Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

Abstract

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Unique protonation states of aspartate and topaquinone in the active site of copper amine oxidase

“…The results of the Full state were already reported in our previous study. 17 In the present paper, instead, other protonation states, along with their H-bond formations, are mainly discussed. The states are labelled according to their relative stability and the total charge of the QM region.…”

“…These distances are clearly beyond any O-H chemical bond, thus it seems that this proton remains unbounded between Asp298 and TPQ. 17 During our QM/MM calculations, we always failed to obtain the unusual H-bonded state of the ND structure, even if the starting positions of the H atom are changed. This result is clearly unable to explain the ND observed proton position.…”

“…The initial structure of AGAO was taken from the recently determined ND structure (PDBID: 6L9C). 17 Atomic positions with higher occupation factor in the crystal structure were selected for the construction of the disordered residues, and the protonation states of titratable residues were followed to the ND structure. A one-subunit model containing one TPQ and one Cu atom was prepared.…”

“…This result is clearly unable to explain the ND observed proton position. 17 Rationalizing this uncommon isolated H atom position observed by ND is hard challenge also at a theoretical level. An alternative possibility if that this ND-identied site is not a proton but rather an alkali metal atom cation (M) trapped between the negatively charged Asp298 and TPQ.…”

“…We recently provided an accurate determination of the positions of H atoms in AGAO by neutron diffraction (ND) with a resolution of 1.72Å. 17 On the basis of the TPQ protonation state, the neutron scattering length density map revealed the presence of a keto form TPQ and the enolate form, in which the enolate form has been believed to be the sole chemical form in TPQ ox state. In addition, one H atom was identied at a central position among three protonatable atoms, namely the OD1 and OD2 atoms in Asp298 and O(5) of TPQ.…”