Enzymatic Metal–Hydrogen Atom Transfer with a Cobalt Protoporphyrin Cofactor

Introduction of unnatural cofactors in biocatalysis may open the door to new reactive enzymatic intermediates, and in turn, new biochemical reactions. Here, we employed a de novo biosynthesized, non-natural cofactor, cobalt protoporphyrin IX (CoPPIX), to generate a mononuclear cobalt hydride in the active site of CYP119, a model P450 enzyme. We show that this cobalt hydride intermediate engages in metal-hydrogen atom transfer (MHAT) reactivity, a well-studied and highly utilized reactivity pattern in synthetic chemistry, but which is not known to operate in Nature. We paired convenient in vivo CoPPIX biosynthesis with a colorimetric screen to enable rapid directed evolution. Thus, we engineered CYP119 for MHAT-mediated deallylation of nitrophenols, with the goal of generating not one prolific catalysis, but a diverse set of MHAT-compatible enzymes. Because many silanes hydrolyze quickly, we additionally sought enzymes that accelerate metal-hydride formation from a more persistent silane. This evolution yielded 80 diverse active site recombinants that catalyze MHAT. Serendipitously, we found many variants reduced the aromatic ring of the colorimetric probe, a reaction not previously known. Detailed mechanistic analysis established this is a radical, MHAT-mediated reductive dearomatization that occurs efficiently under aerobic conditions, albeit on a limited suite of nitrophenyl ethers. These results lay a framework for further engineering and study of biocatalytic MHAT and the unique role of metal substitution to tune reactivity.