How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

Vanadium haloperoxidases (VHPOs) are unique enzymes inbiologythat catalyze a challenging halogen transfer reaction and converta strong aromatic C-H bond into C-X (X = Cl, Br, I)with the use of a vanadium cofactor and H2O2. The VHPO catalytic cycle starts with the conversion of hydrogenperoxide and halide (X = Cl, Br, I) into hypohalide on the vanadatecofactor, and the hypohalide subsequently reacts with a substrate.However, it is unclear whether the hypohalide is released from theenzyme or otherwise trapped within the enzyme structure for the halogenationof organic substrates. A substrate-binding pocket has never been identifiedfor the VHPO enzyme, which questions the role of the protein in theoverall reaction mechanism. Probing its role in the halogenation ofsmall molecules will enable further engineering of the enzyme andexpand its substrate scope and selectivity further for use in biotechnologicalapplications as an environmentally benign alternative to current organicchemistry synthesis. Using a combined experimental and computationalapproach, we elucidate the role of the vanadium haloperoxidase proteinin substrate halogenation. Activity studies show that binding of thesubstrate to the enzyme is essential for the reaction of the hypohalidewith substrate. Stopped-flow measurements demonstrate that the rate-determiningstep is not dependent on substrate binding but partially on hypohalideformation. Using a combination of molecular mechanics (MM) and moleculardynamics (MD) simulations, the substrate binding area in the proteinis identified and even though the selected substrates (methylphenylindoleand 2-phenylindole) have limited hydrogen-bonding abilities, theyare found to bind relatively strongly and remain stable in a bindingtunnel. A subsequent analysis of the MD snapshots characterizes twosmall tunnels leading from the vanadate active site to the surfacethat could fit small molecules such as hypohalide, halide, and hydrogenperoxide. Density functional theory studies using electric field effectsshow that a polarized environment in a specific direction can substantiallylower barriers for halogen transfer. A further analysis of the proteinstructure indeed shows a large dipole orientation in the substrate-bindingpocket that could enable halogen transfer through an applied localelectric field. These findings highlight the importance of the enzymein catalyzing substrate halogenation by providing an optimal environmentto lower the energy barrier for this challenging aromatic halide insertionreaction.