Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H₃ (His₃ ), H₄ (His₄ ), H₂ DH (His₃ Asp with two His and one Asp in the same plane) and H₃ D (His₃ Asp with three His in the same plane) by using the scaffold of the de novo protein GRα₃ D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good Cu^II -bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.