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Title: In vitro studies on cytochrome c biogenesis
Author: Daltrop, Oliver
ISNI:       0000 0001 3402 7496
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2003
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C-type cytochromes are essential for almost all organisms and are mainly involved in electron transport; they are characterised by the covalent attachment of heme to protein through two thioether bonds to a CXXCH peptide motif. This thesis describes the development of in vitro systems to establish chemical aspects of the process of cytochrome c maturation. Initially, the uncatalysed reaction of heme and apocytochrome c from Hydrogenobacter thermophilus cytochrome c552 was studied in vitro, yielding the desired thioether bonds under mild conditions and in the absence of any biosynthesis apparatus. The reaction proceeded via a b-type cytochrome intermediate, showing the ability of the apoprotein to bind heme noncovalently prior to thioether bond formation. It was also determined that the two cysteine residues of the CXXCH motif can form a disulfide. Optimal reaction conditions for thioether bond formation required both the heme and the cysteine residues to be reduced. Mechanistic insights were gained by showing that the thioether bonds can form independently from one another. Furthermore, it was shown that, in the case of H. thermophilus apocytochrome, thioether bonds can form stereoselectively with respect to the α, γ mesoaxis of heme. These findings were extended to a broader range of apocytochromes. It was discovered that mitochondrial apocytochromes c from horse heart and yeast also formed thioether bonds with heme, as well as the bacterial Paracoccus denitrificans apocytochrome c550. It was established that apocytochromes show a trend to bind hydrophobic ligands or heme to yield b-type cytochromes. Thioether bonds can form in vitro in these heme-protein complexes. In the second part of this thesis the heme chaperone CcmE, which is part of the cytochrome c biogenesis apparatus in many Gram-negative bacteria, was studied as an extension to the establishment of in vitro systems. It was discovered that CcmE can bind heme initially non-covalently and then covalently upon reduction of the heme. However, CcmE seems to have a preference for ferric rather than ferrous heme. The involvement of the vinyl groups of heme is suggested. Mutation of the heme-binding histidine residue of CcmE established the involvement of this residue in the covalent binding of heme for the in vitro reaction; mechanistic insights were gained from the observation that a histidine to cysteine mutant could still bind heme covalently in vivo and in vitro. Effects of the presence of a His tag on CcmE were shown and are discussed. Furthermore, in vitro heme transfer from CcmE to certain apocytochromes c was achieved. All these in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during the complex process of c-type cytochrome maturation in vivo.
Supervisor: Ferguson, Stuart John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cytochromes ; Heme Molecular biology Cytology Genetics