A De Novo‐Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity
Fabio Pirro, Salvatore La Gatta, Federica Arrigoni, Antonino Famulari, Ornella Maglio, Pompea Del Vecchio, Mario Chiesa, Luca De Gioia, Luca Bertini, Marco Chino, Flavia Nastri, Angela Lombardi
Abstract
Abstract 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.