Abstract
In Escherichia coli, heme is delivered to cytochrome c in a process involving eight proteins encoded by the ccmABCDEFGH operon. Heme is transferred to the periplasmic heme chaperone CcmE by CcmC and from there to apocytochrome c. The role of CcmC was investigated by random as well as site-directed mutagenesis. Important amino acids were all located in periplasmic domains of the CcmC protein that has six membrane-spanning helices. Besides the tryptophan-rich motif and two conserved histidines, new residues were identified as functionally important. Mutants G111S and H184Y had a clear defect in CcmC-CcmE interaction, did not transfer heme to CcmE, and lacked c-type cytochromes. Conversely, mutants D47N, R55P, and S176Y were affected neither in interaction with nor in delivery of heme to CcmE but produced less than 10% c-type cytochromes. A strain carrying a CcmCE fusion had a similar phenotype, suggesting that CcmC is important not only for heme transfer to CcmE but also for its delivery to cytochrome c. Co-immunoprecipitation of CcmC with CcmF was not detectable although CcmE co-precipitated individually with CcmC and CcmF. This contradicts the idea of CcmCEF supercomplex formation. Our results favor a model that predicts CcmE to shuttle between CcmC and CcmF for heme delivery.
Highlights
The covalent attachment of heme to the CXXCH signature motif of apocytochromes is a critical step during cytochrome c biosynthesis
CcmC contains a conserved tryptophan-rich signature motif (WGXWXWDXRLT, where represents an aromatic amino acid residue) [1, 3, 19, 20] that resides in the second periplasmic domain (Fig. 1) and shares a high degree of similarity with the tryptophan-rich motif of CcmF, NrfE, and the CcsA homologues of type II cytochrome c maturation [21]
In E. coli, the heme chaperone CcmE plays a crucial role in this process by binding heme transiently in a covalent manner that is still not understood in detail
Summary
The covalent attachment of heme to the CXXCH signature motif of apocytochromes is a critical step during cytochrome c biosynthesis. The ␥-proteobacterium Escherichia coli requires eight cytoplasmic membrane proteins encoded by the ccmABCDEFGH operon [5, 6] These proteins catalyze the covalent attachment of heme to the conserved CXXCH motifs of apocytochromes c. In Paracoccus denitrificans, a ccmC mutant in addition to deficiencies in cytochrome c maturation and siderophore production showed intolerance to rich medium [17]. The connection between these processes is not known. Two absolutely conserved histidines are present in the first and third periplasmic domains of CcmC Mutational analysis of these motifs of the E. coli CcmC protein revealed that they are functionally important [22]. SupE44 ⌬lacU169 (⌽80lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 hsdR mcrB araD139 ⌬(araABC-leu)7679 ⌬lacX74 galU galK rpsL thi MC1061 ⌬ccmA-H::kan MC1061 ⌬ccmC TG1 ⌬dmsABC::kan MC1061 ⌬dmsABC::kan EC28 ⌬dmsABC::kan endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac mutD5 mutS mutT Tn10( tetr) recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lacFЈ proAB lacIqZ⌬M15Tn10( tetr)͔
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