Artificial Peroxidase\nBased on the Biotin–Streptavidin\nTechnology that Rivals the Efficiency of Natural Peroxidases

Heme peroxidases represent an important category of heme-containing\nmetalloenzymes that harness peroxide to oxidize a diverse array of\nsubstrates. Capitalizing on a well-established catalytic mechanism,\ndiverse peroxidase mimics have been widely investigated and optimized.\nHerein, we report on the design, assembly, characterization, and genetic\nengineering of an artificial heme-based peroxidase relying on the\nbiotin–streptavidin technology. The crystal structures of the\nwild-type and the best-performing double mutant of artificial peroxidases\nprovide valuable insight regarding the nearby residues strategically\nmutated to optimize the peroxidase activity (i.e., Sav S112E K121H).\nWe hypothesize that these two residues mimic the polar residues in\nthe second coordination sphere, involved in activating the bound peroxide\nin two very widely studied peroxidases: chloroperoxidase (CPO) (i.e.,\nGlu 183 and His 105) and horseradish peroxidase (i.e., Arg 38 and\nHis 42). Despite the absence of a tightly bound axial ligand, which\ncan exert a “push effect”, the evolved artificial peroxidase\nexhibits best-in-class activity for oxidizing two standard substrates\n(TMB and ABTS) in the presence of hydrogen peroxide.