Fig. 6: Heme binding to the ancestral glycosidase.

a UV–VIS spectra for preparations of the ancestral glycosidase showing the protein absorption band at about 280 nm and the absorption bands due to the heme (the Soret band at about 400 nm and the Q bands at higher wavelengths). Black color is used for the protein obtained using the original purification procedure without the addition of hemin or hemin precursor. Blue and red are used to refer, respectively, to preparations in which hemin and 5-aminolevulinic acid (the metabolic precursor of heme) were added to the culture medium. b Binding of heme to the ancestral glycosidase in vitro as followed by changes in VIS spectrum. Spectra of a heme solution 1 μM in the absence (black) or presence (red) of a similar concentration of ancestral protein. The “flat” Soret band of free heme is linked to its self-association in solution, while the bound heme is monomeric and produces a sharper Soret band. c Kinetics of binding of heme to the ancestral glycosidase as followed by the increase in enzyme activity (rate of hydrolysis of 4-nitrophenyl-β-glucopyranoside; see Methods for details). In the three experiments shown a heme to protein molar ratio of 1.2 was used. The protein concentration in each experiment is shown. Note that activity increase is detected even with concentrations of 50 nM, indicating that binding is strong. The lines are meant to guide the eye and thus have no quantitative purpose. (d) Plot of enzyme activity versus [heme]/[protein] ratio in solution for a protein concentration of 1 μM. Activity was determined after a 5 min incubation and the plot supports a 1:1 binding stoichiometry. v/[E]₀ stands for the rate over the total enzyme concentration.