Researcher(s)
- Donna Price, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Molly Sutherland, Biological Sciences, University of Delaware
Abstract
Cytochromes c are highly conserved proteins found in nearly all organisms including humans, plants, and bacteria. Cytochrome c is unique among other cytochromes because of the requirement of covalently attached heme for proper protein folding and function. Cytochrome c is a crucial component of cellular respiration via the electron transport chain, where heme’s unique redox properties are critical in electron transport. Cytochrome c contains a conserved motif (CXXCH) where heme is covalently attached via thioether bonds between the cysteine thiols and heme vinyl groups. Cytochromes c are well studied, but little is known about their biogenesis (i.e. heme attachment). Cytochrome c biogenesis can be accomplished by three different systems: System I (bacteria), System II (bacteria), and System III (eukaryotes). This study focuses on the E. coli System I cytochrome c biogenesis pathway, which involves eight integral membrane proteins (CcmABCDEFGH) and operates in two stages. Initially, CcmABCD facilitates the transmembrane trafficking and attachment of heme to CcmE. Subsequently, CcmE transfers heme to CcmFH, the holocytochrome c synthase responsible for heme attachment to cytochrome c. We hypothesized that CcmC serves as the heme receptor in this pathway, requiring interaction with heme on the cytoplasmic face of the protein. To test this hypothesis, we introduced single and double amino acid alanine substitutions at key residues in CcmC and CcmD to investigate their interactions with heme. These variants were co-expressed with apocytochrome c in recombinant Escherichia coli, and an in vivo cytochrome c biogenesis assay was conducted. To determine the significance of these residues in cytochrome c biogenesis, the results were analyzed by heme stain to compare the amount of holocytochrome c matured by these alanine variants against the wild type.