoglobin, Myoglobin, and Photoactive yellow protein 

PNAS 102 (2005) 7145; Nat. Methods 5 (2008) 881; J. Phys. Chem. B 113 (2009) 13133; Chem. Commun. 47 (2010) 289; J. Phys. Chem. Lett. 2 (2011) 350 

Professor Ihee demonstrates tracking of protein structural changes with pump-probe X-ray protein liquidography with picosecond time resolution. We investigated the tertiary and quaternary conformational changes of hemoglobin and myoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. By providing insights into the structural dynamics of proteins functioning in their natural environment, pump-probe X-ray protein liquidography complements and extends results obtained with time-resolved optical spectroscopy and X-ray Laue crystallography. We also identify a complex chemical mechanism and all atomic structures of five distinct structural intermediates of the blue light photoreceptor photoactive yellow protein (PYP) using time-resolved X-ray Laue crystallography.

Revealing an unknown reaction pathway of photocatalyst; Ru3(CO)12 

Angew. Chem. Int. Ed. 47 (2008) 5550; J. Am. Chem. Soc. 132 (2010) 2600 

TRXL was used to probe the photolysis of Ru3(CO)12 in cyclohexane, and a new intermediate (Ru3(CO)10 with terminal CO only) was identified besides the two μ-CO intermediates known from ultrafast IR spectroscopy. Three intermediates, Ru3(CO)10 with terminal CO only, Ru3(CO)11(μ-CO) and Ru3(CO)10(μ-CO), undergo different photodissociation pathways at 260 nm and 390 nm.

The reaction mechanism of metathesis probed by time-dependent fluorescence quenching; Metal-based catalysts 

J. Am. Chem. Soc. 130 (2008) 16506; J. Am. Chem. Soc. 132 (2008) 12027 

Quantitative catalyst-substrate association relationships of diverse metathesis Mo and Ru catalysts to their substrates are determined directly by a general method based on FRET principle. The determined substrate preferences exhibit the order of alkyne > alkene > allene for Schrock or Schrock-Hoveyda Mo catalysts, allene > alkene > alkyne for Grubbs or Crubbs-Hoveyda 1st generation Ru catalysts, and alkyne > allene > alkene for Grubbs or Grubbs-Hoveyda 2nd generation Ru catalysts.

Determining molecular structures of reaction intermediates in solution: Capturing the bridged structure of C2H4I 

Science 309 (2005) 1223; J. Phys. Chem. A 109 (2005) 10451; J. Chem. Phys. 124 (2006) 124504

Professor Ihee and his research group have used time-resolved X-ray Liquidography (TRXL) to identify the intermediates of the elimination reaction of haloethane (C2H4I2). Upon irradiation at 267 nm, haloethane dissociates into a haloethyl radical and an iodine atom. Of particular interest is the molecular structure of the haloethyl radical because a bridged structure rather than open classical structure (anti and gauche) had been proposed to explain the observed stereoselectivity of certain chemical processes but had never been directly observed. According to the TRXL result, the bridged structure is strongly favored rather than open classical structure and is also predicted by quantum mechanical calculation.

Publications (Selected)