Selective Probing of Non-Native Cardiolipin-Bound Conformations of Ferricytochrome C via Ferrocyanide-Mediated Photoreduction

Abstract

Upon binding to cardiolipin (CL), ferricytochrome c replaces its native Met80 ligand and ultimately gains peroxidase activity, a vital step towards its dissociation from the inner mitochondrial membrane and its subsequent initiation of apoptosis. Multiple binding studies involving ferricytochrome c and CL-containing liposomes have resulted in a variety of partially conflicting binding models, but little quantitative insight into the co-existence of native and partially unfolded conformations of ferricytochrome c has been obtained thus far. While multiple groups have put forth binding models to explain this phenomenon, further validation of these models and characterization of the involved conformational states is still outstanding. Resonance Raman (RR) spectroscopy is perfect for this task due to the high sensitivity of the heme vibrational modes to ligation, oxidation, and spin state changes of the heme iron. By utilizing ferrocyanide-mediated photoreduction, a normally undesired phenomenon that is inhibited for ferricytochrome c conformations lacking Met80 ligation, we selectively probed the non-native states of ferricytochrome c bound to CL-containing liposomes both in the presence and absence of NaCl. The thus elucidated concentrations of these non-native states at different lipid concentrations and ionic strengths were consistent with a previously reported model, where different conformations of the protein were calculated based on a global thermodynamic fit to a proposed [CL] dependent equilibrium between a native-like (prone to photoreduction) and a partially unfolded bound state for which photoreduction is inhibited. RR spectral evidence suggests a dominance of hexacoordinated low spin states even at high lipid content. Only trace evidence for the presence of a high spin state could be inferred from Raman spectra, but the presence of a charge transfer band at 625 nm in the optical spectrum might point to the presence of a quantum-mixed state.