How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by N δ -Methyl Histidine Affect Its Properties and Functions? A Computational Study

Heme peroxidases have important functions in nature related to the detoxification of H O . They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H O complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered N -methyl histidine-ligated cytochrome peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially N -methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the N -methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the N -methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by N -methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on N -methyl histidine-ligated cytochrome peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.