A Nonheme Iron Peroxidase Mimic Enabled by Histidine Methylation and Supramolecular Self-Assembly

Histidine plays a key regulatory role in nonheme iron enzymes, as coordination through the imidazole δ- or ε-nitrogen can strongly influence iron reactivity; however, isolating the individual contributions of these nonequivalent nitrogen sites in native protein systems remains challenging. In this study, we present a peroxidase-mimetic catalyst formed through the supramolecular self-assembly of fluorenyl-modified histidine (Fmoc-His) with Fe²⁺, in which selective methylation at the N_ε or N_δ position of histidine modulates tautomeric preferences and thereby tunes Fe²⁺-mediated catalysis. Catalytic studies with multiple substrates show that δ-position methylation markedly enhances catalytic activity. Spectroscopic and kinetic analyses reveal that δ-methylation promotes compact supramolecular assembly, strengthens Fe²⁺-scaffold interactions, and is consistent with a ternary-complex-type catalytic pathway involving Fe²⁺, H₂O₂, and the substrate, with multiple reactive oxygen species collectively contributing to substrate oxidation. Theoretical calculations further suggest that δ-methylation may facilitate transient colocalization of reactants within the supramolecular assembly, while the catalyst maintains good structural stability during catalysis. This study demonstrates that histidine methylation provides an effective means to regulate the reactivity of iron-centered supramolecular catalytic systems and offers insights into the rational design of supramolecular metalloenzymes.