Researcher(s)
- Evelyn McQuaid, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Molly Sutherland, Biological Sciences, University of Delaware
Abstract
Cytochrome c is an important protein found in nearly all organisms from humans to bacteria. It plays a crucial role in the electron transport chain (ETC), mediating important processes such as cellular respiration and detoxification. To perform its function, cytochrome c must have a heme molecule covalently attached at a conserved CXXCH motif, a process called cytochrome c biogenesis. This occurs via one of three pathways: Systems I, II, and III. My project is focused on the prokaryotic System II pathway from Bacteroides thetaiotaomicron, which is composed of two proteins CcsB and CcsA. It is known that the active site is made up of a heme-handling WWD domain that positions heme for attachment to apocytochrome c. However, we theorize additional interactions outside of this active site mediate the specificity of heme attachment. Recently, a computational study identified a ‘beta-cap’ region in the periplasmic domain of CcsBA that is proposed to block the active site when heme is not present. We hypothesize the beta-cap region of CcsBA plays a role in the specificity of the CcsBA-cytochrome c interaction. To test this, my project was to perform QuikChange site-directed mutagenesis in the beta-cap region, mutating conserved residues in the region to alanine (F136A, Y141A, N171A, F136A/Y141A, Y142A/N171A, F136A/Y141A/N171A), in order to create a set of CcsBA variants that will be used in future functional assays to determine if beta-cap residues impact heme attachment. If these mutants affect cytochrome c biogenesis, it suggests that the beta-cap region contributes to the specificity of the CcsBA-cytochrome c interaction. Ultimately, this region could be a novel antibiotic target as the prokaryotic System II pathway is essential in a number of pathogenic organisms and its active site differs that of human cytochrome c synthase.