Figure 1.
All four Cys residues of WhiB1 are required for iron-sulfur cluster acquisition.
(A) Photograph depicting anaerobic reconstitution of WhiB1 and variants with the indicated single Cys substitutions after removal of unincorporated components by chromatography on heparin Sepharose. (B) UV-visible spectra of WhiB1 and variants after anaerobic reconstitution of iron-sulfur clusters. The black line shows the spectrum of wild-type WhiB1 (4.5 µM); the blue line, WhiB1-C9A (2.5 µM); the red line, WhiB1-C37A (3.7 µM); the brown line, WhiB1-C40A (3.6 µM); the orange line, WhiB1-C46A (3.6 µM). The green line shows the spectrum of apo-WhiB1 (2.3 µM). The buffer was 25 mM Tris-HCl pH 7.4 containing 0.5 M NaCl and 10% glycerol. (C) All four WhiB1 Cys variants bind whiB1 promoter DNA (PwhiB1). Radiolabeled PwhiB1 DNA was incubated with increasing concentrations of the indicated WhiB1 proteins before separation of protein-DNA complexes in electrophoretic mobility shift assays. Lanes 1, no protein; lanes 2–6 contain, 1, 2, 5, 10 and 15 μM WhiB1, respectively. The locations of PwhiB1 and PwhiB1-WhiB1 complexes are indicated.
Figure 2.
The WhiB1-D13A variant has properties similar to the wild-type protein.
(A) Spectroscopic properties and reactivity of the WhiB1-D13A iron sulfur cluster. The UV-visible spectra of WhiB1-D13A (18.8 μM): after reconstitution (solid line); after 60 min exposure to 110 μM O2 (long-dashed line; mostly superimposed on the solid line); and after 5 min exposure to a 20-fold molar excess of NO (short-dashed line). The buffer was 25 mM Tris-HCl pH 7.4 containing 0.5 M NaCl, 1 mM DTT and 10% glycerol. (B) Apo-WhiB1-D13A binds PwhiB1. Electrophoretic mobility shift assays were as follows: lanes 1, no protein; lanes 2–7, 0.5, 1, 2, 4, 8 and 16 μM WhiB1-D13A, respectively. The locations of PwhiB1 and PwhiB1-WhiB1-D13A complexes are indicated.
Figure 3.
DNA-binding and iron-sulfur cluster acquisition by WhiB1 variants with amino acid substitutions in the C-terminal region.
WhiB1 proteins with the indicated amino acid substitutions were incubated with radiolabeled PwhiB1 DNA and complexes were separated by electrophoresis. (A) Amino acid substitutions in the putative β-turn of WhiB1. (B) Amino acid substitutions in two conserved amino acid motifs located downstream of the predicted β-turn of WhiB1. Lanes 1, no protein; lanes 2–7, 2.5, 5, 7.5, 10, 12.5 and 15 μM of the indicated WhiB1 protein, respectively. The locations of PwhiB1 and PwhiB1-WhiB1 complexes are indicated. (C) Representative UV-visible spectra of holo- WhiB1-G61E (26 μM; A420∶A280 ratio 0.19) and WhiB1-R74E (13 μM; A420∶A280 ratio 0.16).
Figure 4.
Locations of amino acids required for iron-sulfur cluster acquisition and DNA-binding by WhiB1.
The locations of: the four conserved Cys residues that are required for iron-sulfur cluster acquisition (white text on black background); the predicted β-turn and helix in the C-terminal region; the WhiB1 conserved motifs downstream of the predicted β-turn (boxed); and amino acids that impair DNA-binding in WhiB1 (bold) or WhiBTM4 (underlined) are shown.