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.

Keywords: X-ray diffraction; enzymatic mechanisms; metalloproteins; neutron protein crystallography; protonation; radiation damage.

Figure 1

Incoherent neutron scattering cross sections and coherent neutron scattering lengths for selected elements. Relative incoherent scattering cross sections are represented by a disc coloured on a grey scale (dark, high incoherent cross section; light, low incoherent cross section), and relative coherent scattering lengths are represented by red and green discs. The red discs for hydrogen and manganese indicate the negative sign of their scattering lengths, while those shown in green are positive.

Figure 2

The active site of copper nitrite reductase (PDB entry 6gtj, perdeuterated; Halsted et al., 2019 ▸). (a) A D₂O molecule is bound to the active site with a neutral His256 (His_CAT). 2F _o − F _c NSLD map (σ = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively. (b) An OD⁻ ion is bound to the active site with a positively charged His211 (His_CAT; PDB entry 6l46, H/D exchanged; Fukuda et al., 2020 ▸). NSLD 2F _o − F _c density (σ = 1.50) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.

Figure 3

The active site of copper amine oxidase. The cofactor TPQ is present in its ketone and enol forms and a proton is shared between TPQ and the active-site Asp298 (PDB entry 6l9c, H/D exchanged; Murakawa et al., 2020 ▸). F _o − F _c NSLD omit map (σ = 3.00) is displayed as a green mesh for selected D atoms; H and D atoms are displayed in white and turquoise, respectively.

Figure 4

The active site of the heme centre of ascorbate peroxidase (APX). (a) Compound II of APX with a positively charged His42 (PDB entry 5jpr, H/D exchanged; Kwon et al., 2016 ▸). 2F _o − F _c NSLD map (σ = 1.50) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively. (b) A neutral Arg38 residue in ascorbate-bound APX (PDB entry 6xv4, perdeuterated; Kwon et al., 2020 ▸). 2F _o − F _c NSLD map (σ = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.

Figure 5

The active site of LPMO. The second-shell His157 is neutral in the copper(II) resting state (PDB entry 5tki, H/D exchanged; O’Dell et al., 2017 ▸). 2F _o − F _c NSLD map (σ = 1.50) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.

Figure 6

The active site of manganese superoxide dismutase. (a) The oxidized active site with a doubly protonated Gln143 (PDB entry 7kks, perdeuterated; Azadmanesh et al., 2021 ▸). 2F _o − F _c NSLD map (σ = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively. F _o − F _c NSLD omit map is displayed as a mesh with σ = 3.00 in orange and σ = 3.50 in magenta for selected D atoms. (b) The reduced active site with a singly protonated Gln143 (PDB entry 7kkw, perdeuterated; Azadmanesh et al., 2021 ▸). 2F _o − F _c NSLD density (σ = 1.00) is displayed as a blue mesh; D atoms are displayed in turquoise, respectively. F _o − F _c NSLD omit map is displayed as a mesh with σ = 2.50 in yellow and σ = 3.00 in orange for selected D atoms.

Figure 7

The active site of chlorite dismutase. The active-site Arg127 remains fully protonated in the outward conformation (PDB entry 5nku, H/D exchanged; Schaffner et al., 2017 ▸). 2F _o − F _c NSLD map (σ = 1.40) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.

Figure 8

The active site of amicyanin and a strong hydrogen bond to an unexchanged tryptophan (PDB entry 3l45, H/D exchanged; Sukumar et al., 2010 ▸). (a) Conserved active-site residues coordinating the copper cofactor. (b) The hydrogen bond between the non-exchanged Trp45 and Tyr90. 2F _o − F _c NSLD map (σ = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.

Figure 9

The active site and protonation states of glutamate residues with backbone carbonyl O atoms (PDB entry 6kk8, H/D exchanged; Yamada et al., 2019 ▸). 2F _o − F _c NSLD map (σ = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively. (a) Manganese catalase active site with the bridging O atoms coordinated to the two manganese cofactors. Bridging atoms O₁₀₀₄ and O₁₀₀₅ have alternate conformations, which are shown in red and pale red. (b) Single protonation of Glu167 forming a hydrogen bond to the Ala279 carbonyl group oxygen. (c) Single protonation of Glu280 forming a hydrogen bond to the Phe116 carbonyl group oxygen.