Interaction between hemin and the SpyTag/SpyCatcher system: towards an artificial bioinspired metalloprotein

The challenging design of artificial metalloenzymes can be achieved by bioconjugation based on the targeted insertion of synthetic metal coordination motif on a peptide that can subsequently be covalently attached to a protein scaffold. The Spy system, consisting of a protein domain, SpyCatcher, which reacts spontaneously with a short peptide, SpyTag, forming a covalent isopeptide bond, can be used for this purpose. In this work, a SpyTag peptide functionalized with a histidine-based domain is engineered to bind hemin both as a free peptide and after assembly with SpyCatcher. The binding between SpyTag and hemin occurs successfully with micromolar affinity, but the coordination of hemin with reconstituted Spy protein (the SpyTag/SpyCatcher adduct) is not maintained with the same strength. The hemin-SpyTag peptide complex displays enhanced reactivity towards the activation of hydrogen peroxide in oxidative reactions, while the SpyTag/SpyCatcher adduct does not significantly affect hemin reactivity. Neither system exhibits substantial catalase-like activity. All tested components act as ROS scavengers, likely due to oxidative modifications undergone by the peptide/protein backbone. Overall, the study provides a solid experimental and methodological basis and highlights the need to improve hemin binding within the Spy system for future optimization and development of more advanced artificial hemoproteins.