Researcher(s)
- Tyana Spindle, Animal Science, University of Delaware
Faculty Mentor(s)
- Molly Sutherland, Department of Biological Sciences, University of Delaware
Abstract
Cytochromes c are proteins found in almost every form of life and are important for producing energy through cellular respiration. They work as critical components in electron transport chains, a process important for making ATP.
Cytochromes c are unique because they need a heme group to be covalently attached to a specific amino acid sequence (CXXCH motif) to function correctly. There are many different types of cytochromes c, which are made by one of three pathways (System I, II, or III).
My project focused on System I, which is mainly found in bacteria. It’s composed of eight integral membrane proteins (CcmABCDEFGH) which function in two steps to transport heme across the bacterial inner membrane to attach to cytochrome c. CcmFH is the enzyme that attaches heme to apocytochrome c. We hypothesize that CcmFH helps reduce heme to be attached to cytochrome c; we believe this requires two tryptophans moving the electron from the quinol pool to the WWD heme.
We mutated tryptophans to alanine, which resulted in lower levels of heme co-purification. CcmF has two histidines in the periplasm that ligand heme. In my project, I mutated these histidines into alanine to remove the WWD heme. This allows us to see which heme(s) were lost in the original tryptophan mutants. This mutation was made through site-directed mutagenesis, which involved designing a primer encoding for alanine instead of histidine. The primer was used in a PCR reaction to introduce the mutation into CcmF. Successful clones for both histidines were confirmed via sequencing. Since CcmF makes cytochrome c and cytochrome c works in the electron transport chain to produce energy for bacteria, getting rid of CcmF would deprive harmful bacteria from energy. Thus, making it a good target for new antibiotics. Thus, understanding CcmF is necessary for the development of new medications.