Halogenated phenols, such as 2,4-dichlorophenol (2,4-DCP) and 4-bromophenol (4-BP) are pollutants generated by a various industrial sectors like chemical, dye, paper bleaching, pharmaceuticals or in an agriculture as pesticides. The use of Horseradish peroxidase (HRP) in the halogenated phenols treatment has already been mentioned, but it is not well understood how the different phenolic substrates can bind in the peroxidase active site nor how these specific interactions can influence in the bioremediation potential. In this work, different removal efficiencies were obtained for phenolic compounds investigated using HRP as catalyst (93.87 and 59.19% to 4BP and 2,4 DCP, respectively). Thus, to rationalize this result based on the interactions of phenols with active center of HRP, we combine computational and experimental methodologies. The theoretical approaches utilized include density functional theory (DFT) calculations, docking simulation and quantum mechanics/molecular mechanics (QM/MM) technique. Michaelis Menten constant (Km) obtained through experimental methodologies were 2.3 and 0.95 mM to 2,4-DCP and 4-BP, respectively, while the specificity constant (K_cat/K_m) found was 1.44 mM⁻¹ s⁻¹ and 0.62 mM⁻¹ s⁻¹ for 4-BP and 2,4-DCP, respectively. The experimental parameters appointed to the highest affinity of HRP to 4-BP. According to the molecular docking calculations, both ligands have shown stabilizing intermolecular interaction energies within the HRP active site, however, the 4-BP showed more stabilizing interaction energy (−53.00 kcal mol⁻¹) than 2,4-dichlorophenol (−49.23 kcal mol⁻¹). Besides that, oxidative mechanism of 4-BP and 2,4-DCP was investigated by the hybrid QM/MM approach. This study showed that the lowest activation energy values for transition states investigated were obtained for 4-BP. Therefore, by theoretical approach, the compound 4-BP showed the more stabilizing interaction and activation energy values related to the interaction within the enzyme and the oxidative reaction mechanism, respectively, which corroborates with experimental parameters obtained. The combination between experimental and theoretical approaches was essential to understand how the degradation potential of the HRP enzyme depends on the interactions between substrate and the active center cavity of the enzyme.

卤代苯酚，例如2,4-二氯苯酚（2,4-DCP）和4-溴苯酚（4-BP）是由各种工业部门产生的污染物，例如化学，染料，纸张漂白，制药或农业中的农药。已经提到过辣根过氧化物酶（HRP）在卤代酚类化合物处理中的用途，但人们还不了解不同的酚类底物如何在过氧化物酶活性位点结合，以及这些特定的相互作用如何影响生物修复潜力。在这项工作中，使用HRP作为催化剂研究的酚类化合物获得了不同的去除效率（分别为4BP和2,4 DCP的93.87％和59.19％）。因此，为了使基于酚与HRP活性中心相互作用的结果合理化，我们将计算和实验方法相结合。使用的理论方法包括密度泛函理论（DFT）计算，对接模拟和量子力学/分子力学（QM / MM）技术。通过实验方法获得的Michaelis Menten常数（Km）对2,4-DCP和4-BP分别为2.3和0.95 mM，而特异性常数（K_cat / K _m）对于4-BP和2,4-DCP分别为1.44 mM ^-1  s ^-1和0.62 mM ^-1  s ^-1。实验参数指定了HRP对4-BP的最高亲和力。根据分子对接计算，两个配体均显示出稳定的HRP活性位点之间的分子间相互作用能，但是4-BP的稳定相互作用能（-53.00 kcal mol ^-1）比2,4-二氯苯酚（-49.23 kcal）摩尔^-1）。除此之外，采用混合QM / MM方法研究了4-BP和2,4-DCP的氧化机理。这项研究表明，对于4-BP，获得了研究的过渡态的最低活化能值。因此，通过理论方法，化合物4-BP分别显示出与酶内相互作用和氧化反应机理相关的更稳定的相互作用和活化能值，这与所获得的实验参数相符。实验方法与理论方法之间的结合对于理解HRP酶的降解潜力如何取决于底物与酶的活性中心腔之间的相互作用至关重要。