Doctoral theses
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| Identifier |
000036333 |
| Title |
To O2 και το ΝΟ στην Αναπνοή: Time-Resolved FTIR και Resonance Raman Mελέτη τηςΚυτοχρωμικής c Οξειδάσης και της Αναγωγάσης του Μονοξειδίου του Αζώτου |
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Author
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Πινακουλάκη, Ευτυχία
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Thesis advisor
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Βαρώτσης, Κωνσταντίνος
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| Abstract |
The aim of this phD thesis was to study 1) cytochrome c oxidase (CcO), which is the enzyme that catalyses the reduction of dioxygen to H2O in the aerobic respiration of mammals and bacteria, and 2) nitric oxide reductase (NOR) that catalyses the reduction of nitric oxide in the anaerobic respiration of bacteria. The active center of CcO is a binuclear site consisting of a high spin heme and a copper atom, CuB. The catalytic site of NOR consists of a high spin heme and a non-heme iron, FeB. A major aim of the study of CcO was to determine the structural and functional role of Tyr280, which is covalently bound to one of CuB ligands, namely His276. We have used resonance Raman and FTIR spectroscopy to characterize cytochrome aa3 and the Y280H mutant from P. denitrificans in the oxidized and fully reduced state, as well as the CO- complexes of the enzymes. Heme a3 retains the same proximal environment, spin, and coordination state in the wild type and Y280H mutant enzyme. The catalytic site retains its active configuration that allows dioxygen binding to heme a3, thus the stability of the site is not compromised by absence of the covalent link between Tyr280-His276. We have studied the formation of the 607 and 580 nm species of the catalytic cycle of CcO by investigating the reaction of the fully oxidized cytochrome aa3 from P. denitrificans with hydrogen peroxide, at room temperature, using optical and resonance Raman spectroscopy. The role of Y280 in the formation of the 607 and 580 nm species was also examined by comparing the reactions of the oxidized wild type enzyme/H2O2 and the mixed- valence enzyme/O2, to the respective reactions of the Y280H mutant. We have observed the formation of the same species in the reactions of the wild type and the Y280H mutant enzyme, and thus, suggested that Y280 is not involved in the generation of these intermediates. FTIR and time-resolved step scan FTIR have been applied for the investigation of the structure and dynamics of the binuclear site of cytochrome caa3 from Thermus thermophilus, which displays significant activity in the reduction of nitric oxide. Cytochrome caa3 is the only member of the heme-copper oxidase family, whose binuclear center consists of an a3- type heme of the β-form and a CuB atom of the α-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the α-form are not required for enzymatic activity. A model describing the photodissociation/association of ligands in the active site of the enzyme has been proposed. We have also used the same experimental approach for the comparative study of the alternative oxidases of P. denitrificans, cytochromes aa3 and ba3. We have studied the structure of the dinuclear heme/non-heme iron center of nitric oxide reductase from P. denitrificans in both the oxidized and reduced states of the enzyme, as well as in the CO-complex of NOR. The two distinct νas(Fe-O-Fe) modes we have observed suggest the presence of two different conformations (open and closed) of the catalytic site of the oxidized enzyme. The proximal histidine is not ligated to the heme b3 in the oxidized state of NOR. The transition to the reduced state of the enzyme results in the formation of the heme b3-His bond. Heme b3 becomes low spin and six-coordinate in the CO- complex. Resonance Raman and FTIR spectroscopy have been applied for the investigation of the reaction of both oxidized and reduced NOR with nitric oxide. The interaction of oxidized NOR with NO results in the formation of a heme b3 ferric-six-coordinate low-spin NO- complex, which we propose that is an intermediate of the catalytic cycle. The product of reaction of fully reduced NOR/NO indicates that the formation of the N-N bond in the denitrification process occurs with an intact His-heme b3 bond. The reduction of dioxygen by NOR has been investigated, as NOR is considered to be a distant member of the heme-copper oxidase superfamily, and compared to the respective reaction of cytochrome cbb3 from P. stutzeri, which is the heme-copper oxidase that displays the higher homology with NOR. We have also studied the reaction of the fully reduced cytochrome cbb3 with NO. Cytochrome cbb3 displays the highest activity in nitric oxide reduction among the members of the heme-copper oxidase family. Addition of NO to the reduced state of cytochrome cbb3 causes the disruption of the heme b3-His bond, resulting in the formation of a five coordinate b3 2+-NO complex. A model describing the oxidation of the enzyme after the addition of NO has been proposed.
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| Language |
Greek |
| Issue date |
2002-11-01 |
| 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/c/9/8/metadata-dlib-2002pinakoulaki.tkl
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| Views |
285 |
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