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

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 O2‐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.

Engineering bio‐brick protein scaffolds for organizing enzyme assemblies

“…The formation of larger assemblies for FDH/AlaDH@S-CAB C may be originated by the covalent nature of the disulfide staple that shifts the equilibrium towards higher ordered assemblies as illustrated in Figure 5d. In contrast, FDH/AlaDH@SCAB H assemblies are mediated by weaker non-covalent interactions dominated by the equilibrium with a K D in the order of μM for copper:bihistidine interactions (Pirro et al, 2023), which, as shown experimentally results in discrete assemblies.…”

Mechanism of non-phenolic substrate oxidation by the fungal laccase Type 1 copper site from Trametes versicolor: the case of benzo[a]pyrene and anthracene

“…This theoretical scheme has already been tested and successfully adopted in previous DFT investigations carried out in our laboratory on various bioinorganic copper systems. [72][73][74]…”

Controlling Reactivity and Electron Transfer in De Novo Designed Artificial Cu Proteins by Systematic Primary, Secondary, and Outer Sphere Modulation

“…Indeed, recent success has been achieved in creating functional arti cial metalloenzymes (ArMs) within de-novo scaffolds. [39][40][41][42][43][44][45][46][47][48] One such example is the novo-designed arti cial Cu proteins (ArCuPs) that function as peroxidases and peroxygenases featuring Cu(His) 3 active sites within trimeric (3SCC) self-assemblies. 45,46 The 3SCC ArCuP activates H 2 O 2 45 and electrocatalyzes the 2eand 4eoxidation and peroxygenation.…”