Your browser does not support JavaScript!

Home    Time-Resolved FTIR και Ηλεκτροχημικές Μελέτες των cbb3, aa3-600 και ba3 Oξειδασών  

Results - Details

Add to Basket
[Add to Basket]
Identifier 000039987
Title Time-Resolved FTIR και Ηλεκτροχημικές Μελέτες των cbb3, aa3-600 και ba3 Oξειδασών
Alternative Title Time-resolved FTIR and electrochemical studies of cbb3, aa3-600 and ba3 oxidases
Author Σταυράκης, Σταύρος
Thesis advisor Βαρώτσης, Κωνσταντίνος
Abstract The aim of the present dissertation is the characterization of the structural and dynamic properties of the binuclear center upon binding of exogenous ligands as well as the determination of reduction potentials of cytochrome and quinol oxidases. FTIR and time-resolved step scan FTIR have been applied for the investigation of the structure and dynamics of the binuclear site of cytochrome cbb3- CO complex. The rates for the CuB-CO dissociation (t1/2 = 120 μs) and heme b3-CO recombination (t1/2= 150 μs) we have determined in this study suggest that the rate limiting step for the heme b3 recombination of CO is the half-life of the CuB-CO complex. The time-resolved FTIR data reveal that in this cbb3-type oxidase the decay of the transient CuB-CO complex is concurrent with the formation of the heme b3-CO complex, and the ν(CO) of CuB at 2064 cm-1, despite the lack of the cross-link tyrosine 280, is similar to that observed in cytochrome aa3. As a first step in understanding the cytochrome cbb3/NO reaction is the characterization of the binuclear center upon NO binding. In this work we report the NO-bound form of oxidized cbb3 by Fourier tranform infrared spectroscopy (FTIR) and compare it with that of the heme b33+-NO complex of NOR, and the NO complex of metmyoglobin. The data reported here demonstrate that NO binding occurs exclusively at the heme b3 center and not at CuB. The absence of a negative charge in the binuclear center of heme b3-CuB in conjunction with a more open pocket than in other heme-copper oxidases, lowers the energy barrier optimizing the efficiency of NO escape We report the first evidence for the formation of a mixed valence heme b Fe3+/Cu1+-CO complex at ambient temperature. This bimetallic center is the catalytic site of heme-copper oxidases where the O2, and in some cases, the NO reductions take place. We have used cytochrome oxidase cbb3 to demonstrate that addition of O2 to the fully reduced CO bound heme b Fe+2 enzyme oxidizes the heme b Fe and forms a Cu+1-CO complex. The ν(CO) frequency at 2047 cm-1 of the novel Cu+1-CO species is 16 cm-1 lower than that of the transient (t1/2=160 ms) heme Fe3+/Cu1+-CO (ν(CO)= 2063 cm-1) species that is formed upon photodissociaton of CO from heme b Fe+2. The formation of such novel equilibrium mixed valence heme b Fe3+/Cu1+-CO complex is observed for the first time in any structurally known heme-copper oxidases and heme-Cu model compounds. Τime-resolved step scan FTIR has been applied for the investigation of the structure and dynamics of the binuclear site of αα3-600 from Bacillus subtilis. The results illustrate that that the dynamics of the heme a3 proximal pocket in not a possible explanation for the dissociation of CO from CuB and that distal effects play the dominant role in the recombination process. Our results are consistent with an activation barrier which prevents CO recombination to heme a3 and ligand removal from CuB occurs via escape into the surrounding solvent before binding to the heme a3. A model describing the photodissociation/association of ligands in the active site of the enzyme has been proposed. The spectroelectrochemical technique has been applied in order to determine the potentials of each of the redox centers of cytochrome cbb3. Moreover a comparison of the redox potential of heme b3 is being made between cytochrome cbb3 and the NO reductase so as to draw conclusions with regard to the heme environment before the reaction with the NO. The results show that complete reduction of the binuclear center of cytochrome cbb3 is thermodynamically favorable. This means that the mechanism of activation of NO occurs with a fully reduced binuclear center b3/CuB. On the contrary the potential of heme b3 of NOR declares that the reduction of the binuclear center b3/FeB is not favorable from the physiologic donors of electrons. The spectroelectrochemical technique has been applied in order to determine the potentials of each of the redox centers of cytochrome ba3. The mechanism by which cytochrome ba3 reduces NO to N2O has not yet been clarified. As a first step in investigating this complicated mechanism, we have used the spectroelectrochemical technique in order to study the fully reduced ba3-NO complex. Knowing the number of electrons that we electrochemically give or extract, we are in a position to monitor spectroscopically the possible forms of ferrous heme a3, during the electrochemical oxidation of the fully reduced ba3-NO complex. The results indicate that cytochrome ba3 catalyzes the two-electron reduction of NO to N2O and that the oxidation of heme b is the rate-limiting step for the reduction of NO. These results may help us to elucidate the catalytic cycle of NO reduction by cytochrome ba3.
Language Greek
Issue date 2004-09-24
Collection   School/Department--School of Sciences and Engineering--Department of Chemistry--Doctoral theses
  Type of Work--Doctoral theses
Views 257

Digital Documents
No preview available

Download document
View document
Views : 9