Abstract
BackgroundThe invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. Scope of reviewRecent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusionsXFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significanceXFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.
Highlights
X-ray crystallography using synchrotron radiation is currently the most powerful method in determining high-resolution structures of biological macromolecules
This review describes the X-ray free-electron laser (XFEL)-based crystallographic studies, especially time-resolved X-ray crystallography, on three metalloproteins performed at SACLA, an XFEL facility in Japan [21]
The MneMn distances determined in the synchrotron radiation (SR) structure of the oxygen evolving complex (OEC), are slightly longer (0.1–0.2 Å) than those obtained by extended X-ray absorption fine structures (EXAFS) [34,35] or theoretical calculations based on the SR structure [36,37,38]
Summary
NO reduction is involved in denitrification by microorganisms growing in oxygen-limited environments [78]. P450nor active center can be reduced with hydride (H−) directly from NADH [93]. The resting state of P450nor has a ferric heme with a water molecule and a Cys thiolate as axial ligands of iron [98]. The ferric NO complex is reduced with hydride (H−) from NADH, producing the second intermediate, designated as intermediate-I (I) [95,97]. I is a two-electron reduced product of the ferric NO complex, whether it is a singly or doubly protonated form is not clear. Mechanism of NO-activation by hydride transfer from NADH, and the relationship between the electronic state of the protonated NO ligand and its reactivity for being attacked by the second NO molecule are interesting topics to be elucidated
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