FIGURE 5.

Heme co-purifies together with wild type HrrS (solid line) but not with the triple mutant HrrS^{Y 112A-F115A-F118A} (dotted line). HrrS (solid line) and HrrS^{Y 112A-F115A-F118A} (dotted line) were purified from E. coli (BL21) cells as described in the Section “Materials and Methods.” The cells were grown for 16 h, expressing the kinases. No additional heme/hemin was added to the medium or lysate. The purified protein (12 μM) was conducted to UV-vis spectroscopy in a range from 280 to 600 nm. Both spectra were normalized to the absorption of the proteins (at 280 nm) and background absorption of buffer with micelles was subtracted from both spectra. An arrow indicates a small soret peak at 406 nm in the spectrum of wild type HrrS (solid line) which is not observable in the triple mutant (dotted line). The box shows a zoom into the peak area.