Structure of Mb NMH H64V, V68A mutant complex with EDA

Abstract

Non-canonical amino acid ligands are useful for fine-tuning the catalytic properties of metalloenzymes. Here, we show that recombinant replacement of the histidine ligand proximal to haem in myoglobin with Nδ-methylhistidine enhances the protein’s promiscuous carbene-transfer chemistry, enabling efficient styrene cyclopropanation in the absence of reductant, even under aerobic conditions. The increased electrophilicity of the modified Fe(iii) centre, combined with subtle structural adjustments at the active site, allows direct attack of ethyl diazoacetate to produce a reactive carbenoid adduct, which has an unusual bridging Fe(iii)–C–N(pyrrole) configuration as shown by X-ray crystallography. Quantum chemical calculations suggest that the bridged complex equilibrates with the more reactive end-on isomer, ensuring efficient cyclopropanation. These findings underscore the potential of non-canonical ligands for extending the capabilities of metalloenzymes by opening up new reaction pathways and facilitating the characterization of reactive species that would not otherwise accumulate. A haem–carbenoid has been proposed to be involved in abiological enzymatic reactions. Now, Hilvert and co-workers provide crystallographic evidence for a haem–carbenoid intermediate as the reactive species in an olefin cyclopropanation reaction catalysed by an artificial metalloenzyme.