Effects of x-ray free-electron laser pulse intensity on the Mn K<b><i>β</i></b><sub>1,3</sub> x-ray emission spectrum in photosystem II—A case study for metalloprotein crystals and solutions
Thomas Fransson, Roberto Alonso‐Mori, Ruchira Chatterjee, Mun Hon Cheah, Mohamed Ibrahim, Rana Hussein, Miao Zhang, Franklin D. Fuller, Sheraz Gul, In‐Sik Kim, Philipp S. Simon, Isabel Bogacz, Hiroki Makita, Casper de Lichtenberg, Sanghoon Song, A. Batyuk, Dimosthenis Sokaras, Ramzi Massad, Margaret Doyle, Alexander Britz, Clemens Weninger, Athina Zouni, Johannes Messinger, Vittal K. Yachandra, Junko Yano, Jan Kern, Uwe Bergmann
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 Mn 4 CaO 5 cluster in photosystem II (PS II), the catalytic site of photosynthetic water oxidation. The combination of serial femtosecond x-ray crystallography and Kb 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 Mn 4 CaO 5 cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the Mn 4 CaO 5 cluster are very subtle, it is critical to consider the potential effects of the intense XFEL pulses on the Kb XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn Kb 1,3 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 10 15 to 5 10 17 W/cm 2 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.