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

Molly C. Sutherland, Joshua M. Jarodsky, Sergey Ovchinnikov, David Baker, Robert G. Kranz

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Although many putative heme transporters have been discovered, it has been challenging to prove that these proteins are directly involved with heme trafficking in vivo and to identify their heme binding domains. The prokaryotic pathways for cytochrome c biogenesis, Systems I and II, transport heme from inside the cell to outside for stereochemical attachment to cytochrome c, making them excellent models to study heme trafficking. System I is composed of eight integral membrane proteins (CcmA–H) and is proposed to transport heme via CcmC to an external “WWD” domain for presentation to the membrane-tethered heme chaperone, CcmE. Herein, we develop a new cysteine/heme crosslinking approach to trap and map endogenous heme in CcmC (WWD domain) and CcmE (defining “2-vinyl” and “4-vinyl” pockets for heme). Crosslinking occurs when either of the two vinyl groups of heme localize near a thiol of an engineered cysteine residue. Double crosslinking, whereby both vinyls crosslink to two engineered cysteines, facilitated a more detailed structural mapping of the heme binding sites, including stereospecificity. Using heme crosslinking results, heme ligand identification, and genomic coevolution data, we model the structure of the CcmCDE complex, including the WWD heme binding domain. We conclude that CcmC trafficks heme via its WWD domain and propose the structural basis for stereochemical attachment of heme.

Original languageEnglish
Pages (from-to)1065-1080
Number of pages16
JournalJournal of Molecular Biology
Issue number8
StatePublished - Apr 13 2018


  • CcmC structure
  • cytochrome c biogenesis
  • heme
  • heme binding site
  • heme trafficking


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