Fig. 4: Mutation of key histidine residues alters heme coordination.
a UV-Vis absorption spectra of Dri1 and amino acid variants with hemin. b Heme-binding curve of Dri1 and variants with increasing concentration of hemin. c-d, SAXS-MD best fit model superimposed onto the crystal structures (cyan) of Dri1 His21Ala (c) and Dri1 His79Ala-Arg90Ala (d) and overlaid onto DENSS envelopes calculated from SAXS to qmax = 1.0 Å-1. Model to data fit for the SAXS-driven MD models and crystal structures are shown in Supplementary Fig. 9d and j, respectively. e Superposition of dimeric structures obtained from crystallography: Dri1 (green), Dri1 His21Ala (light pink), and Dri1 His79Ala-Arg90Ala (purple). Respective hemes at the dimer interface are shown in stick representation. f–h Selected side chains of Dri1 and variants involved in heme binding and interactions are shown (sticks) and labeled. Structures were determined from crystals. f, Heme iron is axially ligated by His79 which in turn is coordinated by a tetrahedrally bound zinc ion to form the WT Dri1+heme complex. g Substitution of His21 results in a shifted interaction and a rotated heme, preventing His79 from axially ligating the heme Fe. Unlike the 2 Å distance between His79 and Fe in Dri1, the nearest side chain to the heme iron in Dri1 His21Ala is Asp20 (~4 Å). h Dri1 His79Ala-Arg90Ala shows a similar organization to Dri1, although loss of the Arg90 side chain leads to 180° rotation of the heme and orients the propionate groups toward Arg12 near the N-termini. His21 and His16 residues weakly interact with heme Fe ~4 Å away. Source data are provided as a Source Data file.
