Incorporating Co-Center Heme Mimics Into Engineered Biocatalysts: Expanding the Catalyzing Capabilities of P450s

Cytochrome P450s (P450s), a superfamily of enzymes with a heme active site, are effective catalysts for a variety of regio/stereoselective, late-stage modifications on non-specific substrates. Recently, a bioengineering focus has been put on P450s in attempts to tap into the rich functionalization properties of the natural biocatalysts. Research groups have shown an evolved species of P450s can catalyze enantioselective C-N and C-C bond formations. Some cobalt (Co) complexes have been shown to effectively catalyze these reactions as well or better than the iron counterparts. These Co complexes have also been shown to perform O-H, B-H, and C-H insertions, typically difficult for Fe catalysts, but lack stereoselectivity. We hypothesize that Co-cofactors, with a similar planar porphyrin ring structure to heme, may be substituted into engineered P450s to efficiently catalyze a variety of reactions with stereoselectivity dictated by the P450s. To confirm proper incorporation of these Co-cofactors into the evolved P450s, as well as visualize intermediates of potential mechanisms, various techniques, including Raman spectroscopy, Electron Paramagnetic Resonance (EPR), and Inductively Coupled Plasma Mass Spectrometry (ICP-MS), were used to evaluate the spin state, oxidation state, and coordination of the metal center. By validating the incorporation of these Co complexes into P450s, a new class of reactions can be explored by P450s with applications to drug development and human health.