Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3+ peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process. The Co(II) analogue of the designed peptide Zn(II)(TRIL23H)3 is made as a spectroscopic probe for the carbonic anhydrase activity exhibited by this de novo designed system. The coordination geometry of Co(II) and Zn(II) are vastly different at high and low pH. Nonetheless, the Co(II) analogue at high pH exceeds the catalytic efficiency of the parent Zn(II) protein. image

While many life-critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three-stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His)3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper-catalyzed nitrite reductase activity (CuNiR). In contrast, the well-established zinc-catalyzed carbonic anhydrase activity (p-nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non-coordinating and non-acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(II)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(II)A2B heterotrimers, while d sites provide pure Pb(II)C2D or Pb(II)CD2 scaffolds. Altering the metal from Pb(II) to Hg(II) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(II)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

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: H-3 (His(3)), H-4 (His(4)), H2DH (His(3)Asp with two His and one Asp in the same plane) and H3D (His(3)Asp with three His in the same plane) by using the scaffold of the de novo protein GR alpha D-3. 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.