Catalytic Reaction Mechanism of Oxalate Oxidase (Germin). A Hybrid DFT Study.

The mechanism of the catalytic reaction for oxalate oxidase has been investigated with the hybrid density functional method B3LYP. The models used in the calculations comprise of the manganese ion, three imidazoles, and one acetate, which model the active-site Mn(II) and its first-shell protein ligands. Moreover, the reactants, i.e., singly protonated oxalate and dioxygen, have been explicitly considered. The computational results suggest that the enzyme-oxalate complex can adopt two conformations, one with bidentate oxalate and 6-coordinate manganese and the second one with monodentate substrate and coordinatively unsaturated Mn(II). This second species reacts with dioxygen on the quartet potential energy surface, and in a rate-limiting step yields one CO2 molecule and a reactive intermediate, in which Mn(III) is coordinated by HOO(-) and a formyl radical anion. A subsequent fast spin transition, from the quartet to the sextet spin state, allows an electron transfer from the formyl radical anion to Mn(III) and leads to the product-enzyme complexes. It is proposed that the final step of the catalytic cycle involves protonation of these species and release of products. Taken together, the mechanistic proposal presented in this work agrees well with the available experimental data and provides an explanation for the very efficient coupling between the two-electron dioxygen reduction and oxalate oxidation performed by oxalate oxidase.