Engineered hemoproteins containing non-canonical cofactors toward artificial metalloenzymes

Hemoproteins have emerged as versatile scaffolds for the construction of artificial metalloenzymes. Through directed evolution via random and/or site-saturation mutagenesis, these proteins can be repurposed to catalyze abiological transformations. Their catalytic scope can be further expanded by introducing non-canonical molecular components. One approach involves the incorporation of non-canonical amino acid residues, such as methylhistidine, into the protein scaffold. Another strategy replaces the native heme with synthetic cofactors. While natural heme cofactors are generally restricted to porphyrins, synthetic chemistry has enabled access to a variety of porphyrin derivatives and artificial porphyrinoids with diverse core structures and peripheral functionalities. This review highlights recent efforts in designing such non-canonical cofactors and engineering complementary protein mutants to achieve challenging transformations, including C–H hydroxylation/amination and olefin cyclopropanation. Expanding the chemical space of hemoproteins through the integration of non-canonical cofactors represents a promising direction toward artificial metalloenzymes with novel and valuable catalytic functions. Engineered myoglobins containing non-canonical cofactors catalyze abiological transformations such as cyclopropanation, C–H amination, and hydroxylation. Rational cofactor and protein design enables precise control of reactivity and selectivity, expanding the catalytic potential of hemoproteins beyond their native functions. • Non-canonical cofactors expand hemoprotein reactivity beyond natural functions. • Redox-tuned artificial hemes enable aerobic cyclopropanation of inert olefins. • Myoglobin-porphycene catalysts achieve TON over 50,000 in C–H bond amination. • Hybrid catalysis integrates enzymes and nanoparticles for dioxygen-driven oxidation. • Molecular dynamics-based protein design enhances enantioselective C–H hydroxylation.