A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O-2-dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.

Automated summary

De novo metalloprotein design is a remarkable method for shaping protein structures to achieve specific functions. In this study, a protein called DR1 was designed and characterized to harbor a di-copper site, mimicking copper-containing polyphenol oxidases. The di-copper site was engineered into a four-helix bundle scaffold through hierarchical design of metal coordination spheres. Spectroscopic, thermodynamic, and functional analyses demonstrated that DR1 exhibited the characteristics of a Type 3 copper site, supporting different copper redox states and catalyzing the oxygen-dependent oxidation of catechols. Careful design of substrate access site residues endowed DR1 with substrate recognition capability.