Volume 279, June 2026, 113268Author links open overlay panel, , , Highlights•

A de novo cobalt(II) metalloprotein enables spectroscopic evaluation of anion binding.

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Anion binding perturbs six-coordinate octahedral geometry at the cobalt(II) site.

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Protein matrix defines anions affinity and selectivity, showing primary sequence effects.

Abstract

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.

Graphical abstractA de novo designed cobalt(II) metalloprotein mimicking carbonic anhydrase reveals distinct anion recognition trends. Spectroscopic studies show how different anions modulate metal coordination geometry, highlighting the role of the protein environment in shaping metal–anion interactions.Download: Download high-res image (172KB)Download: Download full-size imageKeywords

De novo designed metalloproteins

Three-stranded coiled coil

Cobalt(II) coordination chemistry

Anion binding

© 2026 The Authors. Published by Elsevier Inc.