Engineering Single Amino Acid Mutations 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 a covalently bound heme to the CXXCH motif of apocytochrome c  for proper folding and maturation to holocytochrome c. Cytochrome c biogenesis can be accomplished via one of three pathways: System I (Prokaryotes), System II (Prokaryotes) and System III (Eukaryotes). My project focuses on the System II pathway, which is composed of two proteins, CcsA & CcsB, which helps in the transportation of  heme across the bacterial membrane and its attachment to the CXXCH motif.  CcsBA has two heme interaction domains, one on the transmembrane of the protein, composed of two conserved histidines and anotherin the periplasmic face of the protein composed of two conserved histidines and the WWD domain. Heme must be transported between these two domains, yet the path of heme trafficking is unknown. To test the hypothesis that a heme channel exists between these domains, cysteine/heme crosslinking was utilized. My project was to engineer single amino acid cysteine variants  in Helicobacter hepaticus CcsBA in the periplasmic face. QuikChange site directed mutagenesis was used to generate 10 cysteine variants of CcsBA. The clones will be used to further analyze this channel by trapping heme via crosslinking in CcsBAs from Helicobacter hepaticus.