Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography

Abstract

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.

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

“…36,37 Neutron protein crystallography is also a non-destructive technique ideally suited for the study of radiation-sensitive metalloproteins for characterization of catalytic intermediates. 38 The application of cryo-neutron protein crystallography to study the protonation states of activated oxygen catalytic intermediates has been demonstrated by Raven, Moody and coworkers for the characterization of compound I and compound II. 39,40 In particular, a neutron crystallography study of cytochrome c peroxidase (CcP) resulted in cryo-capture and characterization of a deprotonated Fe( iv )O compound I species in a 2.5 Å resolution structure.…”

Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

“…This makes the structure determination of the native/resting state difficult. One way of circumventing photoreduction is to use neutrons instead of X-rays for crystallographic studies of metalloproteins (Schro ¨der & Meilleur, 2021), though this imposes severe limitations on the systems that can be studied, as much bigger crystals are required for neutron diffraction. Furthermore, reduction of transition metals by X-rays may be a convenient reaction trigger for studies of enzymatic reactions in crystals (Bourgeois & Weik, 2009;Bourgeois & Royant, 2005).…”

The Biochemistry and Effectiveness of Antioxidants in Food, Fruits, and Marine Algae

“…Likewise, in the humans’ case, the conversion of D-Glucuronate to L-Gulonate occurs mainly through aldehyde reductase (GR) and to a smaller extent with aldose reductase’ (AR) contribution ( Figure 5 ) [ 51 , 65 , 66 ]. Considering that the common molecular mechanism of the body’s limited ability to synthesize vitamin C is the absence of GULO [ 68 , 69 , 70 , 71 ], genetically, it is considered that the loss of synthesizing the ascorbic acid is likely due to the complete loss of the L-gulono-γ-lactone oxidase (GULO) gene.…”