Structural transitions of cytochrome c : implication for apoptotis and peroxidase actvity.

The discovery of new functions of cytochrome c was a good example to study the evolutionary processes and it is a stunning example of the collapse of an old dogma:”one gene, one protein, one function”. Cytochrome c is a small heme protein, a soluble protein and an essential component of the mitochondrial respiratory chain because of its capability to transfer electrons between the III and IV complexes. Beyond the role as electron carrier in the respiratory chain, cytochrome c is involved in the triggering of apoptosis “programmed cell death”. Cytochrome c, released from the inner mitochondrial membrane in response to specific signals, binds to Apaf-1, nucleotides (dATP or ATP) and procaspase 9 to form the apoptosome in the cytosol. All these important biological activities are realized in its native structure. Studies from several laboratories document that the unfolding of cytochrome c reveals a new function as a peroxidase. With this in mind, the aim of the present thesis was to define the role of the binding ATPcytochrome c and to demonstrate the importance of the ATP as an allosteric effector, in regulating structural transitions among different conformations and different oxidation states of cytochrome c, endowed or not with apoptotic activity. The present study suggests a physiological role for ATP binding to cytochrome c at low millimolar concentrations in the cytosol, beyond the regulatory role previously reported in the oxidative phosphorylation in mitochondria. For the experiments, it has been used the horse heart cytochrome c, a single polypeptide chain organized in five smallhelices and three omega loops. The prosthetic group is linked to the polypeptide chain by means of two tioether bonds formed by cysteins in position 14 and 17. The heme-iron is also axially coordinated with the two residues His18 and Met80. In the present work, the structural and functional properties of single mutants K88E, E62N, R91N and double mutant K88E/E62N have been compared, because in previous studies the binding site of ATP had been identified by docking simulations. By performing NMR measurement it has been demonstrated that ATP interacts with a site (S1) formed by K88, R91 and E62. Glu62 and Lys88 are replaced by Asn and Glu, present in the iso-1 cytochrome c sequence that is not able to interact with ATP. R91N has been analyzed because it‟s conserved in all species of cytochrome c and it is implicated in the binding of ATP to cytochrome c. The apoptotic activity of cytochrome c variants has been studied by using a “cell-free” activation assay. In the second part of this thesis it has been studied the peroxidase activity of non-native variants of cytochrome c. Native cytochrome c is characterized by a compact tertiary structure and is thus unavailable for binding of any other compound. The bond between the methionine residue and the heme iron, however, is rather weak so that it‟s easily broken and facilitates the access of small molecules, such as H2O2, in the heme pocket. In particular, the access of H2O2 determines the change of cytochrome c into Compound I-type peroxidase. At the aim of investigating the peroxidase activity of non-native cytochrome c, different forms of the protein have been analyzed in order to correlate their structural features with the acquired enzymatic activity. We studied the structural features of cytochrome c bound to cardiolipin (CL)-containing liposomes and Y67H and H26Y cytochrome c mutants. Histidine in position 26 was changed with a tyrosine (H26Y) because it was highlighted the contribution of the hydrogen bond linking the histidine in position 26 (Ωloop 20s) to the carbonyl group of the residue 44 (Ω-loop 40s), to the stability, folding, and functional properties of cytochrome c. The other mutation was concerning with Tyr67, which was replaced by a histidine (Y67H), with the aim of introducing a general acid–base catalyst, a fundamental element of the peroxidase mechanism, in the heme cavity. The spectroscopic features of cytochrome c/CL complex, H26Y and Y67H mutants were analyzed and the biological significance of the observed conformational changes was inferred by the relationship with their peroxidase activities and apoptogenic properties. To gain further insights on the capability of ATP to function as allosteric regulator of cytochrome c conformational transitions, its effect on refolding and its modulation of the peroxidase activity of cytochrome c variants were also investigated. Moreover, the peroxidase kinetic parameters of the non native conformers here investigated were measured also in the presence of minocycline, an antibiotic endowed with antioxidant properties reported to act as peroxidase inhibitor and neuroprotective agent that delays progression of neuron degeneration.