Mutations to sidechains prone to binding metal ions. These residues may compete or synergize with Glu4 and His7 for metal binding to create novel metal sites. A8C. The A8C mutation introduces an ionizable thiol group that has a propensity to coordinate with soft or borderline ions like Co(II). Analysis of SC-XRD data of the blue crystals reveals that Co- MAHF-9 A8C contains trigonal pyramidal 2His-2Cys ligated Co(II) centers, where all Glu4 residues are non-interacting (Figure ). The 2His-2Cys ligation is found in most zinc finger proteins. The Co-S bond distances of 2.2 and 2.3 Å, which are consistent with a Co(II)-thiolate bond. Since no other cations were identified in the crystal structure, this implies that the Glu4 residues are protonated and neutral. Even though the thiol group on Cys has a pKa ~8 while the carboxylic acid group of Glu has a pKa of ~5, the strength of the metal-thiolate coordination can drive the deprotonation of the thiol.