Engineering Single Amino Acid Cysteine Variants to Analyze Heme Transport in System II Cytochrome c biogenesis

• Aeila Chesley, Biological Sciences, University of Delaware

• Molly Sutherland, Biological Sciences, University of Delaware

Cytochromes c are highly conserved proteins found in humans, plants, bacteria, and archaea. They have several functions with key roles in respiration, photosynthesis, and apoptosis. The key feature of cytochromes c is the requirement for covalent binding of heme to the CXXCH motif of apocytochrome c for proper folding and maturation to holocytochrome c. All cytochrome c biogenesis is accomplished by three pathways: System I & II (Prokaryotes) and System III (Eukaryotes). My project focuses on System II, which is composed of two proteins, CcsA CcsB, which transport heme across the bacterial membrane and attach it to the CXXCH motif.  CcsBA has two heme interaction domains. One on the cytoplasmic face of the protein composed of two conserved histidines and one in the periplasm composed of two conserved histidines and the WWD domain. Heme must be transported between the two domains, yet the path of heme trafficking is unknown. To test our hypothesis that a heme channel exists between these domains, cysteine/heme crosslinking will be utilized. Cysteine has a natural propensity to form a covalent bond to heme when in proximity. Cysteine/heme crosslinking exploits this to identify residues that are important for heme handling. My project is to engineer single amino acid cysteine variants in Helicobacter hepaticus   CcsBA. QuikChange site directed mutagenesis was used to clone X cysteine variants into CcsBA. I designed primers for mutagenesis, performed PCR, transformed E. coli, isolated plasmids and confirmed mutations via Sanger DNA sequencing. Tania Yeasmin will use the clones to further analyze key sites in CcsBAs from Helicobacter hepaticus, Campylobacter jejuni, & Helicobacter pylori.