Electronic and Nuclear Structural Snapshots in Ligand Dissociation and Recombination Processes of Iron Porphyrin in Solution: A Combined Optical/X-ray Approach

The photodissociation and recombination of CO and 1-methylimidazole (Im) from iron protoporphyrin IX (FePP–ImCO) dissolved in a 30% v/v aqueous solution of Im was studied using ultrafast optical transient absorption (TA) and X-ray transient absorption (XTA) spectroscopies. FePP–ImCO was shown to lose the CO ligand upon excitation at the Q bands, with 3.8 ps vibrational cooling and 21.6 ps intersystem crossing time constants derived from optical TA experiments, followed by ligation of a second Im on the nanosecond time scale. The penta-coordinate FePP–Im intermediate which forms following CO dissociation adopts a square pyramidal geometry with a “domed” iron center that is reminiscent of that formed upon loss of CO from carbon­monoxy­myoglobin (MbCO). Unlike MbCO, which typically retains its newly generated penta-coordinated geometry until CO recombination, FePP can adopt a hexa-coordinate geometry by binding an additional Im ligand (FePP–(Im)₂), allowing the porphyrin to exist in the low-spin electronic state even without the CO attached. The second Im ligand remains bound until CO recombination occurs with a time constant of 283 μs. The photodissociated states of FePP–ImCO and MbCO 100 ps after photoexcitation have similar iron site geometries, implying that the protein matrix in MbCO maintains minimum potential energy in the heme center despite the large-scale reorganization in the protein secondary and tertiary structure that arises from the dynamic active site/matrix interaction

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX