Dynamic features of a heme delivery system for cytochrome C maturation.
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.
Ahuja
Umesh
U
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Thöny-Meyer
Linda
L
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins; D002455:Cell Division; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D005838:Genotype; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000235:genetics / Q000502:physiology*; D006639:Histidine / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D013347:Subcellular Fractions; D014364:Tryptophan / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C492058:CcmC protein, E coli; C116246:CcmC protein, bacteria; C415989:CcmD protein, E coli; C115384:CcmE protein, E coli; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008565:Membrane Proteins; D009841:Oligonucleotides; D006418:Heme; D006639:Histidine; D014364:Tryptophan; D045304:Cytochromes c; D000409:Alanine