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

Enzymes with active sites involving histidine selectively utilize either the δ- or ε-nitrogen atom (N or N) of the histidine imidazole for catalysis. However, evaluating the impact of N and N 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 N or N of histidine is methylated to modify the tautomeric preferences, thereby tuning hemin catalysis. UV-vis spectra, H-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-His/hemin exhibiting stronger binding than Fmoc-His/hemin. Theoretical simulation results suggest that His and His ligation produce a saddled structure and planar structure of hemin, respectively, stemming from the disparity of steric hindrance at the N and N positions. The significant inhibition of hemin's oxidative activity by Fmoc-His is observed, likely due to the strong binding of Fmoc-His, potentially hindering access of the substrate, HO, to the hemin iron. Conversely, Fmoc-His enhances hemin catalysis, surpassing even Fmoc-His alone. This differential impact of Fmoc-His and Fmoc-His on hemin activity is further corroborated by apparent activation energy and kinetic parameters ( , / ). This study sheds light on the heterogeneous biological effects at the nitrogen positions of histidine imidazole and offers insights into designing supramolecular metalloenzymes.