Directed evolution enhances the peroxidation activity of myoglobin reconstituted with an iron corrole complex

Engineered hemoproteins incorporating synthetic metal porphyrinoids with non-natural tetrapyrrole frameworks are known to exhibit unique catalytic properties which are not observed in native hemoproteins. We previously reported the reconstitution of myoglobin with an iron corrole complex (Mb-FeCor) leading to unique catalytic activity in H2O2-dependent peroxidation. To optimize the amino acid sequence of the active site of myoglobin for this noncanonical corrole complex, we performed genetic engineering of Mb-FeCor based on the directed evolution methodology. After 3 rounds of directed evolution, a triple mutant Mb(L89F/H97F/I107F)-FeCor was obtained as a best-performing catalyst, exhibiting a 24-fold higher initial rate for ABTS peroxidation relative to the original Mb-FeCor. Furthermore, the kcat/KM value of this engineered variant was calculated to be 16 μM–1s−1, exceeding that of horseradish peroxidase (HRP, 0.12 μM–1s−1). These results demonstrate that combining synthetic porphyrinoid incorporation with directed evolution of a protein scaffold provides an efficient strategy for improving the catalytic activity of hemoprotein-based artificial metalloenzymes.