Enzymatic metal-hydrogen atom transfer with a cobalt protoporphyrin cofactor

Biocatalysts are prized for their selectivity, tunability, and their compatibility with environmentally-friendly reaction conditions. Introduction of unnatural cofactors opens the door to new reactive enzymatic intermediates, and in turn, the possibility for new biochemical reactions. In the present study, we employed a de novo biosynthesized, non-natural cofactor, cobalt protoporphyrin IX,1 to generate a mono-nuclear cobalt hydride intermediate in the active site of a common P450 scaffold. We show that this cobalt hydride intermediate engages in metal-hydrogen atom transfer (M-HAT) reactivity, a well-studied and highly utilized reactivity pattern in synthetic chemistry,2 but which is not known to operate in nature. Because the required cofactor is fully biosynthesized and incorporated into proteins in vivo, the catalysts are highly amendable to directed evolution. We leveraged the ability to quickly access these new artificial metalloenzymes with a colorimetric screen and evolved new variants for M-HAT-mediated deallylation of phenols. We showed how common silanes have a propensity to hydrolysis that can be overcome with directed evolution by accelerating metal-hydride formation from a bulky, water stable silane. During this evolution, we discovered that variants were catalyzing HAT to the colorimetric probe itself, resulting in a unique reductive dearomatization reaction. This radical process occurs efficiently under aerobic conditions and reactions of this type have not been observed previously. These discoveries demonstrate how the tunability of biocatalytic systems can enable innovations in synthetic chemistry. We anticipate that further engineering and study of M-HAT biocatalysts will prompt new questions about hydrogen atom transfer reactivity and enable the adoption of biocatalysts for numerous synthetically useful transformations.