Engineering a Functional Histidine Brace Copper-Binding Site into a De Novo -Designed Protein Scaffold

D protein, our design has integrated rational and computational strategies to optimize coordination shell residues. Circular dichroism and analytical ultracentrifugation experiments indicate that the folding and dimerization state is driven by copper binding. A detailed characterization by UV-Vis and EPR revealed that miniLPMO replicates the spectroscopic features of natural histidine brace sites. Finally, the designed metalloprotein catalyzes the cleavage of glycosidic bonds upon hydrogen peroxide activation, mimicking the activity of natural lytic polysaccharide monooxygenases (LPMOs). This study establishes the feasibility of integrating peculiar catalytic metal-binding sites into scaffolds unrelated to the native protein and designed entirely from scratch.