Abstract
De novo metalloprotein design has contributed to tremendous advances in bioinorganic chemistry by allowing the manufacturing of proteins with unique structures and functionalities that go beyond evolutionary constraints. Among the array of metal sites that can be engineered within de novo scaffolds, the design of catalytic copper centers is particularly challenging but still harder to achieve due to the versatile coordination environment and redox properties of the copper ion. Here, we present miniLPMO, a fully de novo protein, incorporating a functional histidine brace copper-binding site. Starting from a four-helix-bundle scaffold based on the designed homodimeric α(2)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.