Figure 2

Spectroscopic characterization of heme extraction from Hb and semiHb by wt IsdB and IsdB^Y440A. (A) Absorption spectra of 1.5 µM MetHb before (brown) and after (green) mixing with 7.5 µM IsdB. Inset: time-dependence of absorbance at 406 nm upon mixing IsdB with OxyHb (red) or MetHb (brown). (B) Stoichiometric titration of MetHb with IsdB. (C) Absorption spectra of 1.5 µM MetHb before (brown) and after (orange) mixing with 7.5 µM IsdB^Y440A. Inset: time-dependence of absorbance at 406 nm upon mixing IsdB^Y440A with MetHb. (D) Absorption spectra of 1.5 µM α-semiHb (dark blue) or β-semiHb (pink) before (continuous line) and after (dashed line) mixing with 7.5 µM IsdB. Inset: time-dependence of absorbance at 406 nm upon mixing IsdB with α-semiHb (dark blue) or β-semiHb (pink). Black lines through kinetic traces are the fitting to a biexponential equation with parameters reported in Table 1.