Fig. 8. Evolutionary degradation of ancestral heme binding.

a Section of the phylogenetic tree used for the Bayesian analysis of family 1 glycosidases. The nodes in the evolutionary trajectory from node 72 to Halothermothrix orenii (labeled “MODERN”) are highlighted and labeled. See Fig. S1 for a complete and detailed version of the phylogenetic tree. b, c Ratio of absorbance (Abs) at the maximum of the heme Soret band to the absorbance at the maximum of the protein aromatic absorption band for ancestral (see panel A) and modern family 1 glycosidases. Data in (b) correspond to protein preparations in which 0.4 mM 5-aminolevulinic acid (the metabolic precursor of heme) was added to the culture medium and the protein was purified by Ni-NTA affinity chromatography and further passage through a PD10 column (see Figs. S20 and S21 for further detail). For the data in (c), heme-free protein samples at ∼5 μM were incubated for 1 h at pH 7 with a 5-fold excess of heme and free heme was removed through size exclusion chromatography (2 passages through PD10 columns) before recording the UV–VIS spectra (Fig. S23). d Profiles of protein concentration (bicinchoninic acid method³³), heme concentration (pyridine hemochrome spectrum³) and glycosidase activity (with 0.25 mM 4-nitrophenyl-β-D-glucopyranoside) upon elution from gel filtration chromatography (HiLoad 16/600 Superdex 200 pg GE Healthcare). For these experiments, heme was gradually added to ∼30 μM samples of ancestral (N72, N73, and N74) and modern (Halothermothrix orenii) glycosidases up to a ∼5-fold excess and, after several hours, free heme was removed using PD10 columns. The elution volume for the main protein concentration peak is consistent with the monomeric association state (Fig. S3).