Progress Toward Time-Resolved X-ray Spectroscopy of Metalloproteins

Metalloproteins, or proteins with a metal ion cofactor, are essential for biological function of both lower and higher level organisms. These proteins provide a multitude of functions from molecular transport, such as the hemoglobin transport of oxygen, to biologically important catalytic processes. As an example case, photosystem II (PSII) is studied as a representative metalloprotein. It was chosen based on the potential impact in the energy sector due to its ability to perform water oxidation using solar based energy. Understanding mechanisms by which the Mn₄Ca cluster inside PSII, also known as the oxygen evolving complex (OEC), can store energy as redox equivalents for splitting water will be essential for future development of analogous artificial systems. By using time resolved x-ray spectroscopy, the electron structure of the metal in the protein was probed through the catalytic cycle. While the applications mentioned herein are based on PSII from spinach, the developments in time-resolved x-ray spectroscopy techniques are also applicable to other metalloproteins.

By creating a new x-ray spectrometer we were able to capture the difference in x-ray emission spectra between two compounds differing in a single metal bound ligand, i.e. Mn^IV-OH and Mn^IV=O. This both establishes the functionality of the x-ray emission spectrometer and provides useful insight into the expected changes upon an oxygen double bond formation. This change in spectroscopic signal is discussed in context of the OEC which has been hypothesized to form a Mn^IV=O state.

A new sample delivery system and further developments to the x-ray spectrometer enabled both time-resolved x-ray absorption and time-resolved x-ray emission of PSII. These experiments show the potential of synchrotron sources for time-resolved x-ray spectroscopy. From our x-ray absorption measurements we were able to follow the electronic structure changes in time using a single incident photon energy. From the kinetic traces obtained, we show possible alternative interpretations of previous results showing a delay in reduction during the final step in water oxidation. From the x-ray emission spectroscopy (XES) measurements of PSII we were able to reproduce previous results within a limited collection time and give estimates for data size requirements for metalloproteins using this spectrometer. Between the results of both these measurements, we show the improved capability for time resolved measurements at synchrotrons.

The development of x-ray free electron lasers (XFELs) has also opened many opportunities for understanding faster electronic dynamics by providing femtosecond x-ray pulse durations with ~10¹² photons per pulse. While theoretical modeling of distortions to crystallographic data have been performed, little to no work has been done to understand under what conditions such an intense pulse will have on an impact on emission spectra. Here an atomistic model was developed, and data collected, to clarify the effects of sequential ionization, i.e. two single photons absorbed by the same atom at different times during a single pulse. Experimentally we found that XFELs easily achieve flux densities that invoke a different response than is classically observed for single photon absorption and emission for Mn^II which was used as a representative case for 3d transition metals in general. We also give parameters by which the onset of this damage can be predicted and an approximation to its effect on 3d transition metals. Additionally this work guides the work of future XFEL facilities as it shows that shorter pulses, currently believed to be able to escape x-ray induced distortions to crystallography data, is not a viable method for overcoming changes in x-ray emission spectra.