Heme Trafficking by the Cytochrome C Biogenesis Pathways

Cytochromes function in electron transport chains to perform critical cellular functions, such as respiration and photosynthesis. Cytochromes c are unique due to their requirement for the covalent attachment of heme via two thioether bonds at a conserved CXXCH motif. Three pathways have been identified for cytochrome c maturation: System I (prokaryotes), System II (prokaryotes) and System III (eukaryotes). System I consists of 8 integral membrane proteins (CcmABCDEFGH), System II is comprised of 2 membrane proteins (CcsBA) and these pathways will be the focus of this presentation. Trafficking of heme from the site of its synthesis (cytoplasm) to the site of attachment to apocytochrome c (periplasm) is critical for cytochrome c biogenesis, yet little direct evidence of heme trafficking exists. The study of heme trafficking has proved elusive in this and most other systems due to tight cellular regulation, as well as the cytotoxic and amphipathic nature of heme. Here, we use key putative heme transporters in the prokaryotic pathways as model systems to develop a novel technique to covalently ‘trap’ heme during the trafficking process. First, the conserved WWD domain, which is predicted to interact with heme in the periplasmic space and is found in both System I and System II pathways, was used to develop the heme trapping technique. Heme was covalently trapped in the WWD domains of CcmC (System I) and CcsA (System II), providing the first direct evidence of how heme is trafficked to the periplasm in cytochrome c biogenesis. Currently, this trapping approach is being used on other integral membrane proteins in the pathway to capture heme intermediates and delineate the exact paths for trafficking in vivo. We envision that this approach will be applicable to other heme transporters and trafficking pathways from prokaryotes through eukaryotes.