A8H. An A8H mutation introduces an additional metal-binding imidazole ring. Analysis of SC-XRD data demonstrates that distorted octahedral metal sites in Co-MAHF-9 A8H are ligated by 4His residues (two from His7 and two from His8) and capped with a κ 2 -acetate (derived from Co(OAc)2) (Figure ). All Glu4 residues are noncoordinating (based on bond valence sum analysis), suggesting that half the Glu residues must be protonated and neutral, yielding a stoichiometry of Co(peptide) 0 (peptide) -(OAc). The 4His ligation is very similar to the one found in the non-heme iron enzyme, EgtB. 37 A8D. The A8D mutant introduces an additional carboxylic acid. Analysis of SC-XRD data of Co-MAHF-9 A8D reveals distorted tetrahedral centers with a 1His-2Asp-1Glu ligation (Figure ). To balance the overall charge, the non-ligated Glu4 and His7 on chain B are proposed to be protonated, resulting in a cationic and neutral sidechain, respectively. Asp8 on chain A and B make H-bonds with His7 of chain B. The 1His-3carboxylate binding has been seen in proteins like the zinc transporter protein, YiiP. 38 A8E. Only pink microcrystalline powder was obtainable for Co- MAHF-9 A8E. The powder is moderately crystalline as evidenced by the peaks visible in its PXRD pattern, however, too few peak positions are visible to allow for accurate unit cell indexing (Figure ). Effect of metal ion. Due to the differences in ionic radii and electronic configurations, different metal ions have preferential coordination numbers and geometries. Various 3d transition metal ions were combined with MAHF A8S to investigate the competing effects of H-bonding and metal preferences on framework structure (Figure ). Mn-MAHF-9 A8S. Substitution of Co(II) for the larger Mn(II) ion (ionic radius of 0.89 Å versus 0.72 Å) in the framework synthesis with MAHF A8S indeed forms a new structure. Analysis of SC-XRD data of Mn-MAHF-9 A8S reveals a square pyramidal geometry, rather than distorted tetrahedral sites as observed in Co-MAHF-9 A8S. Previously observed H-bonding between Ser8 and His7 in the case of Co(II) are absent with Mn(II). This change is likely because Mn(II), being larger than Co(II), prefers a higher coordination number, even if that results in the loss of H-bonding from Ser8. Fe-MAHF-9 A8S. Fe(II), with an ionic radius of 0.77 Å, is in between the size Mn(II) and Co(II), could favor either higher coordination or a derivation similar to Co(II) binding. Interestingly, analysis of SC-XRD data of Fe-MAHF-9 A8S shows a framework containing both 4-and 6-coordinate metal sites. The 4-coordinate sites are very distorted from tetrahedral, with a τ of 0.5, being closer to a see-saw geometry. The 6-coordinate site is octahedral achieved by κ 2 binding of Glu4. The unit cell is also larger featuring 12 chains and 6 distinct Fe sites in the ASU to accommodate both coordination numbers. There is also significant disorder involving entire chain displacements, attesting to the frustration throughout the framework. In the predominate conformation, H-bonding only exists between His7 of chain K and Ser8 of chain J, since the His7/Ser8 contacts in other chains are slightly too far apart (> 3.2 Å cutoff for a Hbond). The unique structure demonstrates that major competing forces of peptide interactions and metal preference influence the formation of frameworks. Cu-MAHF-9 A8S. The strong Jahn-Teller effect of Cu(II) precludes a tetrahedral geometry. As expected, analysis of SC-XRD data shows that Cu-MAHF-9 A4S has square planar Cu(II) sites with a 2His-2Glu ligand environment. This framework lacks direct Hbonding of Ser8 with another sidechain, again demonstrating that metal electronic effects preferences can outweigh H-bonding effects. Zn-MAHF-9 A8S. The metal ion Zn(II) forms a framework with tetrahedral metal nodes, but interestingly, the Zn(II) framework topology is very different from that made with Co(II), even though both contain 4-coordinate sites broadly classified as tetrahedral. The most prominent contrasting features found in the Zn framework are helices bridging metal centers, helices across the channel being parallel, and lack of Ser8-His7 H-bonding. Given that the ionic radii of 4-coordinate Zn(II) and high-spin Co(II) are similar (0.74 and 0.72 Å, respectively), ion size is not likely the main reason for the differences. One possible reason is that the d 10 Zn(II) ion strongly prefers an idealized tetrahedral geometry to minimize d-orbital overlap with ligands, whereas the d 7 Co(II) ion is more tolerant of geometric distortions. This rationale is supported by the τ parameters of the metal centers in the MAHF-9 A8S frameworks, with Zn(II) having a τ of 0.88 and Co(II) having a τ of 0.78.