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.

Heme is a critical co-factor for proteins with essential biological functions including gas sensing and electron transport. However, free or unbound heme is highly toxic to cells. To mitigate toxicity, there must be specific pathways to move heme in cells. While heme transport proteins have been identified genetically, their mechanism of transport remains elusive. The bacterial cytochrome c biogenesis pathways, Systems I and II, are excellent model systems to investigate heme transport. We compare the System I heme transporter, CcmCD, with the System II heme transporter, CcsBA. Analysis of putative transmembrane localized heme axial ligands in CcmCD determined that heme liganding is not required for heme transport. In contrast, CcsBA requires transmembrane heme axial liganding for function. Thus, the bacterial cytochrome c biogenesis heme transporters have different mechanisms, suggesting that additional mechanisms of transmembrane transport may be uncovered via experimental analysis of other heme transporters in prokaryotes and eukaryotes.