Characterization of CcmG mutants. (A) Heme stain of cytochromes produced by CcmG single mutants (30 μg of periplasmic protein). Lane 1, CcmG_Asp107Ala; lane 2, CcmG_Asp129Ala; lane 3, CcmG_Glu86Ala; lane 4, CcmG_Glu145Ala; lane 5, CcmG_Asp162Ala; lane 6, CcmG_wild-type. (B) Characterization of CcmG double (CcmG_{Glu86Ala/Glu145Ala}) and CcmG triple (CcmG_{Glu86Ala/Glu145Ala/Asp162Ala}) mutants. Heme stain of 100 μg of periplasmic protein per lane (upper panel) and Western blot of whole-cell extracts obtained by trichloroacetic acid (TCA) precipitation of 0.5 optical density unit (at 600 nm) of cells per lane (lower panel) by using an antiserum against the CcmG peptide Asn104-Glu118. Lane 1, CcmG_{Glu86Ala/Glu145Ala}; lane 2, CcmG_{Glu86Ala/Glu145Ala/Asp162Ala}; lane 3, CcmG wild type. (C) Heme stain of CcmG N-terminal deletions; heme stain of 50 μg of periplasmic protein (upper panel) and Western blot with anti-His antibody of whole-cell extracts obtained by TCA precipitation of 1.5 optical density units (at 600 nm) of cells per lane (lower panel). Lane 1, CcmGΔ_Leu24-Thr66; lane 2, CcmG_Asp31-Gln67; lane 3, CcmG_His.

CcmG structure and sequence. (a) Sequence alignment of E. coli and B. japonicum CcmGs. Residues mutated in E. coli CcmG are marked by asterisks. The residues deleted to create CcmGΔ_Leu24-Thr66 are italicized, and the residues deleted to create CcmGΔ_Asp31-Gln67 are italicized and underlined. The redox-active cysteines are shown in boldface type. Secondary-structure elements based on the B. japonicum structure are shown, and the predicted transmembrane region is boxed in gray. Sequences are from the Swissprot database. (b) Cartoon showing the structure of CcmG and the positions of the three acidic residues identified as important for function. The redox-active center is indicated by spheres for the sulfur atoms of the cysteines.

Absorption difference spectra of c-type cytochromes. A ΔccmG mutant expressing apocytochrome c was complemented with a plasmid expressing either wild-type CcmG (A), the double mutant CcmG_{Glu86Ala/Glu145Ala} (B), or the triple mutant CcmG_{Glu86Ala/Glu145Ala/Asp162Ala} (C). Periplasmic fractions were prepared from anaerobically grown cells, and protein levels were adjusted to 0.4 mg/ml. Dithionite-reduced spectra minus ammonium persulfate-oxidized spectra were recorded, and the ΔA_551-536 value was used to determine the relative amounts of cytochrome c.