Figure 4.

Phenotypic characterization of mutants with H60A and H184A exchanges of H₆-CcmC. (A) The ΔccmC mutant EC28 was cotransformed with plasmids pEC432 (ccmC; lane 1), pEC422 (H_6-ccmC; lane 2), pEC433 (H₆-ccmC^H60A; lane 3), pEC436 (H₆-ccmC^H184A; lane 4), and pRJ3291 (B. japonicum cycA). Cells were grown anaerobically in the presence of nitrite. Periplasmic proteins (50 μg per lane) were separated by SDS/15% PAGE and stained for covalently bound heme. (B) The Δccm mutant EC06 expressing ccmDE constitutively from plasmid pEC408 was cotransformed with plasmid pEC432 (ccmC; lane 1), pEC422 (H_6-ccmC; lane 2), pEC433 (H_6-ccmC^H60A; lane 3), and pEC436 (H_6-ccmC^H184A; lane 4). Membrane proteins (50 μg per lane) were separated by SDS/15% PAGE and stained for covalently bound heme. (C) Western blot of the same membrane fractions (30 μg per lane) as in B probed with antiserum directed against CcmE. (D) Identical Western blot probed with a monoclonal antiserum directed against a tetrahistidine epitope.