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 for covalently attached heme for proper 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 thiol 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). The goal of this project is to identify the heme receptor domain for System I. System I consists of eight integral membrane proteins (CcmABCDEFGH) and is proposed to function in two steps. First, heme is transported across the membrane by CcmABCD and covalently attached to CcmE. Next, CcmE is released via ATP hydrolysis catalyzed by CcmAB and transfers heme to CcmF/H, the holocytochrome c synthase. Second, CcmF/H attaches heme to apocytochrome c. A major research question for System I is to determine how heme is delivered to the pathway. I hypothesize that CcmC is the heme receptor. To test this hypothesis, I have engineered 8 single amino acid variants, resulting in cysteine point mutations. These variants will be used in cysteine/heme crosslinking, a technique used to identify heme-binding domains. I designed mutagenesis primers and utilized QuikChange site-directed mutagenesis to engineer these variants. Variants were confirmed using Sanger DNA sequencing. Future work will include recombinant expression of proteins, affinity purification, and assessment of cystine/heme crosslink formation via heme stain and UV-vis spectroscopy.