Heme is an important cofactor for a wide array of biological processes across all domains of life. Its redox properties confer roles in electron transport, gas sensing and as a regulator of critical cellular processes. Yet heme is also highly cytotoxic, resulting in tight regulation of its biosynthesis and intracellular levels. As a result, heme trafficking mechanisms are not well understood. The Sutherland lab is developing the bacterial cytochrome c biogenesis pathways as a model system to elucidate mechanisms of heme binding, modification and transport. Cytochromes c are hemoproteins encoded by nearly all organisms that most often function in the context of electron transport chains for cellular energy production. Cytochromes c require the covalent attachment of heme for folding and function, a process called cytochrome c biogenesis. Three pathways for cytochrome c biogenesis are known: System I (prokaryotes), System II (prokaryotes), and System III (eukaryotes). We focus on System I, composed of eight proteins CcmABCDEFGH that function in two steps: CcmABCD transports heme across the bacterial membrane and attaches heme to CcmE. HoloCcmE transports heme to CcmFH for attachment to cytochrome c. We use a recombinant E. coli system to interrogate heme transport and test the hypothesis that CcmCD is a heme transporter. Cysteine scanning coupled with cysteine/heme crosslinking, a novel approach which exploits the propensity of cysteine and heme to form a covalent bond when in close proximity, was used to undertake a structure-function analysis of CcmCD. CcmCD was affinity purified and cysteine/heme crosslink formation was determined by heme stain. Residues identified to interact with heme were mapped onto published cryo-EM structures, revealing a cytoplasmic heme acceptance domain and enclosed transmembrane heme channel. Structural predictions of other bacterial CcmCD proteins suggest variability in the heme acceptance domain and conservation of the heme channel, indicating a common mechanism for heme transport.