Biochemical mapping reveals a conserved heme transport mechanism via CcmCD in System I bacterial cytochrome c biogenesis.

Heme is a redox-active cofactor for essential processes across all domains of life. Heme's redox capabilities are responsible for its biological significance but also make it highly cytotoxic, requiring tight intracellular regulation. Thus, the mechanisms of heme trafficking are still not well understood. To address this, the bacterial cytochrome c biogenesis pathways are being developed into model systems to elucidate mechanisms of heme trafficking. These pathways function to attach heme to apocytochrome c, which requires the transport of heme from inside to outside of the cell. Here, we focus on the System I pathway (CcmABCDEFGH) which is proposed to function in two steps: CcmABCD transports heme across the membrane and attaches it to CcmE. HoloCcmE then transports heme to the holocytochrome c synthase, CcmFH, for attachment to apocytochrome c. To interrogate heme transport across the membrane, we focus on CcmCD, which can form holoCcmE independent of CcmAB, leading to the hypothesis that CcmCD is a heme transporter. A structure-function analysis via cysteine/heme crosslinking identified a heme acceptance domain and heme transport channel in CcmCD. Bioinformatic analysis and structural predictions across prokaryotic organisms determined that the heme acceptance domains are structurally variable, potentially to interact with diverse heme delivery proteins. In contrast, the CcmC transmembrane heme channel is structurally conserved, indicating a common mechanism for transmembrane heme transport. We provide direct biochemical evidence mapping the CcmCD heme channel and providing insights into general mechanisms of heme trafficking by other putative heme transporters. IMPORTANCE: Heme is a biologically important cofactor for proteins involved with essential cellular functions, such as oxygen transport and energy production. Heme can also be toxic to cells and thus requires tight regulation and specific trafficking pathways. As a result, much effort has been devoted to understanding how this important, yet cytotoxic, molecule is transported. While several heme transporters/importers/exporters have been identified, the biochemical mechanisms of transport are not well understood, representing a major knowledge gap. Here, the bacterial cytochrome c biogenesis pathway, System I (CcmABCDEFGH), is used to elucidate the transmembrane transport of heme via CcmCD. We utilize a cysteine/heme crosslinking approach, which can trap endogenous heme in specific domains, to biochemically map the heme transport channel in CcmCD, demonstrating that CcmCD is a heme transporter. These results suggest a model for heme trafficking by other heme transporters in both prokaryotes and eukaryotes.