Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloprotein

Changing the primary metal coordination sphere is a powerful strategy for modulating metalloprotein properties. Taking advantage of this approach, we have replaced the proximal histidine ligand in myoglobin with the histidine analogues N_d-methylhistidine (NMH), 5‑thiazoylalanine (5ThzA), 4-thiazoylalanine (4ThzA) and 3-(3-thienyl)alanine (3ThiA) by amber stop codon suppression using engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein’s promiscuous carbene transfer activity with ethyl diazoacetate. Myoglobin variants with increased reduction potentials (NMH and 5ThzA) proved superior for cyclopropanation and N-H insertion, especially under aerobic conditions, and could even promote these reactions in the absence of reducing agent. In contrast, the variants with the lowest E^o values (4ThzA and 3ThiA) exhibit comparatively high S-H insertion activity even though the respective histidine surrogates do not coordinate the heme iron. Given the important functional roles played by histidine in many enzymes, these genetically encoded histidine analogues represent valuable tools for probing mechanism and enabling new chemistries in metalloproteins.