Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe³⁺ ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe³⁺ is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe³⁺ ions, two residue clusters of OxOx were assigned as potential Fe³⁺ binding sites, the mechanism of the inhibition of Fe³⁺ was delineated. Importantly, the residues that interact with Fe³⁺ ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe³⁺ highlights insight into substrate binding site in OxOx.