Analysis of putative heme ligands in the System I bacterial cytochrome c biogenesis heme transporter, CcmCD

Heme is a key co-factor for biologically important proteins that are required for critical cellular functions such as oxygen transport and energy production. Yet heme is highly cytotoxic and is proposed to require specific transport pathways to move within cells. Despite its importance, mechanisms for heme transport remain largely uncharacterized due to heme’s cytotoxicity and the transient nature of transport. In recent years, bacterial cytochrome c biogenesis pathways, System I and II, have emerged as model systems to probe heme-protein interactions and heme transport across bacterial membranes. In System II, CcsBA is proposed to be a heme transporter based on the identification of two discrete heme interaction domains, one located near the cytoplasm and the other located in the periplasm. These domains each use two histidine residues to ligand the heme. In System I, CcmCD was recently designated as a heme transporter, and the path of heme transport was biochemically defined. Based on the cryo-EM structure of CcmCD, four residues near the cytoplasm could act as axial ligands to heme. Thus, a structure-function analysis of these putative heme ligands was undertaken to determine if CcmCD and CcsBA interact with heme in a similar manner. This analysis revealed that CcmCD does not contain heme axial ligands nor is heme liganding required for CcmCD heme transport. These results provide the first experimental evidence that heme transporters do not interact with heme nor transport heme by a conserved mechanism.