A8T. An A8T mutation, in comparison to A8S, adds a branching methyl group that is expected to impose steric stress, like in the case of the A8V mutant. Analysis of SC-XRD data of Co-MAHF-9 A8T yields similarities and differences relative to Co-MAHF-9 A8S (Figure ). The structure of Co-MAHF-9 A8T exhibits a lower crystallographic symmetry, increasing the number of peptides in the ASU from one to four. While peptides along the channel in Co-MAHF-9 A8S make continuous Ser8-His7 H-bonds, in Co-MAHF-9 A8T, only pairs of helices can achieve Thr8-His7 H-bonds. Overall, this leads to half of the Co(II) sites resembling those in Co-MAHF-9 A8S (tetrahedral 2His-2Glu) and the other half displaying octahedral geometry with 2His-1Glu coordination provided by the framework. At the octahedral site, the cobalt is bound by one κ 1 -Glu and two His ligands in a facial geometry (the other Glu is in the secondary sphere, H-bonded to a Co-water moiety). A8W. The A8W mutant contains a very bulky, bicyclic group that also possesses one H-bond donor. Trp is most related to Phe, and so, the following discussion will compare structures of the A8F and A8W variants. Analysis of SC-XRD data of the pink crystals confirms that Co-MAHF-9 A8W has a coordination site and pore shape that is very different from those observed in Co-MAHF-9 A8F, even though the two have the same helix alignments (Figure ). The metal site is octahedral, with the two peptides in the ASU providing a meridional 2Glu-1His environment. Three water molecules complete the coordination sphere, and one water is H-bonded to His7 of chain B. The extended size of Trp relative to Phe appears to promote T-shaped π-π interactions between Trp8-Trp8 and Trp8-His7 that are responsible for the structural differences. Unexpectedly, the N-H moiety of Trp does not contribute to any H-bonds. A8Y. The A8Y variant has a sidechain similar in size to that of A8F, but with an additional H-bond donating and accepting hydroxyl group. The directions of helices in Co-MAHF-9 A8Y are identical to those in Co-MAHF-9 A8F, but the framework structure is very different (Figure ). Co-MAHF-9 A8Y lacks a continuous channel, though it does have pockets occupied with six water molecules. The Co(II) centers are octahedral, with the framework providing a 2Glu-1His ligand set. The cause of these differences between Co-MAHF- 9 A8Y and Co-MAHF-9 A8F could be attributed to the fact that Tyr8 makes several H-bonds with Glu4 and His7. In particular, Hbond donation from Tyr8 prevents His7 of chain B from coordinating to the Co(II) center. . The A8N mutation introduces a carboxamide capable of multiple H-bond donations and acceptances. The channels of Co- MAHF-9 A8N have helix orientations akin to Co-MAHF-9 A8S (Figure ), however Asn8 only H-bonds with His7 in some instances. Thus, despite the sidechain having more H-bonding groups, the shape of the Asn sidechain in the context of the framework reduces the number of H-bonds made. Half of the helices are μ1 ligands (those that possess Asn8-His7 interactions) and others are μ2 ligands. The metal center is bound by 2His-1Glu in a meridional fashion. A8Q. The A8Q mutation also provides a carboxamide functionality, but it is displayed on a longer sidechain. The helix orientations of Co-MAHF-9 A8Q are the same as Co-MAHF-9 A8N, however the longer sidechain of Gln versus Asn results in new H-bonding networks and metal coordination (Figure ). The extended Gln8 on chain B makes an H-bond with a Co-OH2 moiety instead of His7 in the case of A8N mutant. Because this coordinated water is confined by H-bonding with Gln8, Glu4 lacks adequate space to bridge multiple metal ions, resulting in all μ1 peptides. Each Co center is square pyramidal with a facial 2His-1Glu set from the framework. This coordination sphere is especially important as it is found across many families of mononuclear non-heme iron oxygenases and in metalloproteases like thermolysin. Mutants with cationic sidechains. These mutants have positively charged sidechains. In addition to increasing the net charge of the framework, they are also capable H-bond donors. A8R. The A8R mutant places a cationic guanidium sidechain (pKa ~ 12) in the framework. The cationic Arg8 and anionic Glu4 results in a neutral peptide. Analysis of SC-XRD data reveals that the pores of Co-MAHF-9 A8R contain adventitious sulfate ions recruited by the cationic Arg8 (Figure ). Sulfate is derived from desulfonation of the Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) protecting groups used for arginine during solid-phase peptide synthesis. Thus, the overall stoichiometry is (peptide) 0 Co II (SO4). The octahedral Co(II) sites are coordinated to four waters, three of which are on the same face of the octahedron, in addition to 1His-1Glu. The sulfate ions within the framework make many H-bonds with the facially ligated waters, in addition to the Arg8 sidechain.