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.

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.

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