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The development of redox-active metalloprotein catalysts is a challenging objective of de novo protein design. Within this Perspective we detail our efforts to create a redox-active Cu nitrite reductase (NiR) by incorporating Cu into the hydrophobic interior of well-defined three-stranded coiled coils (3SCCs). The scaffold contains three histidine residues that provide a layer of three nitrogen donors that mimic the type 2 catalytic site of NiR. We have found that this strategy successfully produces an active and stable CuNiR model that functions for over 1000 turnovers. Spectroscopic evidence indicates that the Cu(I) site has a lower coordination number in comparison to the enzyme, whereas the Cu(II) geometry may more faithfully reproduce the NiR type 2 center. Mutations at the helical interface successfully produce a hydrogen bond between an interfacial Glu residue and the Culigating His residue, which allows for the tuning of the redox potential over a 100 mV range. We successfully created constructs with as much as a 120-fold improvement from the original design by modifying the steric bulk above or below the Cu binding site. These systems are now the most active water-soluble and stable artificial NiR catalysts yet produced. Several avenues for improving the catalytic efficiency of later designs are detailed within this Perspective, including adjustment of their resting oxidation state, the use of asymmetric scaffolds to allow for single amino acid mutation within the second coordination sphere, and the design of hydrogen-bonding networks to tune residue orientation and electronics. Through these studies the TRI-H system has given insight into the difficulties that arise in creating a de novo redox active enzyme. Work to improve upon this model will provide strategies by which redox-active de novo enzymes may be tuned and detail how native enzymes accomplish catalytic efficiencies through proton gated redox catalysis.

Development of de Novo Copper Nitrite Reductases: Where We Are and Where We Need To Go