X-ray Emission Spectroscopy as an in Situ Diagnostic Tool for X-ray Crystallography of Metalloproteins Using an X-ray Free-Electron Laser

Abstract

Serial femtosecond crystallography (SFX) using the ultra-short X-ray pulses from a X-ray free-electron laser (XFEL) provides a new way of collecting structural data at room temperature that allows for following the reaction in real time after initiation. XFEL experiments are conducted in a shot-by-shot mode as the sample is destroyed and replenished after each X-ray pulse – monitoring and controlling the data quality by using in situ diagnostic tools is thus critical. To study metalloenzymes, we developed the use of simultaneous collection of X-ray diffraction of crystals along with X-ray emission spectroscopy (XES) data that is used as a diagnostic tool for crystallography, by monitoring the chemical state of the metal catalytic center. We have optimized data analysis methods and sample delivery techniques for fast and active feedback to ensure the quality of each batch of samples and the turnover of the catalytic reaction caused by reaction triggering methods. Here, we describe this active in situ feedback system using Photosystem II as an example that catalyzes the oxidation of H2O to O2 at the Mn4CaO5 active site. We used the first moments of the Mn Kβ1,3 emission spectra, highly sensitive to the oxidation state of Mn, as the primary diagnostics. This approach is applicable to different metalloproteins to determine the integrity of samples and follow changes in the chemical states of the reaction that can be initiated by light or activated by substrates, and offers a metric for determining the diffraction images that are used for the final datasets.

Untangling the sequence of events during the S 2 → S 3 transition in photosystem II and implications for the water oxidation mechanism

“…SFX data were collected using a Rayonix MX340-XFEL or an octal MPCCD detector. XES signal was collected by means of an 16 analyzer crystal array in van Hamos geometry together with an ePIX 100 or a single module MPCCD detector (75). SFX data processing used the cctbx.xfel software framework (41,76).…”

Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

“…Improved resolution approaching 2 Å unambiguously reveals oxygen and metal atomic positions in the catalytic center in each state. Kβ 1,3 X‐ray emission spectra (XES) were simultaneously collected from crystals to provide confirmation of catalytic advancement by probing the changes in the oxidation state of the metal cluster (Kern et al , , , Fuller et al , Fransson et al ), together with the oxidation state changes of the mobile quinone Q B site, evidenced by its electron density changes. Availability of the structural data for each stable intermediate state now enables us to reexamine previous X‐ray spectroscopy data based on the new structural information.…”

Rationalizing the Geometries of the Water Oxidising Complex in the Atomic Resolution, Nominal S3 State Crystal Structures of Photosystem II

“…The low apparent OEC Mn oxidation state change/turnover in their PSII samples, as seen by Mn K β XES, has been explained by Yachandra et al. (e. g. see) as a consequence of the Mn oxidation states being ‘delocalised’ throughout the OEC cluster, compared to model complexes. Their analysis in fact assumed an effective shift of −0.06 eV/(unit redox change), for the OEC, compared to a value of −0.11 eV/(unit redox change), expected from the Berkeley correlation in SI, Figure S1.…”