FIGURE 4.

Role of histidines His-72 and His-149 in HrtR binding to heme and to DNA. A, UV-visible spectra of 30 μm ferric heme (hemin) and MBP-HrtR WT, H72A/H149L, and H72A/H149L (HH). WT and mutant HrtR were bound with 3-fold excess hemin. Unbound hemin was then removed by size exclusion chromatography. UV-visible spectra of the complexes (30 μm in 200 μl) were obtained in a microplate spectrophotometer (Infinite M200, Tecan, Austria). Inset, magnification of the 500–700 nm area. Results are representative of three experiments. B, EMSA of HrtR and histidine mutants binding to the hrtRBA promoter region. Thirty pmol of the hrtRBA promoter fragment were incubated in the presence of 200 pmol of WT and mutated variants as above. Lane 1 was a control without protein. Results are representative of three experiments.