Affiliation:
1. State Key Laboratory of Organic−Inorganic Composites Key Lab of Biomedical Materials of Natural Macromolecules (Ministry of Education) Beijing Laboratory of Biomedical Materials College of Materials Science and Engineering Beijing University of Chemical Technology Beijing China
Abstract
AbstractEnzymes with active sites involving histidine selectively utilize either the δ‐ or ε‐nitrogen atom (Nδ or Nε) of the histidine imidazole for catalysis. However, evaluating the impact of Nδ and Nε is difficult, and directly integrating noncanonical N‐methylated 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 Nε or Nδ of histidine is methylated to modify the tautomeric preferences, thereby tuning hemin catalysis. UV‐vis spectra, 1H‐NMR, and fluorescence experiments elucidate that the N‐methylation 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 Nε and Nδ 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 (kcat, kcat/Km). This study sheds light on the heterogeneous biological effects at the nitrogen positions of histidine imidazole and offers insights into designing supramolecular metalloenzymes.