Preparing and probing DE novo designed metalloenzyme catalysis

The National Institute of General Medical Sciences awarded the Regents of the University of Michigan $2.145 million on September 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to design and test de novo metalloenzymes that replicate the active sites and catalytic functions of natural metalloproteins.

The award funds research into how metal coordination environments affect metal binding and catalysis within designed alpha-helical proteins and coiled-coil scaffolds. The investigator will systematically evaluate metal behavior in well-defined proteins to isolate individual factors governing structure and reactivity, comparing results across different coordination chemistry that would be difficult or impossible to assess using native proteins or water-insoluble small-molecule models. The long-term goal is to develop a comprehensive chemical understanding of metal-protein interactions sufficient to design novel catalysts performing specific reactions—such as superoxide dismutation and peroxidation chemistries—by varying metal identity while maintaining the same protein ligands.

Work is performed in Ann Arbor, Michigan through August 31, 2030. The award is structured as a Project Grant, a mechanism providing about nine months for review and award.

Abstract

Metalloproteins catalyze biochemical reactions that are critical for cellular functions, including energy production, metabolism, and signaling. Despite their importance, the fundamental relationship between the structure of these proteins and their catalytic efficiency remains poorly understood, primarily due to the complex nature of native metalloenzymes and the solvent or synthetic limitations inherent in small molecule models. The interplay between protein structure and catalytic activity forms the foundation of our work. This proposal seeks to bridge these gaps using an intermediate approach with DE novo design of α-helical proteins, providing straightforward, water-soluble scaffolds that not only replicate the active sites of natural metalloproteins but are also amenable to precise modifications. These designed structures offer a robust framework to systematically investigate and compare how different coordination environments affect metal binding and catalysis in aqueous peptide systems, which is challenging with small molecule models, particularly those that are insoluble in water. Our long-term goal is to attain a comprehensive chemical understanding of the interactions between metals and α-helical coiled-coil proteins (e.g., three stranded coiled coils (3SCCS)) to achieve specific metal structures that perform desired catalytic functions such as superoxide dismutation and peroxidation chemistries. Our central hypothesis is that DE novo designed metalloproteins offer a unique opportunity to evaluate metal behavior within well-defined proteins that allow for isolation of individual factors that define structure and reactivity in a manner difficult or impossible to assess using more complex native proteins. These studies form the basis for creating novel catalysts that utilize the same protein ligands but incorporate different metals, allowing for a diverse range of catalytic functions. During the five years of this grant, we will develop mononuclear proteins containing ZN, CO, CU, MN and FE that are capable of enzymatic activities including, but not limited, superoxide dismutation and peptide hydrolysis. We will moderate these activities by altering the location of the metal along the peptide sequence, inserting either non-coded amino acids or 3- and 4- amino acid inserts generating non-canonical helical assemblies and using PB(II) and as(III) to isolate unique heterotrimeric 3SCCS. We are also designing mini-heme enzymes using antiparallel 4SCCS that structural mimic peroxidases and cytochromes P450. These systems allow US to interrogate the impact of our sphere coordination of aggregate stabilities, proximal ligand binding modes and reactivity. This research is both significant and innovative, offering new perspectives on protein-metal interactions and paving the way for the development of novel catalysts. These findings will enhance our understanding of metalloenzymology by creating bioinspired, water-soluble catalysts for use in chemical and pharmaceutical industries.