How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase

Abstract

Conspectus “What I cannot create, I do not understand”Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme. ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto’s acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme’s oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity. To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a “Y”-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron org...

Site specific redox properties in ligand differentiated di-nickel complexes inspired by the acetyl CoA synthase active site

“…This assembly performs the catalytic conversion of CO, CH 3 + , and Coenzyme-A fragments into Acetyl-Coenzyme A (Acetyl-CoA), akin to the industrial Monstanto Process. [8][9][10][11] Fig. 1 depicts the active site of ACS known as the A-Cluster, which consists of two distinct nickel centers bridged to a conventional 4Fe4S cluster by a cysteinyl thiolate.…”

“…1 Active site of acetyl CoA synthase. [8][9][10][11] chemistry, the Ni(CGC) acts as a metallodithiolateligand to a second catalytically active nickel, referred to as Ni p ; i.e., Ni p is proximal to the 4Fe4S cluster. The Ni p is coordinated by three bridging thiolates in its resting state: two originate from the Ni (CGC) and one from a cysteine residue that bridges Ni p to the 4Fe4S cluster.…”

“…While Ni d occupies a tightly bound site within the CGC motif, nature has fine-tuned the ligand environment such that Ni p exhibits greater flexibility with changes in redox levels, expected to be facilitated by the redox-active iron sulfur cluster during catalysis. [8][9][10][11] Our primary objective was to create a platform specifically tailored for integrating redox preferentiality into dinickel complexes related to the ACS active site. By doing so, we aimed to gain a comprehensive understanding of how different coordination environments impact the redox properties of these complexes.…”

“…While Ni d occupies a tightly bound site within the CGC motif, nature has fine-tuned the ligand environment such that Ni p exhibits greater flexibility with changes in redox levels, expected to be facilitated by the redox-active iron sulfur cluster during catalysis. 8–11…”