In vitro studies on cytochrome c biogenesis
<p><em>C</em>-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.</p> <p>This thesis describes...
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| Format: | Thesis |
| Language: | English |
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2003
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| _version_ | 1826315612834496512 |
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| author | Daltrop, O Oliver Daltrop |
| author2 | Ferguson, S |
| author_facet | Ferguson, S Daltrop, O Oliver Daltrop |
| author_sort | Daltrop, O |
| collection | OXFORD |
| description | <p><em>C</em>-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.</p> <p>This thesis describes the development of <em>in vitro</em> systems to establish chemical aspects of the process of cytochrome <em>c</em> maturation. Initially, the uncatalysed reaction of heme and apocytochrome <em>c</em> from <em>Hydrogenobacter thermophilus</em> cytochrome <em>c</em><sub>552</sub> was studied <em>in vitro</em>, yielding the desired thioether bonds under mild conditions and in the absence of any biosynthesis apparatus. The reaction proceeded via a <em>b</em>-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 <em>H. thermophilus</em> apocytochrome, thioether bonds can form stereoselectively with respect to the α, 𝛾 mesoaxis of heme.</p> <p>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 <em>Paracoccus denitrificans</em> apocytochrome <em>c</em><sub>550</sub>. It was established that apocytochromes show a trend to bind hydrophobic ligands or heme to yield <em>b</em>-type cytochromes. Thioether bonds can form <em>in vitro</em> in these heme-protein complexes.</p> <p>In the second part of this thesis the heme chaperone CcmE, which is part of the cytochrome <em>c</em> biogenesis apparatus in many Gram-negative bacteria, was studied as an extension to the establishment of <em>in vitro</em> 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 <em>in vitro</em> reaction; mechanistic insights were gained from the observation that a histidine to cysteine mutant could still bind heme covalently <em>in vivo</em> and <em>in vitro</em>. Effects of the presence of a His tag on CcmE were shown and are discussed. Furthermore, <em>in vitro</em> heme transfer from CcmE to certain apocytochromes <em>c</em> was achieved.</p> <p>All these <em>in vitro</em> results mimic, and thus have implications for, the molecular pathway of heme transfer during the complex process of <em>c</em>-type cytochrome maturation <em>in vivo</em>.</p> |
| first_indexed | 2024-03-06T18:42:30Z |
| format | Thesis |
| id | oxford-uuid:0d602386-e848-4f7a-9a0e-b6dd275b85b5 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:29:23Z |
| publishDate | 2003 |
| record_format | dspace |
| spelling | oxford-uuid:0d602386-e848-4f7a-9a0e-b6dd275b85b52024-12-01T12:07:00ZIn vitro studies on cytochrome c biogenesisThesishttp://purl.org/coar/resource_type/c_db06uuid:0d602386-e848-4f7a-9a0e-b6dd275b85b5CytochromesHemeEnglishPolonsky Theses Digitisation Project2003Daltrop, OOliver DaltropFerguson, SFerguson, S<p><em>C</em>-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.</p> <p>This thesis describes the development of <em>in vitro</em> systems to establish chemical aspects of the process of cytochrome <em>c</em> maturation. Initially, the uncatalysed reaction of heme and apocytochrome <em>c</em> from <em>Hydrogenobacter thermophilus</em> cytochrome <em>c</em><sub>552</sub> was studied <em>in vitro</em>, yielding the desired thioether bonds under mild conditions and in the absence of any biosynthesis apparatus. The reaction proceeded via a <em>b</em>-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 <em>H. thermophilus</em> apocytochrome, thioether bonds can form stereoselectively with respect to the α, 𝛾 mesoaxis of heme.</p> <p>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 <em>Paracoccus denitrificans</em> apocytochrome <em>c</em><sub>550</sub>. It was established that apocytochromes show a trend to bind hydrophobic ligands or heme to yield <em>b</em>-type cytochromes. Thioether bonds can form <em>in vitro</em> in these heme-protein complexes.</p> <p>In the second part of this thesis the heme chaperone CcmE, which is part of the cytochrome <em>c</em> biogenesis apparatus in many Gram-negative bacteria, was studied as an extension to the establishment of <em>in vitro</em> 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 <em>in vitro</em> reaction; mechanistic insights were gained from the observation that a histidine to cysteine mutant could still bind heme covalently <em>in vivo</em> and <em>in vitro</em>. Effects of the presence of a His tag on CcmE were shown and are discussed. Furthermore, <em>in vitro</em> heme transfer from CcmE to certain apocytochromes <em>c</em> was achieved.</p> <p>All these <em>in vitro</em> results mimic, and thus have implications for, the molecular pathway of heme transfer during the complex process of <em>c</em>-type cytochrome maturation <em>in vivo</em>.</p> |
| spellingShingle | Cytochromes Heme Daltrop, O Oliver Daltrop In vitro studies on cytochrome c biogenesis |
| title | In vitro studies on cytochrome c biogenesis |
| title_full | In vitro studies on cytochrome c biogenesis |
| title_fullStr | In vitro studies on cytochrome c biogenesis |
| title_full_unstemmed | In vitro studies on cytochrome c biogenesis |
| title_short | In vitro studies on cytochrome c biogenesis |
| title_sort | in vitro studies on cytochrome c biogenesis |
| topic | Cytochromes Heme |
| work_keys_str_mv | AT daltropo invitrostudiesoncytochromecbiogenesis AT oliverdaltrop invitrostudiesoncytochromecbiogenesis |