New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC.

Maturation of c-type cytochromes in Escherichia coli is a complex process requiring eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE is a mediator of haem delivery. It binds haem transiently at a conserved histidine residue and releases it for directed transfer to apocytochrome c. CcmC, an integral membrane protein with six transmembrane helices, is necessary and sufficient to incorporate haem covalently into CcmE. CcmC contains a highly conserved tryptophan-rich motif, WGXXWXWD, in its second periplasmic loop. Here, we present the results of a systematic mutational analysis of this motif. Changes of the non-conserved T121 and W122 to A resulted in wild-type CcmC activity. Changes of the single amino acids W119A, G120A, W123A, W125I and D126A or of the spacing within the motif by deleting V124 (DeltaV124) inhibited the covalent haem incorporation into CcmE. Enhanced expression of ccmD suppressed this mutant phenotype by increasing the amounts of CcmC and CcmE polypeptides in the membrane. The DeltaV124 mutant showed the strongest defect of all single mutants. Mutants in which six residues of the tryptophan-rich motif were changed showed no residual CcmC activity. This phenotype was independent of the level of ccmD expression. Our results demonstrate the functional importance of the tryptophan-rich motif for haem transfer to CcmE. We propose that the three membrane proteins CcmC, CcmD and CcmE interact directly with each other, establishing a cytoplasm to periplasm haem delivery pathway for cytochrome c maturation.

Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB.

Cytochrome c maturation in Escherichia coli requires the ccm operon, which encodes eight membrane proteins (CcmABCDEFGH). CcmE is a periplasmic heme chaperone that binds heme covalently and transfers it onto apocytochrome c in the presence of CcmF, CcmG, and CcmH. In this work we addressed the functions of the ccmABCD gene products with respect to holo-CcmE formation and the subsequent ligation of heme to apocytochrome c. In the absence of the ccmABCD genes, heme is not bound to CcmE. We report that CcmC is functionally uncoupled from the ABC transporter subunits CcmA and CcmB, because it is the only Ccm protein that is strictly required for heme transfer and attachment to CcmE. Site-directed mutagenesis of conserved histidines inactivates the CcmC protein, which is in agreement with the hypothesis that this protein interacts directly with heme. We also present evidence that questions the role of CcmAB as a heme exporter; yet, the transported substrate remains unknown. CcmD was found to be involved in stabilizing the heme chaperone CcmE in the membrane. We propose a heme-trafficking pathway as part of a substantially revised model for cytochrome c maturation in E. coli.