Binding of Fe3+ ferrichrome to FtsB. (A) Co-migration of Fe3+ ferrichrome with FtsB. FtsB (0.3 ml of 0.18 mM) was incubated with 0.9 mM Fe3+ ferrichrome for 20 min at room temperature, loaded onto a Sephadex G-25 column (1.5 × 18 cm), and eluted with 20 mM Tris-HCl buffer, pH 8.0. Eluant was collected as fractions of 0.7 ml. A280 and A425 of each fraction were used to monitor FtsA and Fe3+ ferrichrome, respectively. (B) MtsA, as a control, did not bind Fe3+ ferrichrome. The experiment was performed under the conditions as those in panel A. (C) The absorption spectra of the protein peaks (fraction 9) in panel A (solid curve) and panel B (short dashed curve) and free Fe3+ ferrichrome (long dashed curve). The three samples contained 46 μM FtsB, 45 μM MtsA, and 100 μM Fe3+ ferrichrome, respectively. (D) Co-migration of partially 55Fe-labeled Fe3+ ferrichrome with FtsB. Fe3+ ferrichrome contained radioactive 55Fe3+ at 1.1% of total Fe3+. A280 and 55Fe3+ radioactivity of each fraction are presented.