Fransson Thomas, Alonso-Mori Roberto, Chatterjee Ruchira, Cheah Mun Hon, Ibrahim Mohamed, Hussein Rana, Zhang Miao, Fuller Franklin, Gul Sheraz, Kim In-Sik, Simon Philipp S, Bogacz Isabel, Makita Hiroki, de Lichtenberg Casper, Song Sanghoon, Batyuk Alexander, Sokaras Dimosthenis, Massad Ramzi, Doyle Margaret, Britz Alexander, Weninger Clemens, Zouni Athina, Messinger Johannes, Yachandra Vittal K, Yano Junko, Kern Jan, Bergmann Uwe
Department of Theoretical Chemistry and Biology, KTH Royal Institute of Technology, Stockholm, Sweden.
Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.
Struct Dyn. 2021 Nov 22;8(6):064302. doi: 10.1063/4.0000130. eCollection 2021 Nov.
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 MnCaO 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 MnCaO cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the MnCaO 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 Kβ 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 × 10 to 5 × 10 W/cm 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.
在过去十年中，X射线自由电子激光（XFEL）已成功用于在室温下使用包括X射线衍射、散射和光谱学在内的各种技术对金属蛋白进行表征。其方法是利用飞秒（fs）X射线脉冲来克服辐射损伤。一个重要且对损伤敏感的活性金属中心的例子是光系统II（PS II）中的MnCaO簇，即光合水氧化的催化位点。串联飞秒X射线晶体学和Kβ X射线发射光谱（XES）的结合已被证明是一种强大的多模态方法，可在整个催化（Kok）循环中同时探测MnCaO簇的整体蛋白质结构和电子状态。由于在MnCaO簇中观察到的光谱变化非常细微，因此考虑强XFEL脉冲对Kβ XES信号的潜在影响至关重要。我们在此报告了一项系统研究，该研究使用微晶和溶液PS II样品，在七次不同的实验运行中收集的广泛脉冲参数范围内，研究了XFEL峰值功率、光束聚焦和剂量对PS II中Mn Kβ XES光谱的影响。我们的研究结果表明，对于脉冲长度约为35 fs、光束强度范围从~5×10到5×10 W/cm的情况，与在Kok循环中不同S态之间观察到的光谱效应相比，光谱效应较小。我们的结果为其他基于XFEL的金属蛋白XES研究提供了一个基准，证实了这种方法的可行性。
