Neutron Crystallography of a Semiquinone Radical Intermediate of Copper Amine Oxidase Reveals a Substrate-Assisted Conformational Change of the Peptidyl Quinone Cofactor

Determining precise locations of hydrogen atoms in enzyme active sites, especially those in the reaction intermediates, provides important information for understanding the structure-function relationships, such as a correlation between pH-dependency and protonation/deprotonation states of dissociable groups. To experimentally determine the coordinates of hydrogen atoms, we solved the neutron crystallographic structure of a catalytic intermediate of copper amine oxidase, containing a peptidyl quinone cofactor, topa quinone (TPQ), which is converted to a semiquinone radical form by anaerobic reaction with an amine substrate. Neutron diffraction data at 1.67 A resolution revealed the protonation/ deprotonation state of the active-site residues, including TPQ. The semiquinone form was doubly deprotonated at the 2-OH and 4OH positions. The surrounding hydrogen-bond network and the CH center dot center dot center dot p and NH center dot center dot center dot pi-like interactions with both sides of the TPQ ring were identified, affording a stabilization mechanism for the semiquinone radical structure. The pH-dependent conformational change of TPQ from an 'off-copper' aminoresorcinol to an 'on-copper' semiquinone was accompanied by protein/solvent proton exchange in the main-chain peptide bond of TPQ. Moreover, the neutron diffraction data disclosed the number of deuterium/ hydrogen atoms covalently attached to the terminal heavy atom of the ligand bound in a hydrophobic pocket adjacent to TPQ, which led to the conclusion that the product aldehyde was replaced by an amine substrate in the pocket. The amino group of the bound substrate interacted with the deprotonated side chain of a conserved aspartic acid residue that acts as a catalytic base. These findings demonstrate that the additional substrate binding triggers the conformational change of TPQ in the reductive half-reaction and makes the enzyme catalysis proceed into the subsequent oxidative half-reaction.

Automated summary

Determining the precise locations of hydrogen atoms in enzyme active sites is crucial for understanding the structure-function relationships. This study solved the neutron crystallographic structure of a catalytic intermediate of copper amine oxidase and revealed the protonation/deprotonation state of active-site residues. The results showed that substrate binding triggers conformational changes in the enzyme and facilitates subsequent reactions.