N-methylation of histidine to tune tautomeric preferences in histidine-heme coordination and enzyme-mimetic catalysis

Abstract

Enzymes with active sites involving histidine selectively utilize either the δ- or ϵ-nitrogen atom (N_δ or N_ϵ) of the histidine imidazole for catalysis. However, evaluating the impact of N_δ and N_ϵ is difficult, and directly integrating noncanonical Nmethylated histidine within enzymes poses risks due to laborious procedures. In this study, we present the self-assembly of Fmoc-Histidine (Fmoc-His) with hemin to create a peroxidase-mimetic catalyst, in which either the N_ϵ or N_δ of histidine is methylated to modify the tautomeric preferences, thereby tuning hemin catalysis. UV-vis spectra, ¹H-NMR, and fluorescence experiments elucidate that the Nmethylation of histidine alters the self-assembly propensity of Fmoc-His, and affects the binding affinity of histidine to hemin iron, with Fmoc-_δmHis/hemin exhibiting stronger binding than Fmoc-_ϵmHis/hemin. Theoretical simulation results suggest that _ϵmHis and _δmHis ligation produce a saddled structure and planar structure of hemin, respectively, stemming from the disparity of steric hindrance at the N_ϵ and N_δ positions. The significant inhibition of hemin’s oxidative activity by Fmoc-_δmHis is observed, likely due to the strong binding of Fmoc-_δmHis, potentially hindering access of the substrate, H₂O₂, to the hemin iron. Conversely, Fmoc-_ϵmHis enhances hemin catalysis, surpassing even Fmoc-His alone. This differential impact of Fmoc-_ϵmHis and Fmoc-_δmHis on hemin activity is further corroborated by apparent activation energy and kinetic parameters (k_cat, k_cat/K_m). This study sheds light on the heterogeneous biological effects at the nitrogen positions of histidine imidazole and offers insights into designing supramolecular metalloenzymes.

Keywords

heme-dependent enzyme / histidine / self-assembly / tautomeric preference

Author summay

Ruikai Du, Yunbo Lv and Haifeng Wu contributed equally to this work.