Dataset Information

Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis.

ABSTRACT: The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: (i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD) and (ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is “trapped”, purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence that P-His1 and P-His2 function as heme-binding ligands is presented. These results show that heme in holoCcmE is essential for complex formation with CcmF and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex [Richard-Fogal C, Kranz RG. The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J Mol Biol 2010;401:350–62] and suggest common mechanistic and structural aspects.

SUBMITTER: San Francisco B

PROVIDER: S-EPMC3943652 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis.

San Francisco Brian B Kranz Robert G RG

Journal of molecular biology 20140201 3

The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: (i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD) and (ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome ...[more]

PMID: 24513106

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Project description:Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes. The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm. To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E. coli. By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant. Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm. Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells. Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space. Such an imbalance could lead to defects in the synthesis of heme-liganded proteins. The heme reporters were also used to analyze strains of E. coli with a defect in genes encoding homologs of a different ABC transporter (helABC). The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R. G. Kranz, J. Bacteriol. 171:456-464, 1989; D. L. Beckman, D. R. Trawick, and R. G. Kranz, Genes Dev. 6:268-283, 1992). This locus was shown to be essential in E. coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis. Consistent with these and previous results, it is proposed that the HelABC transporter is specifically involved in heme export for ligation (hel). This class of periplasmic cytochromes is proposed to require heme liganding before undergoing correct folding.

Dataset Information

Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis.

Publications

Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis.

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