Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature

Abstract

Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.

Fig. 1

Setup of simultaneous X-ray spectroscopy and crystallography experiment at the CXI instrument of LCLS. The crystal suspension is electric-field focused into a microjet that intersects the X-ray pulses. XRD data from a single crystal are collected downstream with a CSPAD detector and XES data from the same crystal are collected at ~90° to the beam via a multi-crystal XES spectrometer and a compact position sensitive detector (PSD). A Nd-YLF laser (527 nm) is used to illuminate the crystals. The timing protocol (inset, left) consists of a fixed time of flight Δt between optical pump and X-ray probe. The schematic of the energy dispersive spectrometer, Mn^II and Mn^IV oxide Kβ_1,3 spectra, and energy level diagram for XES are shown (inset, bottom right).

Fig. 2

Structure deduced from diffraction of micron-sized crystals of PS II using sub 50 fs X-ray pulses at room temperature at LCLS. (A) 2mF_o-DF_c electron density map for the PS II complex in the dark S₁ state obtained at LCLS; one monomer of the protein is shown in yellow and the electron density is contoured at 1.2 σ (blue mesh) shown for a radius of 5 Å around the protein. (B) Detail of the same map in the area of the Mn₄CaO₅ cluster in the dark S₁ state, with mesh contoured at 1.0 σ grey) and 4.0 σ (blue). Selected residues from subunit D1 are labeled for orientation; Mn is shown as violet spheres, and Ca as an orange sphere (metal positions taken from pdb file 3bz1).

Fig. 3

Femtosecond XES of PS II. (A) The 2D Kβ_1,3 X-ray emission spectra from microcrystals of PS II collected with a position sensitive detector at the CXI instrument using sub 50 fs pulses of about 2-3×10¹¹ photons/(pulse μm²). (B) X-ray emission spectra of a solution of PS II (green) and single crystals of PS II (red dashed) in the dark state, both collected at the CXI instrument, obtained from the 2D plot in panel (A) by integration along the horizontal axis. (C) X-ray emission spectra of PS II solutions in the dark state collected at the CXI instrument at RT (green) or collected using SR under cryogenic conditions with low dose (“8K intact”, light blue) and using SR at RT under photo-reducing conditions (“RT damaged”, pink). The spectrum from Mn^IICl₂ in aqueous solution collected at RT at the CXI instrument is shown (grey) for comparison.

Fig. 4

Characterization of the illuminated PS II sample. (A) On-line MIMS measurements of light-induced O₂ yield detected as mixed labeled ¹⁶O¹⁸O species after illumination of photosystem II from T. elongatus. The data shows that >73% of the sample occupies the S₂ state after one illumination. (B) XFEL XES of PS II in the S₂ state. The Kβ_1,3 XES data collected from 362 micro crystals of PS II in the first illuminated S₂ state are shown in blue (*). The XFEL spectrum of microcrystals of PS II in the dark stable S₁ state is shown as a green line. For comparison an X-ray emission spectrum of completely photo-reduced (“damaged”) PS II collected at RT at a synchrotron is shown in pink. (C) Isomorphous difference map between the XFEL-illuminated (S₂ state) and the XFEL-dark (S₁ state) XRD dataset in the region of the Mn₄CaO₅ cluster, with F_o-F_o difference contours shown at +3 σ (green) and –3 σ (red); histogram analysis indicates that this map is statistically featureless (Fig. S6). Metal ions of the Mn₄CaO₅ cluster are shown for orientation as violet (Mn) and orange (Ca) spheres, subunits are indicated in yellow (D1), orange (D2), pink (CP43) and green (PsbO).

Scheme 1

Reaction cycle of water oxidation at the Mn₄CaO₅ cluster in PS II.