Superoxo and hydroperoxo intermediates were cryotrapped at the copper active site of lytic polysaccharide monooxygenase using neutron protein crystallography.
Neutron diffraction is exquisitely sensitive to the positions of H atoms in protein crystal structures. IMAGINE is a high-intensity, quasi-Laue neutron crystallography beamline developed at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. This state-of-the-art facility for neutron diffraction has enabled detailed structural analysis of macromolecules. IMAGINE is especially suited to resolve individual H atoms in protein structures, enabling neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of less than 1 mm and unit-cell edges of less than 150 Å. Beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position, and variable short- and long-wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. This review gives an overview of the IMAGINE beamline at the HFIR, presents examples of the scientific questions being addressed at this beamline, and highlights important findings in enzyme chemistry that have been made using the neutron diffraction capabilities offered by IMAGINE.
Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.
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