FIGURE 6.
Heme-binding studies with single- and triple mutants of HrrS and ChrS. For all samples, 50 μM hemin was added to the cleared E. coli BL21 (DE3) lysate of cells overproducing either HrrS, one of the three HrrS single mutants (A) or the HrrS triple mutant (B). Additionally, ChrS and ChrSY 87A-F90A-F94A were analyzed in an analog experiment (C). After incubation of the lysate with 50 μM hemin for 1 h, ultracentrifugation, solubilization, and purification were carried out as described in the Section “Materials and Methods.” Purified protein samples (12 μM) were conducted to UV-vis spectroscopy and the resulting spectra were normalized to the absorption of the proteins at 280 nm. Background absorption of buffer with DDM micelles was subtracted.