Abstract
In the last ten years, x-ray free-electron lasers (XFELs) have been successfully employed to characterize metalloproteins at room temperature using various techniques including x-ray diffraction, scattering, and spectroscopy. The approach has been to outrun the radiation damage by using femtosecond (fs) x-ray pulses. An example of an important and damage sensitive active metal center is the Mn4CaO5 cluster in photosystem II (PS II), the catalytic site of photosynthetic water oxidation. The combination of serial femtosecond x-ray crystallography and Kβ x-ray emission spectroscopy (XES) has proven to be a powerful multimodal approach for simultaneously probing the overall protein structure and the electronic state of the Mn4CaO5 cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the Mn4CaO5 cluster are very subtle, it is critical to consider the potential effects of the intense XFEL pulses on the Kβ XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn Kβ1,3 XES spectra in PS II over a wide range of pulse parameters collected over seven different experimental runs using both microcrystal and solution PS II samples. Our findings show that for beam intensities ranging from ∼5 × 1015 to 5 × 1017 W/cm2 at a pulse length of ∼35 fs, the spectral effects are small compared to those observed between S-states in the Kok cycle. Our results provide a benchmark for other XFEL-based XES studies on metalloproteins, confirming the viability of this approach.
Highlights
X-ray spectroscopy is a powerful element-sensitive technique for probing the local structure of the active site in metalloproteins and model inorganic complexes.1–9 In metalloproteins, it has been widely used for the mechanistic understanding of the metal catalytic centers using x-rays at synchrotron facilities
The case study we report here provides important information regarding the choice of experimental conditions for the application of x-ray spectroscopy to metalloenzyme studies at x-ray free-electron lasers (XFELs), while understanding the effect of intense XFEL pulses to the x-ray emission spectroscopy data of these systems
Our analysis focuses on the solution experiments with the strongest (R1) and weakest (R3) XFEL beam intensities, and the microcrystal experiment with the strongest (R4) XFEL beam
Summary
X-ray spectroscopy is a powerful element-sensitive technique for probing the local structure of the active site in metalloproteins and model inorganic complexes. In metalloproteins, it has been widely used for the mechanistic understanding of the metal catalytic centers using x-rays at synchrotron facilities. X-ray spectroscopy is a powerful element-sensitive technique for probing the local structure of the active site in metalloproteins and model inorganic complexes.1–9 In metalloproteins, it has been widely used for the mechanistic understanding of the metal catalytic centers using x-rays at synchrotron facilities. One of the main challenges in the several decades of synchrotron-based x-ray spectroscopy and diffraction studies of the OEC has been the modification of the geometric and the electronic structures caused by Mn metal reduction and protein modification due to the synchrotron x-ray beams. These synchrotron radiation-induced damage processes are related to migration of radicals and other relatively slow processes in the range of $20 ps.. The problem of radiation damage has not been fully solved for synchrotron-based soft x-ray studies of the Mn L-edge (635–655 eV) of the OEC.
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