An artificial hemoprotein with inducible peroxidase- and monooxygenase-like activities
Résumé
A new inducible artificial metalloenzyme was obtained by covalent attachment of a Mn(III)-tetraphenylporphyrin to the artificial bidomain repeat protein (A3A3')Y26C. The biohybrid was characterized by MALDI-ToF MS, circular dichroism and UV-Vis spectroscopies. Its peroxidase activity was tested as well as its monooxygenase activity for the oxidation of thioanisole into sulfoxide by H 2 O 2 . Catalytic tests were performed on the original scaffold and with several co-catalysts a) additional imidazole, b) a specific αRep bA3-2 that induces the opening of the (A3A3') interdomain region and c) a αRep bA3-2 bearing a His6-Tag (His6-bA3-2), and the best activity was obtained with His6-bA3-2. The mechanism was rationalized by a molecular modeling study including protein-ligand docking and large scale molecular dynamics. It appears that: 1) the hydrophobic Mn-porphyrin fits well into the (A3A3')Y26C cavity, becomes protected by the protein and fully soluble in water; 2) the biohybrid alone does not display any catalytic activity, even in the presence of imidazole because of a closed conformation of the system; 3) opening of the binding site by bA3-2 leads to a noticeable activity and, 4) the best activity is obtained in the presence of the His6-Tag with one or two of the histidines able to coordinate to the metal acting as co-catalyst in the reactions. To the best of our knowledge, this constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.