Artificial Peroxidase Based on the Biotin–Streptavidin Technology that Rivals the Efficiency of Natural Peroxidases

Abstract

Heme peroxidases represent an important category of heme-containing metalloenzymes that harness peroxide to oxidize a diverse array of substrates. Capitalizing on a well-established catalytic mechanism, diverse peroxidase mimics have been widely investigated and optimized. Herein, we report on the design, assembly, characterization, and genetic engineering of an artificial heme-based peroxidase relying on the biotin–streptavidin technology. The crystal structures of the wild-type and the best-performing double mutant of artificial peroxidases provide valuable insight regarding the nearby residues strategically mutated to optimize the peroxidase activity (i.e., Sav S112E K121H). We hypothesize that these two residues mimic the polar residues in the second coordination sphere, involved in activating the bound peroxide in two very widely studied peroxidases: chloroperoxidase (CPO) (i.e., Glu 183 and His 105) and horseradish peroxidase (i.e., Arg 38 and His 42). Despite the absence of a tightly bound axial ligand, which can exert a “push effect”, the evolved artificial peroxidase exhibits best-in-class activity for oxidizing two standard substrates (TMB and ABTS) in the presence of hydrogen peroxide.