Agnese Bertinelli, Carmela Pacilli, Silvia De Caro, Chiara Bacchella, Gloria Spagnoli, Ylenia Ciummo, Valentina Borghesani, Davide Cavazzini, Angelo Bolchi, Stefania Nicolis, Enrico Monzani, Matteo Tegoni, Simone Dell'Acqua
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.