Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Heme trafficking is crucial for cellular respiration and requires specific protein machinery.
• Prokaryotic systems I and II for cytochrome c biogenesis offer models for studying heme transport.
• The CcmCDE complex is implicated in transporting heme outside the cell for cytochrome c attachment.

Purpose of the Study:

• To develop and apply a cysteine/heme crosslinking technique to map heme binding sites in CcmC and CcmE.
• To elucidate the structural basis of heme trafficking and stereochemical attachment in System I.
• To model the structure of the CcmCDE complex.

Main Methods:

• Engineered cysteine residues in CcmC and CcmE to facilitate crosslinking with heme's vinyl groups.
• Utilized single and double crosslinking strategies for detailed heme site mapping.
• Integrated crosslinking data with heme ligand identification and genomic coevolution analysis.

Main Results:

• Successfully trapped and mapped endogenous heme within CcmC (WWD domain) and CcmE.
• Defined specific heme-binding pockets in CcmE, including "2-vinyl" and "4-vinyl" regions.
• Developed a structural model of the CcmCDE complex, detailing the WWD heme binding domain.

Conclusions:

• CcmC directly traffics heme through its WWD domain.
• The study provides a structural framework for the stereospecific attachment of heme to cytochrome c.
• The novel crosslinking approach is valuable for studying protein-heme interactions in vivo.