Interestingly, the closest residue to the active site, H236, is not conserved among these enzymes. In fact, the only enzymes in which residue 236 is histidine are GRLMR and SeR --the two Se specialized reductases present in this analysis. This implicates H236 in selectivity of chalcogenide substrates -both glutathione ligated and oxyanions --where GRLMR out-performs GSHR in terms of catalytic efficiency for the metal(loid) reduction. Similar to residue 236, the mutation in the SeR variant -L314H also occurs at a non-conserved amino acid position. Sequence alignments for these enzymes can be found in the Supporting Information. This analysis reveals that SeR, interestingly, positions 2 histidine residues within 5Å of the enzyme active site (H236, H314). This location of two histidine residues proximal to the enzyme active site is likely to be significant for catalytic efficiency toward Se substrates. Histidine is an aromatic residue that is well-known to interact with metal cations (e.g., sequential His residues form the basis for immobilized metal affinity chromatography ). As an aromatic residue it participates in π-π interactions; addition of a nearby aromatic residue in SeR, coupled with the H236 already present in GRLMR (and retained in SeR) facilitates π-π interactions between Se substrates and the enzyme. The two active-site proximal histidine residues may help to position the SeO3 2-and other Se substrates through π-Se interactions in a way that makes both Km (substrate binding) and kcat (substrate->product rate) significantly more favorable . Furthermore, the size increase in a mutation of leucine to histidine may disfavor the active-site approach of GSSG and GS-Se-SG, which are significantly larger substrates than selenite. This 'steric bulk' hypothesis is supported by the enzyme kinetic analysis, where changes in Km are significant in the evolved enzyme for GS-Se-SG and GSSG, whereas the changes in kcat for these substrates are antagonistic but small. Combined the L314H mutation appears to underlie all of the observed changes in SeR catalytic efficiency relative to SeO3 2-, GSSG and GS-Se-SG.