Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme**

Abstract

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N_δ -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced E^o values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.