Cardiolipin Membranes as Photoreduction Inhibitors in Ferricytochrome C: A Resonance Raman Study with Soret Band Excitation

Abstract

The binding and behavior of ferricytochrome c to cardiolipin (CL) containing membranes is important to characterize to fully understand the protein's involvement in apoptosis. Upon binding, it has been shown that the protein loses its Met80 ligand and ultimately gains peroxidase activity, a vital step in its dissociation from the inner mitochondrial membrane (IMM). Multiple binding studies have been done on the protein binding to CL containing liposomes, which serve as IMM mimics, using spectroscopic techniques including UV-CD, UV-VIS absorption, and fluorescence, among others. These techniques, however, provide rather limited information about the heme group which serves as the active site for the protein's peroxidase activity. Resonance Raman (RR) spectroscopy on ferricytochrome c allows us to selectively probe the heme vibrational modes, which are very sensitive to changes in the heme's conformation and changes in the ligation, spin, and oxidation states. However, it has been found that Soret bands excitation leads to photoreduction of the protein proving proper analysis very difficult. We investigated whether the binding of ferricytochrome c to CL containing liposomes results in the inhibition of photoreduction due to a sharp decrease in redox potential arising from the dissociation of the Met80 ligand. It was found that the extent of photoreduction is lowered by increasing cardiolipin concentrations, with most spectra obtained at low cardiolipin concentrations showing a clear mixture of bands assignable to both the photoreduced and oxidized protein. There was little to no photoreduction at high lipid concentrations, along with a clear disappearance of the low frequency RR modes, a sign of the heme becoming more planar.