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Identifier

000369503

Title

Φασματοσκοπικός χαρακτηρισμός της αλληλεπίδρασης του N3, CN, CO με το διπυρηνικό κέντρο του κυτοχρώματος c αα3 από το P. denitrificans και του κινολικού κυτοχρώματος bo3 από το E.coli

Alternative Title

Spectroscopic characterization of the interaction of N3, CN, CO with the binuclear center of cytochrome caa3 from the P. denitrificans and quinol cytochrome bo3 from E.coli

Author

Βάμβουκα, Μαγδαληνή

Thesis advisor

Γανωτάκης, Δημήτριος

Abstract

Cytochrome and quinol oxidases are the terminal enzymes of the respiratory chain. These enzymes catalyze the fully reduction of molecular oxygen, and use the free energy released in this reaction to translocate protons across the cell membrane. The proton gradient that is produced drives the synthesis of ATP. The aim of this work is the study of the structure and dynamics of the binuclear center of cytochrome and quinol oxidases by using ultraviolet-visible and infrared spectroscopy. We studied the binding of azide ion to fully oxidized cytochrome aa₃ the P. denitrificans bacterium. The results presented here show that azide binds in two phases, a high affinity and a low affinity phase. Furthemore, the FTIR suggest that, in the low affinity phase, azide binds in two different enzyme conformations, both forming bridging complexes with the oxidized heme Fe-CuB binuclear center. We also examined the interaction of cyanide ion with the cytochrome c oxidase (CcO) from mitochondria and the cytochrome aa₃-600 from B. subtilis and cytochrome bo₃ from E. coli quinol oxidases. This work indicate that cyanide binds as bridging ligand within the binuclear center in CcO, cytochrome aa₃-600 and cytochrome bo₃. It also shows that, at pH 8.4, addition of cyanide to CcO causes the reduction of heme a and CuB. We proposed that under these conditions cyanide forms terminal complexes with Fe_a3³⁺. Based on our results and previous studies we propose static and time resolved UV-Vis and FTIR experiments with the aim 1) to fully characterize the binding of external ligands to the binuclear center of the terminal oxidases and describe how the binding properties depend on the pH and the redox state of the enzyme and 2) to understand the role of the reduction site into the mechanism of proton translocation.

Language

Greek

Subject

Binuclear center

Cytochrome oxidases

FTIR spectroscopy

Ligand binding

Quinol oxodases

UV-VIS spectroscopy

Δέσμευση υποκατάστατων

Διπυρηνικό κέντρο

Κινολικές οξειδάσες