Structural basis of cuproenzyme nitrite reduction at the level of a single hydrogen atom

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 nitrite (NO₂^-) and not as protonated HNO₂. Our X-ray data and quantum chemical calculation show that NO₂^- is in an electron-localized state that can facilitate N-O bond cleavage after receiving an electron. The catalytic residues, Asp^CAT and His^CAT, are deprotonated and protonated, respectively, suggesting that His^CAT 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^CAT-to-Asn mutants, which mimic the protonated state of Asp^CAT, were also determined to investigate the involvement of protonated Asp^CAT in the reaction. Our crystallographic data and quantum chemical calculations reveal in detail the first step of the CuNIR reaction.