FIG 3.

FhtR binds heme. (A) UV-visible absorption spectra of MBP-FhtR as purified from E. coli. UV-visible spectra of 30 μM (in 200 μl) MBP-FhtR was obtained in a microplate spectrophotometer (Spark; Tecan) and normalized to an OD₂₈₀ of 1. (Inset) Photograph of the purified MBP-FhtR. Results are representative of three independent experiments. (B) UV-visible spectra of apoMBP-FhtR complexed with hemin. MBP-FhtR was purified from E. coli (hemA::kan) strain as an apoprotein (dashed line) that was mixed with equimolar concentration of hemin. Spectra was obtained as described above for panel A with 20 μM complex and normalized to an OD₂₈₀ of 1. (Inset) Magnification of the 500- to 700-nm region. Results are representative of three independent experiments. (C) Size-exclusion chromatography of apo- and holo-MBP-FhtR. MBP-FhtR was purified and complexed with hemin as described above for panel B. 40 μM of the complex in 100 μl was loaded on a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) in 20 mM HEPES (pH 7), 300 mM NaCl buffer. Protein and heme content were analyzed at OD₂₈₀ and OD₄₁₀. The results are representative of three independent experiments. (D) Titration of MBP-FhtR with hemin followed by fluorescence and absorbance (inset). For the fluorescence experiment, 50 nM ApoMBP-FhtR purified from E. coli (hemA::kan) as described for panel B were titrated with increasing increments of hemin. Fluorescence intensity (in arbitrary units [a.u.]) was recorded and plotted against hemin concentration. The experiment was repeated three times, fitted using the nonlinear regression function of GraphPad Prism 4 software, and gave a K_d of 310 nM. The inset depicts the absorbance at 407 nm of ApoMBP-FhtR plotted against hemin concentration. The curve is representative of 10 independent experiments and was fitted using the nonlinear regression function of GraphPad Prism 4 software, which determined that the stoichiometry of the FhtR-hemin complex was 1:1.