Probing chemical dynamics and catalytic mechanisms in metalloenzymes with advanced X-ray spectroscopy techniques - Project Summary/Abstract The goal of this proposal is to develop new methods for X-ray absorption spectroscopy (XAS) that can use the ultra-short, femtosecond pulses from X-ray free electron lasers (XFELs) to help us understand the mechanism of redox-active metalloenzymes by following the catalytic reaction, in real time, at room temperature. Unlike X-ray emission spectroscopy (XES), which has been used at XFELs, XAS is much more versatile for studying the electronic structure with X-ray near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) for determining metal-metal, and metal-ligand distances. Unlike crystallography, XAS can be conducted using solution samples and often yields higher resolution for specific metal-distances than X-ray diffraction. Nevertheless, XAS has so far seen only very limited use for room temperature time-resolved studies of metalloenzymes using XFELs. The challenges of XAS at XFELs arise from the requirement to scan a wide energy range of incident X-rays; significant shot-to-shot spectral, temporal, and intensity fluctuations in the pulses; and data collection of dilute metalloenzyme systems with limited sample volumes. While progress has been made overcoming these impediments, the most important issue that has not been addressed adequately until now is how to mitigate the contribution of background scattering, which leads to very poor S/N ratios. This is largely because the high count rate of photons at XFELs leads to pulse pile-up and does not allow us to use the energy discriminating, single-photon counting detectors that are usually employed in synchrotron facilities to discriminate the signal from background photons at XFELs. In this proposal, we will focus on the remaining obstacle to XAS at XFELs by minimizing background scattering through designing and building a robust and versatile spectrometer, with a large solid angle and energy resolution of ~10 eV, to discriminate between the background scatter and signal photons and capture the entire metal Ka fluorescence from the metal, which will be collected using a position-sensitive detector. This instrument will be tested using model compounds and then will be used to collect time-resolved XANES and EXAFS data from two important metalloenzymes: the Mn4Ca complex in photosystem II involved in the oxidation of water to oxygen, and the ribonucleotide reductase class of enzymes with binuclear Fe/Fe, Mn/Fe, or Mn/Mn active sites involved in the conversion of ribonucleotides to deoxy-ribonucleotides, the building blocks of DNA. In PS II, which will be triggered by light flashes, we will focus on the time-points in the S3 to S0 transition reached after illumination with three flashes, the last step in the catalytic cycle where the critical step of O-O formation occurs. The XANES and EXAFS data from these timepoints will be important for unravelling the mechanism of the photosynthetic water oxidation reaction. In RNR, which will be triggered by in situ O2 addition, we will focus on the intermediate state, known as ‘X’, which is transiently generated and whose exact structure is not known and has been the subject of much study.
