FIG 3.

Conserved histidines are required for CcsBA function and heme ligand formation. (A) The CcsBA His variants were coexpressed with cyt c₄ in E. coli Δccm. The ability of variants to mature cyt c₄ was monitored by cell lysis, separation by SDS-PAGE, and heme staining. WT, wild type. (B) Chemical complementation of CcsBA His variants with imidazole. Strains were supplemented with 10 mM imidazole during growth, and cyt c₄ maturation was monitored as described above. (C and D) Quantitation of the data in panel A and B with the wild-type levels normalized to 100%. Data represent results from three triplicate experiments. (E and, F) Variants were subjected to affinity purification using a GST tag, and 10-µg volumes were separated via SDS-PAGE and visualized with Coomassie total protein stain. (G and H) Quantification of heme copurification via UV-vis Soret (410 nm) with 100 µg of protein. Abs, absorbance.