X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kß emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Performance of quartz- and sapphire-based double-crystal high-resolution (∼10 meV) RIXS monochromators under varying power loads.

In the context of a novel, high-resolution resonant inelastic X-ray scattering spectrometer, a flat-crystal-based quartz analyzer system has recently been demonstrated to provide an unprecedented intrinsic-energy resolution of 3.9 meV at the Ir L3 absorption edge (11.215 keV) [Kim et al. (2018) Sci. Rep. 8, 1958]. However, the overall instrument resolution was limited to 9.7 meV because of an 8.9 meV incident band pass, generated by the available high-resolution four-bounce Si(844) monochromator. In order to better match the potent resolving power of the novel analyzer with the energy band pass of the incident beam, a quartz(309)-based double-bounce, high-resolution monochromator was designed and implemented, expected to yield an overall instrument resolution of 6.0 meV. The choice of lower-symmetry quartz is very attractive because of its wealth of suitable near-backscattering reflections. However, it was found that during room-temperature operation typical levels of incident power, barely affecting the Si monochromator, caused substantial thermal distortions in the first crystal of the quartz monochromator, rendering it practically unusable. Finite-element analyses and heat-flow analyses corroborate this finding. As a high-flux, lower resolution (15.8 meV) alternative, a two-bounce sapphire(078) version was also tested and found to be less affected than quartz, but notably more than silicon.

X-ray Emission Spectroscopy of Biomimetic Mn Coordination Complexes.

Understanding the function of Mn ions in biological and chemical redox catalysis requires precise knowledge of their electronic structure. X-ray emission spectroscopy (XES) is an emerging technique with a growing application to biological and biomimetic systems. Here, we report an improved, cost-effective spectrometer used to analyze two biomimetic coordination compounds, [MnIV(OH)2(Me2EBC)]2+ and [MnIV(O)(OH)(Me2EBC)]+, the second of which contains a key MnIV═O structural fragment. Despite having the same formal oxidation state (MnIV) and tetradentate ligands, XES spectra from these two compounds demonstrate different electronic structures. Experimental measurements and DFT calculations yield different localized spin densities for the two complexes resulting from MnIV-OH conversion to MnIV═O. The relevance of the observed spectroscopic changes is discussed for applications in analyzing complex biological systems such as photosystem II. A model of the S3 intermediate state of photosystem II containing a MnIV═O fragment is compared to recent time-resolved X-ray diffraction data of the same state.

X-ray Spectroscopic Characterization of Co(IV) and Metal-Metal Interactions in Co4O4: Electronic Structure Contributions to the Formation of High-Valent States Relevant to the Oxygen Evolution Reaction.

The formation of high-valent states is a key factor in making highly active transition-metal-based catalysts of the oxygen evolution reaction (OER). These high oxidation states will be strongly influenced by the local geometric and electronic structures of the metal ion, which are difficult to study due to spectroscopically active and complex backgrounds, short lifetimes, and limited concentrations. Here, we use a wide range of complementary X-ray spectroscopies coupled to DFT calculations to study Co(III)4O4 cubanes and their first oxidized derivatives, which provide insight into the high-valent Co(IV) centers responsible for the activity of molecular and heterogeneous OER catalysts. The combination of X-ray absorption and 1s3p resonant inelastic X-ray scattering (Kß RIXS) allows Co(IV) to be isolated and studied against a spectroscopically active Co(III) background. Co K- and L-edge X-ray absorption data allow for a detailed characterization of the 3d-manifold of effectively localized Co(IV) centers and provide a direct handle on the t2g-based redox-active molecular orbital. Kß RIXS is also shown to provide a powerful probe of Co(IV), and specific spectral features are sensitive to the degree of oxo-mediated metal-metal coupling across Co4O4. Guided by the data, calculations show that electron-hole delocalization can actually oppose Co(IV) formation. Computational extension of Co4O4 to CoM3O4 structures (M = redox-inactive metal) defines electronic structure contributions to Co(IV) formation. Redox activity is shown to be linearly related to covalency, and M(III) oxo inductive effects on Co(IV) oxo bonding can tune the covalency of high-valent sites over a large range and thereby tune E(0) over hundreds of millivolts. Additionally, redox-inactive metal substitution can also switch the ground state and modify metal-metal and antibonding interactions across the cluster.

Spherical analyzers and monochromators for resonant inelastic hard X-ray scattering: a compilation of crystals and reflections.

Resonant inelastic X-ray scattering (RIXS) experiments require special sets of near-backscattering spherical diced analyzers and high-resolution monochromators for every distinct absorption-edge energy and emission line. For the purpose of aiding the design and planning of efficient RIXS experiments, comprehensive lists of suitable analyzer reflections for silicon, germanium, α-quartz, sapphire and lithium niobate crystals were compiled for a multitude of absorption edges and emission lines. Analyzers made from lithium niobate, sapphire or α-quartz offer many choices of reflections with intrinsic resolutions currently unattainable from silicon or germanium. In some cases these materials offer higher intensities at comparable resolutions. While lithium niobate, sapphire or α-quartz analyzers are still in an early stage of development, the present compilation can serve as a computational basis for assessing expected and actual performance. With regard to high-resolution monochromators, bandpass and throughput calculations for combinations of double-crystal, high-heat-load and near-backscattering high-resolution channel-cuts were assembled. The compilation of these analyzer and monochromator data is publicly available on a website.

Real-time shape evolution of nanoimprinted polymer structures during thermal annealing.

The real-time shape evolution of nanoimprinted polymer patterns is measured as a function of annealing time and temperature using critical dimension small-angle X-ray scattering (CD-SAXS). Periodicity, line width, line height, and sidewall angle are reported with nanometer resolution for parallel line/space patterns in poly(methyl methacrylate) (PMMA) both below and above the bulk glass transition temperature (T(G)). Heating these patterns below T(G) does not produce significant thermal expansion, at least to within the resolution of the measurement. However, above T(G) the fast rate of loss in pattern size at early times transitions to a reduced rate in longer time regimes. The time-dependent rate of polymer flow from the pattern into the underlying layer, termed pattern "melting", is consistent with a model of elastic recovery from stresses induced by the molding process.