Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Abstract

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redoxâ 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 0.36â V versus the normal hydrogen electrode (NHE). Rationally designed proteins with wellâ defined threeâ dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copperâ binding scaffolds: H3 (His3), H4 (His4), H2DH (His3Asp with two His and one Asp in the same plane) and H3D (His3Asp with three His in the same plane) by using the scaffold of the de novo protein GRα3D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good CuIIâ bound structural mimics of Cuâ 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.Artificial defenders: Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. This work describes the first models of Cuâ only SOD in a de novo designed peptide including structural and kinetic analyses. A comparison of the copper ligand type and position is discussed, highlighting our ability to mediate the metalâ binding environment.