Structure-function analysis of the bifunctional ccsBA heme exporter and cytochrome c synthetase

Although intracellular heme trafficking must occur for heme protein assembly, only a few heme transporters have been unequivocally discovered and nothing is known about their structure or mechanisms. Cytochrome c biogenesis in prokaryotes requires the transport of heme from inside to outside for stereospecific attachment to cytochrome c via two thioether bonds (at CXXCH). The CcsBA integral membrane protein was shown to transport and attach heme (and thus is a cytochrome c synthetase), but the structure and mechanisms underlying these two activities are poorly understood. We employed a new cysteine/heme crosslinking tool that traps endogenous heme in heme binding sites. We combined these data with a comprehensive imidazole correction approach (for heme ligand interrogation) to map heme binding sites. Results illuminate the process of heme transfer through the membrane to an external binding site (called the WWD domain). Using metagenomic data (GREMLIN) and Rosetta modeling programs, a structural model of the transmembrane (TM) regions in CcsBA were determined. The heme mapping data were then incorporated to model the TM heme binding site (with TM-His1 and TM-His2 as ligands) and the external heme binding WWD domain (with P-His1 and P-His2 as ligands). Other periplasmic structure/function studies facilitated modeling of the full CcsBA protein as a framework for understanding the mechanisms. Mechanisms are proposed for heme transport from TM-His to WWD/P-His and subsequent stereospecific attachment of heme. A ligand exchange of the P-His1 for histidine of CXXCH at the synthetase active site is suggested. IMPORTANCE The movement or trafficking of heme is critical for cellular functions (e.g., oxygen transport and energy production); however, intracellular heme is tightly regulated due to its inherent cytotoxicity. These factors, combined with the transient nature of transport, have resulted in a lack of direct knowledge on the mechanisms of heme binding and trafficking. Here, we used the cytochrome c biogenesis system II pathway as a model to study heme trafficking. System II is composed of two integral membrane proteins (CcsBA) which function to transport heme across the membrane and stereospecifically position it for covalent attachment to apocytochrome c. We mapped two heme binding domains in CcsBA and suggest a path for heme trafficking. These data, in combination with metagenomic coevolution data, are used to determine a structural model of CcsBA, leading to increased understanding of the mechanisms for heme transport and the cytochrome c synthetase function of CcsBA.