CcmE is a heme chaperone involved in the periplasmic maturation of c-type cytochromes in many bacteria and plant mitochondria. It binds heme covalently and subsequently transfers it to the apo-form of cytochromes c. To examine the role of the C-terminal domain of CcmE in the binding of heme, in vitro heme binding to the apo-form of a truncated (immediately before P136) version of the periplasmic domain of the heme chaperone from E. coli was studied. Removal of the C-terminal domain dramatically altered the ligation of non-covalently bound heme in CcmE' (the soluble form lacking the membrane anchor) but only slightly affected its affinity for protoporphyrin IX and 8-anilino-1-naphthalenesulphonate. This finding has significant mechanistic implications for in vivo holo-CcmE formation and indicates that the C-terminal region is not required for the recruitment and docking of heme into its binding site, but is likely to contain amino-acid(s) involved in heme iron axial coordination. Removal of the C-domain significantly impaired in vivo heme binding to CcmE and conversion of apo-cytochrome to holo-protein by a similar factor, suggesting that the C-terminal domain of the chaperone is primarily involved in heme binding to CcmE rather than in heme transfer to the apo cytochrome.