Oxidation State and PH Dependence of Cytochrome C Binding to Cardiolipin-Containing Liposomes

Abstract

A combination of UV/visible absorption, circular dichroism and fluorescence spectroscopies was used to probe the binding of cytochrome c to cardiolipin (CL) containing liposomes and concomitant conformational changes as a function of cardiolipin concentration at different pH values. Our group has recently developed a model that describes oxidized cytochrome c binding to CL-containing liposomes as a two-step process where native-like liposome bound conformers convert into more unfolded conformations. We have provided evidence that the M80 ligand in the more unfolded state has been replaced by either a lysine or histidine as the axial ligand. A slightly modified binding model was used to describe the binding of reduced cytochrome c, to account for the redox properties of the heme in the absence and presence of oxygen. In our current study, we explored how lowering the pH to values between 6.0 and 7.0 affects binding and conformation of cytochrome c in both oxidation states. It has recently been proposed that such a protocol would facilitate the so-called L-site binding, which involves the electrostatic interaction of amino acid residues Lys22, −25, −27 and His26 and −33 with the acidic phospholipids on the liposome surface. Our results for binding at pH 6.5 indicate a conversion from a low spin to a hexacoordinated high-spin state at moderate to high cardiolipin concentrations, which leads to a significant blue shift of the Soret band. This very much resembles the behavior of denatured cytochrome c where lowering the pH leads to the protonation of a distal histidine ligand and its replacement by a water ligand. Thus, our observations strongly suggest that histidine rather than lysine is the sixth ligand in the misligated state of cytochrome c on CL-containing liposomes.