A theoretical study on the mechanism of the reductive half-reaction of xanthine oxidase

On the basis of the crystal structure of an aldehyde oxidoreductase, Huber et al. proposed a catalytic mechanism for the reductive half-reaction of xanthine oxidase which involves nucleophilic addition of Mo-bound hydroxide (Moco 1) to the substrate and hydride transfer from the substrate to sulfido group (Mo = S). Density functional theory calculations have been carried out for the oxidation of formaldehyde, acetaldehyde, formamide, and formamidine with Moco 2 to understand more detailed catalytic pathways. Our calculation results indicate that the anionic catalyst model acts as a nucleophile and is reactive for the oxidation of aldehyde substrates, which are reactive for nucleophilic addition. In these cases, a concerted mechanism is found to be more favorable than a stepwise mechanism. The concerted mechanism is further shown to be promoted by the presence of a nearby water molecule, in the active site, which serves as a Lewis acid for the nucleophilic addition of hydroxide. For less reactive formamide and formamidine (a model for xanthine) substrates, the calculated activation energies with the above mechanisms are high. These reactions also do not benefit from the presence of the water molecule. The results indicate that different catalyst forms might be responsible for the oxidation of different substrates, which could be regulated by the enzyme active site environment.