Probing anion recognition in a cobalt(II) de novo designed metalloprotein

Anions such as halides and pseudohalides influence metal-site structure and function. De novo designed metallocoiled coils offer a defined platform for studying how metal centers recognize small anions within an α-helical scaffold. Spectroscopic examination of anion binding to three-stranded coiled coils (3SCCs) using artificial cobalt(II) substituted carbonic anhydrases (CA) is used as an analogue of the zinc(II) center. Scaffolds composed of three equivalents of GRW-H (Ac-GWKALEEKLKALEEKLKALEEKLKALEEKHKALEEKG-NH₂) yield a cobalt(II)(His)₃ site whose visible spectrum can be perturbed by nitrite, azide, and thiocyanate, producing significant ligand-field spectral changes that reveal these ions bind with millimolar affinities. These modifications reflect similar chemistry to that observed for cobalt(II)-substituted CA. X-ray absorption spectroscopy confirms that thiocyanate coordinates through nitrogen, converting a 6-coordinate cobalt(II)(His)₃(H₂O)_3-x(OH^-)_x (with x = 0 or 1) species at pH ≤ 9 to a five-coordinate cobalt(II) center. pH-dependent measurements reveal a factor of 2 affinity increase for thiocyanate binding as solution basicity increases, with a pKa ∼ 8.0 consistent with a single deprotonation event. This strengthening of the binding constant does not arise from thiocyanic acid acidity or cobalt hydrolysis and likely reflects deprotonation of a protein residue(s). In contrast to thiocyanate or azide, halides (chloride through iodide) bind much more weakly. The spectral parameters observed vary with anion properties and reflect distinct cobalt(II) geometries. Overall, these results define how a simple His₃ site embedded in a designed protein scaffold recognizes anions and adopts distinct geometries, providing a foundation for designing metalloproteins that activate small inorganic substrates.