Figure 5.

Kinetics of heme binding to CcmE upon production of H₆-CcmC. The experiment has been repeated at least five times, and similar results have been obtained. The Δccm mutant EC06 was transformed with plasmids pEC408 or pEC410 to constitutively express either ccmD plus ccmE or ccmE alone, respectively. These strains were cotransformed with plasmid pEC422 encoding H₆-CcmC, whose production was induced with arabinose when cells had reached an OD₆₀₀ of 0.7. At different time points 0.5 ml of cells diluted to OD₆₀₀ = 0.7 were precipitated with trichloroacetic acid and separated by SDS/15% PAGE. (A) Activity stain for covalently bound heme. (B) Western blot of the same samples as in A probed with antiserum directed against CcmE. (C) Identical Western blot probed with a monoclonal antiserum directed against a tetrahistidine epitope.