Heme Trafficking by the Cytochrome C Biogenesis Pathways

Trafficking of heme is critical for cellular function (e.g. oxygen transport, energy production), however the tools to directly investigate this process are rudimentary. Here we describe a novel method to trap heme as it is trafficked through the cell, allowing for identification of heme binding domains and heme transporters. The approach was developed using prokaryotic cytochrome c biogenesis pathways as model systems. Cytochrome c biogenesis requires the covalent attachment of heme, via two thioether bonds, to a conserved CXXCH motif and is dependent on the trafficking of heme to the holocytochrome c synthetase. In prokaryotes, two pathways, System I and System II, mature cytochrome c. System I is composed of 8 membrane proteins, CcmABCDEFGH, and System II is composed of two integral membrane proteins, CcsBA. These pathways contain a conserved, tryptophan-rich domain called the WWD domain that is proposed to interact with heme. Here, we crosslink heme in the WWD domains of CcmC and CcsA, demonstrating for the first time that the WWD domain is a heme binding domain. Subsequently, a heme binding domain was defined in CcmE, delineating 2-vinyl and 4-vinyl pockets for the heme vinyl groups. The stereospecific position of heme in these domains was determined and a two-step model for System I heme trafficking is proposed. CcmABCD trafficks heme to the WWD domain of CcmC where it is stereospecifically positioned and attached to the heme binding pockets of CcmE (step 1). CcmE transports heme to the holocytochrome c synthetase, CcmF/H, for stereospecific attachment to apocytochrome c (step 2). We envision that the heme crosslink approach will be applicable to other putative heme transporters to prove that they are directly involved in heme transport. Moreover, we believe that these purified integral membrane proteins are excellent candidates for cryoEM structural studies.