Exploring Oxidation State Dependent Conformational Changes of Cytochrome C on Cardiolipin Containing Liposomes

Abstract

¬A combination of circular dichroism, absorption and fluorescence spectroscopies were used to study the binding of oxidized and reduced cytochrome c to cardiolipin (CL) containing liposomes with different CL content as a function of cardiolipin concentration. Recently, our group provided evidence for a two-step binding process of the oxidized (ferricytochrome) state to CL containing liposomes. The second step of binding converts a native-like to a more unfolded structure in which M80 is replaced either by a lysine of a histidine as axial ligand. The conversion into this more unfolded conformation was found to be partially inhibited in the presence of NaCl. Currently we are investigating whether the initial binding and/or the equilibrium between the two membrane bound conformers depends on pH. Our work with the reduced (ferrocytochrome) state has revealed a dependence on NaCl for stability. In the absence of NaCl and at aerobic environment, the protein becomes oxidized upon binding to CL containing liposomes. When NaCl is added to the solution, it stabilizes the reduced state on the liposome surface and inhibits the auto-oxidation. At anaerobic conditions and in the absence of NaCl, the protein undergoes a notable conformational change, but clearly maintains its reduced state. This shows that the obtained oxidation of the protein involves the reduction of molecular oxygen. We are in the process of exploring the pH dependence of the underlying change of the protein's redox potential.