Designing multifunctional peroxidases by modifying the heme distal site in myoglobin

Significant achievements have been made in the design of functional metalloenzymes. However, more convenient and versatile approaches are still needed to design multifunctional heme enzymes with broader substrate scope for potential applications such as bioremediation. Using myoglobin (Mb) as a model protein, we herein developed a convenient approach by introducing a native Lys/Arg residue in the heme distal site or covalent modification of a genetically introduced Cys residue with a non-native functional group. The engineered Mbs were structurally characterized by X-ray crystallography and functionally by kinetic spectroscopic studies. These artificial enzymes were found to exhibit multifunctional activity, including dye-decolorizing peroxidase and dehaloperoxidase activities, even exceeding those of natural enzymes. This study provides insights into the dynamic structure that controls the catalytic function of the artificial heme enzymes with multiple peroxidase activities. This approach is also applicable for creating other functional metalloenzymes with potential applications in environmental remediation and biocatalysis.