Catching CO oxidation Details of the transition state that forms as carbon monoxide (CO) adsorbed on a ruthenium surface is oxidized to CO2 have been revealed by ultrafast excitation and probe methods. Öström et al. initiated the reaction between CO and adsorbed oxygen atoms with laser pulses that rapidly heated the surface and then probed the changes in electronic structure with oxygen x-ray absorption spectroscopy. They observed transition-state configurations that are consistent with density functional theory and a quantum oscillator model. Science, this issue p. 978 Ultrafast x-ray spectroscopy reveals electronic changes that occur during the oxidation of carbon monoxide on a ruthenium surface. Femtosecond x-ray laser pulses are used to probe the carbon monoxide (CO) oxidation reaction on ruthenium (Ru) initiated by an optical laser pulse. On a time scale of a few hundred femtoseconds, the optical laser pulse excites motions of CO and oxygen (O) on the surface, allowing the reactants to collide, and, with a transient close to a picosecond (ps), new electronic states appear in the O K-edge x-ray absorption spectrum. Density functional theory calculations indicate that these result from changes in the adsorption site and bond formation between CO and O with a distribution of OC–O bond lengths close to the transition state (TS). After 1 ps, 10% of the CO populate the TS region, which is consistent with predictions based on a quantum oscillator model.

/pdf/probing-the-transition-state-region-in-catalytic-co-522ich01rc.pdf

"Probing the Transition State Region in Catalytic CO Oxidation on Ru" data files

BACKGROUND Conformational changes are essential for the correct functioning of biological macromolecules. Time-resolved x-ray crystallography extends an extremely successful method for the structural determination of biomolecules by incorporating time as a fourth dimension. Time-resolved x-ray diffraction studies are performed at room temperature so as to allow the biological reaction to evolve within crystals. This reaction must also be initiated throughout crystals—and x-ray diffraction data must be collected—at least as rapidly as the fastest time point of interest. Mature structural analysis tools of macromolecular crystallography can then be adapted to allow x-ray diffraction data to be analyzed in terms of time-dependent conformational changes. ADVANCES Decades of work using polychromatic x-ray pulses (Laue diffraction) at synchrotron radiation sources laid the experimental foundations underpinning the field of time-resolved macromolecular crystallography. Light-driven biological reactions can be rapidly initiated throughout crystals using short laser pulses and have therefore been a major focus for the field. Serial crystallography was first demonstrated 10 years ago at an x-ray free-electron laser (XFEL). In this approach, x-ray diffraction data are collected from a sequence of microcrystals, typically 10 μm or less in their largest dimension, that are being continuously replaced. X-ray diffraction data from thousands of microcrystals are then merged into a complete dataset. Sample delivery for serial crystallography experiments at an XFEL initially relied on liquid microjets, but many other sample-delivery technologies have since been developed, each with its own strengths and weaknesses. Serial crystallography has overcome many of the technical limitations that constrained time-resolved Laue diffraction and has thereby transformed the field, creating a renaissance of interest in time-resolved macromolecular crystallography. In this Review, we describe how time-resolved x-ray diffraction studies using XFEL pulses a few femtoseconds (10−15 s) in duration have allowed atomic motions in biological samples to be visualized on the time scales at which chemical bonds break or isomerize or at which electrons move. We illustrate the power of time-resolved serial crystallography to yield structural and functional insights on slower time scales by showcasing structural results from two energy-transducing membrane proteins, bacteriorhodopsin and photosystem II, neither of which were amenable to synchrotron-based time-resolved Laue diffraction. We also discuss structural results obtained when using mixing jets to diffuse reactants into microcrystals or when releasing photocaged compounds using a laser flash, which have allowed biological reactions that are not naturally light sensitive to be followed in time. OUTLOOK Time-resolved crystallography is transitioning from a highly technical domain of specialists into a flexible approach for elucidating structural and functional insights from macromolecules in their crystalline state. Although serial crystallography was first developed for XFEL-based studies, the recent transfer of time-resolved serial crystallography to synchrotron radiation facilities is critical for the growth of the user community. Nonspecialist user communities will also drive standardized experimental setups that further lower entry barriers for new users. Structural conclusions drawn from XFEL-based studies of ultrafast structural changes should be consolidated by repeating these experiments using lower power density photoexcitation conditions, and data analysis steps—from processing experimental data through to structural interpretation—need to be streamlined. As further structural insights emerge from an increasingly diverse set of macromolecules, it becomes possible to imagine a time when structural results from time-resolved diffraction experiments become as central to understanding a biological reaction as the resting-state structure of a macromolecule is today.

Advances and challenges in time-resolved macromolecular crystallography.

Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.

Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources.

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 Kβ 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.

/pdf/x-ray-emission-spectroscopy-of-single-protein-crystals-3r5f2k7v.pdf

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 3rd generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ 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. 

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

X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn2+ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron ca...

/pdf/x-ray-emission-spectroscopy-at-x-ray-free-electron-lasers-2m79l3vb08.pdf

X-ray emission spectroscopy at X-ray free electron lasers: limits to observation of the classical spectroscopic response for electronic structure analysis

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X-ray emission spectroscopy at X-ray free electron lasers: limits to observation of the classical spectroscopic response for electronic structure analysis