| Abstract |
Cytochrome and quinol oxidases catalyze the reduction of O2 to water, the final step in the cellular respiration. In addition, these enzymes use the free energy released in this reaction, in order to translocate protons across the membrane in which they are buried, producing a proton gradient that drives the synthesis of ATP. The active site is a spin coupled heme-CuB binuclear center, where O2 binds and reduction occurs. It has been proposed that binuclear center is also involved in proton pumping process via ligand rearrangements. The aim of the present dissertation is the characterization of the structural and dynamic properties of the binuclear center upon the binding of exogenous ligands, by using UV-Vis, resonance Raman and FTIR spectroscopy. In this work, it has been characterized the CO binding to fully reduced aa3-600 quinol oxidase. Despite the presence of two different heme-protein conformations that affect the proximal His376-iron bond stretch in the fully reduced ligand-free form of the enzyme, the CO-bound enzyme exists in a single conformation in which the His376-Fe2+-CO moiety has the same structure as the α form found in bovine cytochrome c oxidase. Moreover, it is demonstrated that an inverse linear correlation exists between the frequencies of the ν(Fe-His) and ν(Fe-CO) stretching modes of the terminal oxidases. It is proposed that the frequencies of both the ν(Fe-CO) and ν(C-O) modes found in heme-CuB oxidases are affected by the proximal His376, which is hydrogen bonded to Gly351, and by distal effects on the bound CO exerted by CuB. It is also hypothesised that the hydrogen bond between His376-Gly351 could regulate the anionic character of the proximal histidine, affecting the stabilization of higher oxidation states of heme Fe4+=O2- which is formed during the catalysis. It has been also studied the binding of azide ion to fully oxidized cytochrome c oxidase from bovine heart and the P. denitrificans bacterium and the bo3 quinol oxidase from the E. coli bacterium. Azide ion binds in two different enzyme conformations, both forming bridging complexes with the oxidized heme Fe-CuB binuclear center. This study also reports for first time a pH-dependent conformational change in bovine cytochrome c oxidase, in the fully oxidized N3-bound form of the enzyme. It is proposed that the transition between the two N3-bound conformations is associated with a polarity change in the distal environment, which affect the position of CuB. Comparison of the resonance Raman and FTIR data of the three enzymes suggests significant alternations in the configuration of the bound azide, resulting from specific structural differences within their respective heme-CuB binuclear pockets. Furthermore, these data suggest that differences in distal environment, adjacent bound azide rather than differences in a trans effect induced by proximal histidine affect the conformation of azide. In this dissertation it has been also investigated the interaction of cyanide ion with the oxidized bovine cytochrome c oxidase and the aa3-600 and bo3 quinol oxidases. Cyanide binds as bridging ligand within the binuclear center. However, in contrast to the case of azide, it is observed a single conformation of the Fe3+-CN-CuB2+ moiety. The correlation of the frequencies of the ν(CN) stretching vibrations observed for the three enzymes and the frequencies of the ν(Fe-His) have been reported previously indicates that the position and/or the acidity of CuB differ between the three enzymes. These features are the key determinants of the frequencies of the ν(CN) modes observed for the three enzymes. FTIR data on cytochrome c oxidase also suggest that, if CuB is reduced, cyanide binds to heme Fe3+ as terminal ligand in two distinct conformations. It is proposed that the two conformations reflect a change in the basicity of proximal His376 as well as differences in the distal environment. The results of azide and cyanide binding indicate that, in the fully oxidized form, CuB has a direct interaction with the heme Fe-bound ligand, forming bridging complexes. These phenomena could present a model for the postulated peroxy intermediate. If it is formed, it is very likely to have a bridging structure (Fe3+-O--O--CuB2+).
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Φασματοσκοπικός χαρακτηρισμός της δέσμευσης εξωγενών ligands στην κυτοχρωμική οξείδαση
