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

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• The periplasmic heme chaperone holoCcmE is crucial for heme transport in cytochrome c biosynthesis (system I).
• System I involves holoCcmE formation by CcmABCD and heme delivery to the CcmFH complex for apocytochrome c modification.
• Previous models suggested holoCcmE interaction with CcmF, but direct evidence was lacking.

Purpose of the Study:

• To investigate and characterize the interaction between holoCcmE and CcmF.
• To determine the role of heme in holoCcmE and CcmF complex formation.
• To identify the specific residues in CcmF involved in binding holoCcmE.

Main Methods:

• Complex formation between holoCcmE and CcmF was induced and stabilized ('trapped').
• The resulting complex was purified and characterized using biochemical techniques.
• Mutagenesis was employed to identify key histidine residues in CcmF essential for interaction.

Main Results:

• A stable complex between holoCcmE and CcmF was successfully trapped, purified, and characterized.
• HoloCcmE must be released from CcmABCD to interact with CcmF.
• The holo-form of CcmE showed significantly higher affinity for CcmF compared to apoCcmE.
• Two conserved histidines (P-His1, P-His2) in CcmF were identified as essential for holoCcmE binding and likely serve as heme ligands.
• The heme within holoCcmE is coordinated by P-His1 and P-His2 in CcmF's WWD domain.

Conclusions:

• Heme carried by holoCcmE is essential for its interaction with CcmF.
• The CcmF WWD domain, specifically P-His1 and P-His2, directly coordinates the heme from holoCcmE.
• This interaction mechanism shares similarities with other heme trafficking complexes, suggesting conserved pathways.