Repurposing a Nitric Oxide Transport Hemoprotein Nitrophorin 2 for Olefin Cyclopropanation

A growing number of heme proteins have recently been repurposed for catalyzing abiological carbene transfer reactions. Herein, we rationally designed an engineered variant of nitrophorin 2 (NP2)─a nitric oxide transport hemoprotein─that catalyzes olefin cyclopropanation with high activity and stereoselectivity. Being a β-barrel protein, the engineered NP2 variant showed a unique substrate preference, in contrast to the mainstream α-helical carbene-transfer heme enzymes like cytochrome P450 enzymes and myoglobin. The catalytic reactions can be carried out on a preparative scale while maintaining the stereoselectivity. The stereoselectivity of the NP2-catalyzed styrene cyclopropanation was further supported by quantum chemical calculations, and the significance of key residues was elucidated. As such, this work establishes NP2 as a robust lipocalin scaffold amenable for carbene-transferase development, complementing the current biocatalytic toolbox.