Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity*

Horseradish peroxidase (HRP) is an important enzyme for industrial purposes due to its ability to oxidize pollutants from wastewater. A previous report indicated that peroxidases can have up to 20% increase in enzymatic activity in an aqueous solution of 1-Ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO₄]) at neutral pH. However, the atomistic details of how the solution affects peroxidase activity remain elusive. Here, we have investigated the structure and dynamics of HRP in pure water and aq. [EMIm][EtSO₄], as well as the reaction mechanism of Cpd II catalyzed conversion of oxoferryl(IV) to hydroxoferryl(III/IV) using molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations with the polarizable AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications) force field. The structural changes in the active site in the ionic liquid solution orient a protonated histidine42 (H42⁺) directly over the ferryl moiety, leading to a direct proton transfer (PT) from H42⁺ to the heme in the active site. Conversely, in neat water, the reaction follows the previously reported mechanism where H42⁺ transfers a proton to the ferryl moiety via an ordered water in the active site. Our QM/MM calculations suggest larger barriers in neat water compared to the ionic liquid solution in neutral and acidic pHs. Analysis of the electric fields with the investigation of the electron localization function in the active site at the critical points along the reaction paths indicates that the aqueous IL medium facilitates the reaction by providing a more favourable enzymatic environment at the active site than the system-solvated in neat water. Overall, these results provide further support for the importance of the role of solvation in enzymatic catalysis.