Biology and Biomedical Sciences: Plant and Microbial Biosciences
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Robert G Kranz
Cytochromes c are proteins that are involved in important redox reactions in organisms from every kingdom of life. C-type cytochromes, uniquely, possess a covalently bound heme. Since cytochromes c are assembled at their site(s) of function: outside of the cytoplasmic membrane in bacteria, in the chloroplast lumen, or in the mitochrondrial intermembrane space), their assembly poses unique challenges to heme trafficking and post translational modification. Three major systems exist in nature for cytochrome c assembly, termed systems I, II, and III. Using recombinant Escherichia coli, aspects of systems I and III were analyzed, with an emphasis on the synthase protein(s) and protein complexes that carry out the covalent attachment of heme to apocytochrome. Studies on system I focused on the integral membrane protein CcmF, the putative cytochrome c synthetase, which was shown to contain a stable and stoichiometric heme b. The ligands to the b-heme were identified: TM-His1 and TM-His2), and the midpoint potential of the heme b was determined and found to be consistent with its involvement in a critical redox reaction underlying the synthetase reaction. Additionally, a long-suspected interaction between CcmF and the periplasmic heme chaperone, holoCcmE, was characterized, with the discovery that heme from holoCcmE is coordinated by two conserved periplasmic histidines in CcmF: P-His1 and P-His2) that flank the conserved WWD domain. It was also shown that CcmF, in complex with CcmH, is the holocytochrome c synthase for system I, able to synthesize cytochromes c in the absence of other Ccm components. Studies on system III, the human HCCS, constituted the first purification and biochemical characterization of an HCCS protein. It was discovered that HCCS purifies with a heme b and forms a stable complex with the human cytochrome c. Using a combination of mutagenesis: of both the apocytochrome and HCCS), functional analysis, and biochemical characterization: of HCCS, alone and in complex with cytochrome c), these studies facilitated an elaboration of the molecular mechanisms for holocytochrome c formation by HCCS.
San Francisco, Brian, "The System I and System III Holocytochrome c Synthases in Cytochrome c Biogenesis" (2013). All Theses and Dissertations (ETDs). 1185.