Fig. 1.
Active site structures of R2lox variants in the non-activated reduced (left panel) and oxidized resting state (right panel). All structures are shown in roughly the same orientation, with site 1 on the left. Mutated residues are highlighted in orange. Residues in alternate conformations are labeled in red. Metal–ligand bonds are indicated by grey lines, hydrogen bonds by dashed blue lines. The insets show mFo-DFc refined omit electron density contoured at 3.0 σ for residues 72 and 162 in oxidized state crystals. a wt-R2lox (reduced state, PDB ID 4HR4; oxidized state, PDB ID 4HR0) [23]: an ether cross-link is formed between the Cβ of V72 and the phenolic oxygen of Y162 in the oxidized state. b V72A-R2lox: no ether cross-link is observed. c V72L-R2lox: no ether cross-link is formed. In the reduced state, E167 is observed in two alternate conformations, one leading to a hexacoordinate Fe ion, the other leaving one coordination site vacant (the metal–ligand bonds of both alternates are indicated). d V72I-R2lox: in the reduced state, both V72I and Y162 are best modeled as adopting two alternate conformations. In the oxidized state, an ether cross-link is formed between the Cβ of I72 and Y162, as indicated by the electron density and verified by mass spectrometry. The bond is unrealistically long because it is not present in all molecules in the crystal and was, therefore, not modeled in the final structure, but is indicated here for clarity. For the same reason, the isoleucine is modeled as a rotamer which best fits the density, but which it is unlikely to adopt in the cross-linked state