A Catalytic Binding Site together with a Distal Tyr in Myoglobin Affords Catalytic Efficiencies Similar to Natural Peroxidases

Functional enzyme design has made tremendous progress, but designer enzymes with activities comparable to those of natural enzymes are still limited. In this study, we rationally engineered a functional peroxidase with a catalytic binding site of guaiacol in a model protein, myoglobin (Mb), by replacing Phe46 with a serine (F46S mutation), together with a distal Tyr in the heme pocket (F43Y mutation). The double mutant F43Y/F46S Mb exhibited an overall catalytic efficiency that exceeds most natural peroxidases and is similar to the most efficient horseradish peroxidase. The catalytic substrate binding site was further confirmed by X-ray crystallography, EPR spectroscopy, and inhibition studies, as well as molecular docking simulations. Remarkably, this study reports an artificial peroxidase with an engineered catalytic binding site whose structure was solved both in the absence and presence of the substrate.