Neutron crystallography for the elucidation of enzyme catalysis

https://doi.org/10.1016/j.sbi.2021.05.007Get rights and content

Highlights

  • Neutron crystallography can observe hydrogen atoms directly, which play essential roles in enzyme catalyzed reactions.

  • Ferric ascorbate peroxidase has a possible proton transfer pathway from ascorbate to the heme iron via arginines and waters.

  • Protonation states in the catalytic dyad of severe acute respiratory syndrome coronavirus 2 main protease are observed.

  • Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified.

Abstract

Hydrogen atoms and hydration water molecules in proteins are indispensable for many biochemical processes, especially enzymatic catalysis. The locations of hydrogen atoms in proteins are usually predicted based on X-ray structures, but it is still very difficult to know the ionization states of the catalytic residues, the hydration structure of the protein, and the characteristics of hydrogen-bonding interactions. Neutron crystallography allows the direct observation of hydrogen atoms that play crucial roles in molecular recognition and the catalytic reactions of enzymes. In this review, we present the current status of neutron crystallography in structural biology and recent neutron structural analyses of three enzymes: ascorbate peroxidase, the main protease of severe acute respiratory syndrome coronavirus 2, and copper-containing nitrite reductase.

Section snippets

Neutron crystallography on structural biology

X-ray crystallography is the technique most commonly used to determine the tertiary structures of biomacromolecules such as proteins and nucleic acids, but it is difficult to observe light elements, especially hydrogen atoms, because the X-ray scattering factors are proportional to the number of electrons (i.e. the atomic number) of an atom. Hydrogen atoms comprise about half of all atoms in proteins and play essential roles in the catalytic reactions of enzymes. X-ray structures determined at

Neutron structural analyses of ascorbate peroxidase

Peroxidases are heme-containing metalloenzymes that catalyze the oxidation of various substrates by reducing hydrogen peroxide (H2O2). Cytochrome c peroxidase (CcP) catalyzes the oxidation of cytochrome c, and ascorbate peroxidase (APX), which is closely related to CcP, oxidizes ascorbate. Peroxidases have in common a catalytic cycle that consists of sequential redox steps involving the formation of two highly oxidized FeIV (ferryl) intermediates (named compound I and compound II) [8]. Two

Neutron structural analyses of the main protease of SARS-CoV-2

The current COVID-19 pandemic is caused by a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [21]. The main protease (Mpro) of SARS-CoV-2 plays an indispensable role in viral replication, making it a promising target for inhibitor (drug) design to prevent SARS-CoV-2 activity [22]. Mpro forms a homodimer, and its overall structure is highly conserved among coronaviruses [23]. Each monomer consists of three domains (domains I–III), and the active site is

Neutron structural analyses of copper-containing nitrite reductase

Copper-containing nitrite reductases (CuNIRs) catalyze a critical step in denitrification, the one-electron reduction of nitrite (NO2) to nitric oxide (NO), which generates gaseous NO. This reaction breaks down terrestrial fertilizer. CuNIRs form a homotrimer, and each monomer contains one type 1 copper (T1Cu) and one type 2 copper (T2Cu) [33, 34, 35, 36]. The T1Cu site is involved in accepting an electron from physiological electron donor proteins, such as cytochrome c551, and then, the

Conclusion and outlook

The use of neutron crystallography in structural biology remains limited, at least in terms of the number of structure determinations, but enables the direct observation of hydrogen atoms, which is extremely difficult by other methods. Neutron crystallography has been used to elucidate the detailed catalytic reactions of several enzymes by precisely determining protonation states and hydration structures.

Technical developments will make neutron crystallography a more common method in structural

Conflict of interest statement

Nothing declared.

Acknowledgements

The authors are grateful to Drs. Andreas Ostermann, Leighton Coates, and Yohta Fukuda for critical reading and valuable comments.

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