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 N_δ-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.

非规范氨基酸配体可用于微调金属酶的催化性能。在这里，我们表明，重组置换的组氨酸配位体近端血红素在肌红蛋白与Ñ _δ -methylhistidine增强蛋白的混杂碳烯-转移化学，使在不存在还原剂的高效苯乙烯环丙烷化，甚至在有氧条件下。修饰的Fe（iii）中心的亲电子性增强，加上活性位点的细微结构调整，使得重氮乙酸乙酯可以直接进攻，生成反应性类胡萝卜素加合物，该桥具有非常规的Fe（iii）桥联）–C–N（吡咯）构型，如X射线晶体学所示。量子化学计算表明，桥联的络合物与反应性更高的端基异构体平衡，从而确保有效的环丙烷化。这些发现强调了非规范配体通过开拓新的反应途径并促进表征原本不会积累的反应性物种来扩展金属酶能力的潜力。