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Title: Interactions of mitochondrial cytochrome c with phospholipids
Author: Rajagopal, Badri S.
Awarding Body: University of Essex
Current Institution: University of Essex
Date of Award: 2012
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Mitochondrial cytochrome c (cyt c) associates with the phospholipid cardiolipin (Cl) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion of an acyl chain into cyt c, resulting in the dissociation of the axial Met-80 haem-iron ligand resulting in a five coordinate cyt c/Cl complex that exhibits peroxidatic properties leading to peroxidation of Cl and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cyt c from the mitochondria into the cytoplasm. Two distinct surface regions on cyt c have been suggested to mediate Cl acyl chain insertion and Chapters 2 and 3 of this thesis report the results of probing one of these regions by constructing a series of alanine mutants in yeast iso-l-cyt c aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, Cl binding, and kinetics of carbon monoxide (CO) binding to the ferrous cyt c/Cl complex have been assessed for each individual mutant. The findings reveal that the majority of mutants are capable of binding Cl in the same apparent stoichiometry as the wild-type protein. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the Cl binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cyt c/CL complex. The second part of this thesis, Chapters 4 and 5, switches focus to human cyt c. A recently discovered natural mutant of human cyt c - G41S, was found to have enhanced apoptotic activity in megakaryocytes leading to thrombocytopenia. An expression construct for WT human cyt c was prepared enabling the G41S mutation to be made and studied. Peroxidase activity in the presence and absence of three phospholipids (TOCl, DOPS and DOPG) was studied and revealed enhanced activity for the G41S mutant in the presence of TOCL. To correlate a mechanism of peroxidase activity in the human cyt c/lipid complex an extensive EPR study was carried out which identifies the spin state changes of haem-iron upon peroxidation of cyt c/lipid complexes. Furthermore, the EPR study identifies that a Tvr radical is formed during peroxide turnover by the cyt c/lipid complex. Identification of which of the five Tyr residues in human cyt c the radical resides has been aided by the crystal structure of human cyt c with Tyr- 46 or Tyr-48 being identified. Further work is required to confirm which of the two Tyr residues act as the radical site.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available