The Use of Genetically Expanded and Site-Specifically Wired P450_BM3 in Bioelectrocatalysis

Cytochrome P450 enzymes (P450s) are a protein superfamily. Within this superfamily, P450_BM3 (CYP102A1) is a model system upon which many protein engineering applications have been carried out. However, this fusion protein is large, and complex making it difficult to design and engineer for practical catalysis. Moreover, this enzyme relies on the acquisition of electrons from expensive cofactors to catalyze the reaction, limiting the largescale usage of this enzyme. So far, modifications of the P450_BM3 heme domain on electrodes have been reported, but none of them exhibited catalytic activity.

In our project, we would like to supply electrons to the P450_BM3 heme domain via an electrode to avoid the use of native cofactors. To attach the enzyme on the electrode, an unnatural amino acid, propargyl-L-lysine (PrK), was site-specifically incorporated into the enzyme as an anchor point. The heme domain attached to the glassy carbon electrode can generate a direct electron transfer between the enzyme and the electrode surface. As a case study, cyclopropanation was catalyzed. If successful, our work would be the first example of P450_BM3 heme domain site-specifically wiring to an electrode for bioelectrocatalysis, allowing improvement in catalytic efficiency. In addition, although some work has been done to elucidate the mechanism of cyclopropanation, it is still not well understood, and we hope that our work will shed light on the catalytic mechanism of this reaction.