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 a 20% increase in enzymatic activity in 0.26 M 1-Ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO₄]) at neutral pH. However, the atomistic details of how the solution affects peroxidase activity remain elusive. In the enzymatic landscape of HRP, an intermediate termed Compound II (Cpd II) plays a key role and involves a histidine (H42) residue that may take different protonation states. Cpd II exists as oxo-ferryl (2a) or hydroxo-ferryl (2b(Fe^IV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(Fe^III) form seen in synthetic complexes, has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aq. [EMIm][EtSO₄] (0.26 M), as well as the reaction mechanism of Cpd II catalyzed conversion of 2a to 2b using polarizable molecular dynamics (MD) and polarizable ab initio quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq. [EMIm][EtSO₄] a significant reduction in the fluctuation of residues 70, 71 and 74 is observed. This reduction is due to the migration of EtSO₄^– ions close to this region, which results in the observed fluctuation decrease, and additional structural changes leading to the dehydration of the active site. These structural changes in the active site orient H42⁺ directly over the ferryl moiety, leading to a direct proton transfer with a significant energy reduction in the reaction barrier. 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. We further investigated an alternative path with the deprotonated form of H42, where the mechanism shifts to HAT, with similar reaction barrier differences between the aqueous and aqueous/IL mixture, albeit with significantly higher relative barriers. Analysis of the electric fields at the active site indicate that the aq. IL medium facilitate the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, our calculations help explain the observed improvement in activity for peroxidases in IL solution, and underscore the importance of favorable electric fields in the active site to promote catalysis.

Jorge Nochebuena

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