LAUSR

Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt‐containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal‐binding proteins through the formation of cobalt‐ligand bonds. Since most of these natural and artificial cobalt‐containing proteins are stable, we believe that these cobalt‐ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS) to directly measure the rupture force of Co(II)‐thiolate bond in Co‐substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling‐based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)n suitable for single‐molecule studies. Thus, we quantified the strength of Co(II)‐thiolate bonds in rubredoxin with a rupture force of ∼140 pN, revealing that it is a mechanostable chemical bond. In addition, the Co−S bond is more labile than the Zn−S bond in proteins, similar to the result from the metal‐competing titration experiment.