Investigating three-dimensional structures of reaction intermediates in solution using time-resolved X-ray solution scattering

Abstract

To understand molecular properties, it is essential to investigate the molecular structure and related chemical reactions. Especially, a molecular structure evolves as a chemical reaction proceeds through metable states on the reaction coordinate called reaction intermediates. Three-dimensional structures of the reaction intermediates determine products of the reaction and the reaction pathway and thus structures of the intermediates can provide fundamental information to understand and control the reaction. Reaction dynamics of a number of chemical reactions have been investigated by various experimental techniques such as X-ray crystallography, electron diffraction, solution scattering, nuclear magnetic resonance (NMR), and spectroscopies, however, it is still challenging to elucidate reaction mechanisms with high spatiotemporal resolutions. Time-resolved X-ray solution scattering has been established recently and it is relevant for probing fast and subtle structural changes in solution. In a time-resolved X-ray solution scattering experiment, a chemical reaction is usually triggered by a laser pulse and subsequent structural changes of the molecule are detected by X-ray scattering signal as a function of time. X-ray scattering signal are dependent on positions of all constituent atoms in a molecule so that it can provide information on global structure of the molecule. It is complementary to spectroscopic techniques that are usually sensitive to structures and energies nearby chromophores. Time-resolved X-ray solution scattering can be applied to various molecules ranging from small molecules to large protein molecules in solution. We employed this technique to proteins such as homodimeric hemoglobin and hemoglobin which exhibit allosteric effects and myoglobin to investigate structural dynamics of them upon photodissociation of carbon monoxide ligands. We also applied the technique to a gold complex to visualize bond formation process. Furthermore, we investigated rotational dephasing process of the gold complex by monitoring evolution of orientational distribution of excited molecules and derived formula for calculating X-ray solution scattering intensity arising from an anisotropic orientational distribution of molecules. Hemoglobin acts as an oxygen carrier in the human body by binding biatomic ligands up to four ligands. Binding affinity of a ligand is altered by ligand-binding states of other subunits and this is called as the allosteric effect. Homodimeric hemoglobin is more convenient molecular system to study the allosteric effect since it has homodimeric structure, simpler than the heterotetrameric structure of hemoglobin. Time-resolved X-ray solution scattering was employed to investigate the allosteric structural transitions in homodimeric hemoglobin. The kinetic and structural analyses on the data revealed detailed structural parameters such as intersubunit rotation angle and heme-heme distance of three reaction intermediates as well as kinetic parameters related to transitions among the intermediates. We extended the study toward F97Y and T72V mutants to figure out effects of the heme movement and water cluster at the subunit interface on structural dynamics of homodimeric hemoglobin. Acceleration of overall reaction kinetics and attenuation of ligand-linked heme movement were observed for the F97Y mutant. We also found that intersubunit communication is increased in the T72V mutant. Along with homodimeric hemoglobin, hemoglobin and myoglobin were investigated as well with time-resolved X-ray solution scattering. From those experiments, reaction kinetics of hemoglobin was identified and tertiary structural information of the earliest myoglobin intermediate was extracted. As X-ray free electron lasers (XFEL) have operated, achievable temporal width of an X-ray pulse has been decreased down to 100 fs. As a result, experimental time resolution of time-resolved X-ray solution scattering has improved up to sub-picoseconds. By making use of femtosecond X-ray pulses generated at an XFEL, we observed the moment of bond formation in a gold trimer complex, $[Au(CN)_2^-]_3$, in solution. Bond formations between the adjacent gold atoms in the gold complex can be triggered by laser excitation and the bond formations proceed without limitations by slow diffusion. This is because the constituent gold atoms are gathered together in a solvent cage by a relativistic effect called aurophilicity. We monitored distances among the gold atoms after laser excitation. As a result, we observed the bond formation as well as a bent-to-linear structural transition within 500 fs. Further contraction of bond lengths and formation of a tetramer complex were observed as well. We also revealed rotational dephasing process of the gold complex by tracking evolution of transient anisotropy generated by a linearly polarized laser. X-ray solution scattering signal has relatively low information content compared to X-ray crystallography owing to random orientation of molecules in solution. One of the compensations for the information loss is to make anisotropic orientational distribution of excited molecules using a linearly polarized laser. By the linearly polarized laser, excited molecules in solution are photoselectively aligned so that arise an anisotropic X-ray scattering pattern. By changing the direction of laser polarization, we can obtain distinct anisotropic X-ray solution scattering patterns, indicating increase of information content. To interpret the experimental anisotropic X-ray solution scattering signal, a methodology for calculating X-ray scattering signal for the anisotropic orientational distribution is required. Furthermore, it is essential to analyze femtosecond X-ray solution scattering data since scattering signal measured at time delays earlier than a rotational dephasing process is not isotropic anymore. We derived a formula for calculating anisotropic X-ray scattering intensities and demonstrated expected amount of the information content.