Doctoral theses
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| Identifier |
000290434 |
| Title |
Bιοενεργητικοί Μηχανισμοί του Ο2 και του ΝΟ με την εφαρμογή Density Functional Theory |
| Alternative Title |
Bioenergetic mechanisms of O2 and NO as revealed by density functional theory |
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Author
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Δασκαλάκης, Ευάγγελος Γ
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Thesis advisor
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Βαρώτσης, Κωνσταντίνος
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| Abstract |
Vibrational and structural characterization of the intermediates in processes like the reduction of molecular oxygen and NO activation by cytochrome c oxidases (CcO) and nitric oxide reductases (Nor), respectively, is a crucial step in the under-standing of O2 and NO bioenergetics. Both O2 and NO act as substrates of respiration and denitrification enzymes, which are metabolic pathways yielding energy in mam-malian and bacterial cells. The first section of this thesis refers to molecular oxygen activation. After identifying the place of terminal oxidases in the chaotic pathways of metabolism, we make an introduction to their structural and functional characteristics (chapter I). The active site of CcO is a binuclear center composed of a high spin heme iron and a copper site (CuB). The second section is consent, additionally, with NO chemistry in biological systems, while the introduction of chapter VII contains a short reference on the denitrification process and nitric oxide reductases. In Nor, the active site is composed of a high spin heme iron and a non-heme FeB. Initially, a DFT study was conducted to probe the dynamics of CuB metal center in cytochrome c oxidases, as this site is the entrance of substrate O2 to the binuclear site. CuB-CO vibrational characterization in relation to changes in the coordination sphere of copper are of high interest. CuB linands (three histidines and the covalently cross-linked tyrosine) remain protonated and coordinated to CuB upon CO addition and at pH 5.5-10 range. cbb3s CuB site lacks the cross-linked tyrosine, but exerts the same behavior. Theoretical characterization of copper metal site in CcO gives insight into the spectroscopically silent metal, proving that its ligands do not take active role in processes like proton translocation in CcO catalytic cycle. Extending theoretical calculations to models of active site containing more than 100 atoms we investigated the presence of two different oxo-ferryl conformations (804 and 790 cm-1) in the catalytic cycle of cytochrome c oxidase. These two species are attributed to two different conformations of the heme iron axial ligand with and without hydrogen bonding interactions in the proximal area. We characterized possi-ble changes in proximal area that yield the 804/ 790 cm-1 species and proposed a mechanistic pathway of oxygen activation by CcO based on the new findings. More-over we drew a possible connection between E278 in the D-proton pathway and proximal area during the P F transition. In correlation to the previous study, we investigated proximal and distal effects that shift ν(Fe-O) vibrational stretching frequency in oxy and hydroxy intermediates of CcO/ O2 reaction. In contrast to oxo-ferryl species, the hydroxy intemediate appears in two different conformations (450/ 475 cm-1) mainly due to a distal hydrogen bond, while oxy intermediate exerts a single ν(Fe-O) at 568 cm-1 consistent with only one conformation. Turning to NO biochemistry in bacterial denitrification we characterized theo-retically the key-intermediate in the catalytic cycle of nitric oxide reductase in the step of ΝΝ bond formation. We proposed a mechanistic pathway of NO activation based on the hyponitrite (HO-N=N-O-) structure determined in the Fea33+/ FeB2+ active site of Nor and we compared it to that of ΝΝ formation in ba3 cytochrome oxidase. The polarizability of O2, CO and NO free gases in enzyme or protein cavities and the way that this is altering their vibrational stretching frequencies is of great im-portance in identifying such structural sites. Hydrogen bonds or electrostatic interac-tions present in these cavitties lead to significant ν(O-O), ν(N-O) and ν(C-O) shifts in a way that in certain cases reassembles the binding of these gases to Fe or CuB. To conclude, its of great importance to understand that enzymatic systems un-der study, like those mentioned above, were modeled with complexes containing up to about 130 atoms and in most cases with two transition metals, forcing pure ab initio calculations to be extremely time-consuming. DFT method was able to intrude in ac-tive sites of heme proteins and extract information in atomic level either enforcing experimental data or determining their interpretation with great impact on enzyme dynamics.
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| Language |
Greek |
| Issue date |
2006-07-20 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Chemistry--Doctoral theses
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Type of Work--Doctoral theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/2/8/b/metadata-dlib-2006daskalakis.tkl
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| Views |
247 |
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