# Enzymatic Metal–Hydrogen Atom Transfer with a Cobalt Protoporphyrin Cofactor

## TL;DR

Researchers introduced a non-natural cobalt cofactor into an enzyme to enable a new type of chemical reaction called metal–hydrogen atom transfer, which could expand the range of biochemical transformations.

## Contribution

The study demonstrates the first enzymatic metal–hydrogen atom transfer using a cobalt hydride intermediate in a P450 enzyme.

## Key findings

- Directed evolution produced 80 active site variants of CYP119 capable of catalyzing MHAT.
- Some enzyme variants unexpectedly reduced aromatic rings via radical-mediated reductive dearomatization.
- The MHAT reactivity was shown to occur efficiently under aerobic conditions on specific nitrophenyl ethers.

## Abstract

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.