How Do Vanadium Chloroperoxidases Generate Hypochlorite from Hydrogen Peroxide and Chloride? A Computational Study

Vanadium haloperoxidases are one of the few enzymes in nature that utilize a vanadium center and catalyze the halogenation of substrates through the biosynthesis of hypohalite. Vanadium chloroperoxidases (VCPOs) bind and activate hydrogen peroxide and in a reaction with chloride convert it into hypochlorite as a precursor for a substrate chlorination reaction. Despite the fact that these enzymes have been studied extensively, surprisingly little is known on their catalytic cycle and particularly on the function of the vanadium atom in the reaction mechanism. In order to gain insight into the intricate details of the catalytic cycle of VCPOs, we performed an extensive computational study using large cluster model complexes, where we tested many possible pathways and active-site protonation states. Our work establishes that the biosynthesis of hypochlorite proceeds in two steps: H2O2 activation on the vanadium center to form an end-on V(V)-hydroperoxo complex, followed by OH* transfer from hydroperoxo to chloride on the vanadium center to form hypochlorite. We show that the initial reaction starts with a proton transfer from H2O2 to the equatorial OH group of the V-V(O)(2)(OH)(2)(-) active site, followed by hydroperoxo binding and water release to form the highly stable vanadium-hydroperoxo-dioxo-hydroxo complex. A further proton transfer from an active-site His or Lys residue can lead to the vanadium-peroxo-hydroxo-oxo complex, which we assign as a dead-end complex unable to react further to hypochlorite products. The mechanisms were considered under various protonation state, and it is shown to be the most effective with His(404) singly protonated. The work shows that vanadium is a spectator ion that does not change its oxidation state during the reaction mechanism but holds and positions the H2O2 substrate and guides its proton-relay steps through its oxo and hydroxo ligands. The effect of the protonation state of first- and second-coordination sphere residues and ligands was tested and shows that the reaction is highly sensitive to local changes in the protonation state. Finally, the computations show that the oxygen atom of HOCl exclusively derives from H2O2.