<a>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<sub>d</sub>-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 <i>E</i><sup>o</sup> 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
metalloprotein</a>s.
An artificial metalloenzyme for carbene transfer based on a biotinylated dirhodium anchored within streptavidin
