9KVM

Neutron and X-ray joint refined structure of a copper-containing nitrite reductase (C135A mutant) in complex with formate

Summary for 9KVM

• Entry DOI: 10.2210/pdb9kvm/pdb
• Descriptor: Copper-containing nitrite reductase, COPPER (II) ION, FORMIC ACID, ... (4 entities in total)
• Functional Keywords: copper, denitrification, oxidoreductase
• Biological source: Geobacillus thermodenitrificans (strain NG80-2)
• Total number of polymer chains: 1
• Total formula weight: 35791.21

Authors

Fukuda, Y.,Lintuluoto, M.,Hirano, Y.,Kusaka, K.,Inoue, T.,Tamada, T. (deposition date: 2024-12-05, release date: 2025-06-11)

Primary citation

Fukuda, Y.,Lintuluoto, M.,Hirano, Y.,Kusaka, K.,Inoue, T.,Tamada, T.
Structural basis of cuproenzyme nitrite reduction at the level of a single hydrogen atom.
J.Biol.Chem., :110290-110290, 2025

PubMed Abstract: Hydrogen (H) atoms account for about half the atoms in biomacromolecules and are essential for their biochemical properties such as enzymatic functions. Obtaining precise enzyme structures that include all the H atoms allows a deeper understanding of their structure-function relationships. Copper-containing nitrite reductases (CuNIRs) catalyze transformation of nitrite to nitric oxide, which has impacts on geochemical, agricultural, and medical health fields. Despite intense research efforts, the dynamics of H atoms during the enzymatic reaction of CuNIRs are unknown and hence the catalytic mechanism remains unclear. We performed neutron crystallography to shoot a single H-atom resolution picture of a CuNIR in complex with nitrite. We found that nitrite binds on the catalytic Cu center as NO and not as protonated HNO. Our X-ray data and quantum chemical calculation show that NO is in an electron-localized state that can facilitate N-O bond cleavage after receiving an electron. The catalytic residues, Asp and His, are deprotonated and protonated, respectively, suggesting that His is the point of departure of the proton transfer sequence. Quantum chemical calculations show that the neutron structure is consistent with the Cu(II) state and that the highly polarized state of the catalytic site is stabilized by the permittivity of solvent molecules filling a water channel. Subatomic resolution X-ray structures of the Asp-to-Asn mutants, which mimic the protonated state of Asp, were also determined to investigate the involvement of protonated Asp in the reaction. Our crystallographic data and quantum chemical calculations reveal in detail the first step of the CuNIR reaction.

PubMed: 40436316
DOI: 10.1016/j.jbc.2025.110290

Experimental method

NEUTRON DIFFRACTION (1.9 Å)
X-RAY DIFFRACTION (1.2 Å)