We employed a combination of fluorescence, visible circular dichroism, and absorption spectroscopy to study the conformational changes of ferricytochrome c upon its binding to cardiolipin-containing small unilamellar vesicles. The measurements were performed as a function of the cardiolipin concentration, the cardiolipin content of the liposomes, and the NaCl concentration of the solvent. The data were analyzed with a novel model that combines a single binding step with a conformational equilibrium between native-like and non-native-like proteins bound to the membrane surface. The equilibrium between the two conformations, which themselves are comprised of structurally slightly different subconformations, shifts to the more non-native-like conformation with increasing cardiolipin concentration. For the binding isotherms described in this paper, we explicitly considered the enthalpic and entropic contributions of molecular crowding to protein binding at low lipid concentrations and high occupancy of the liposome surface. Increasing the CL content of liposomes increases the overall binding affinity but makes the conformational distribution much more susceptible to the influence of sodium and chloride ions, which shifts the equilibrium toward the more native-like state and directly inhibits binding, particularly to liposomes with 100% cardiolipin content. Spectroscopic evidence further suggests that a fraction of the non-native conformers adopts a pentacoordinated state similar to those obtained in class C peroxidases. On the basis of our results, we propose a hypothesis that describes the balance between facilitating and impeding forces controlling the peroxidase activity of cytochrome c in the inner membrane space of mitochondria.

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant protein insertion into the membrane. A global analysis of our data revealed the existence of two binding sites on the protein which causes rather different degrees of protein unfolding. We found that these two modes of interaction between protein and liposome led to conformational changes. While site 1 is relatively unaffected by NaCl, site 2 shows a more native-like state or a higher population of the native state in the presence of NaCl. At the highest utilized concentration of NaCl, there is only a 40% inhibition of the binding to site 2. We interpret our finding for this binding site as reflecting an equilibrium between electrostatically bound proteins with a high degree of unfolding and less unfolded proteins which bind either via H-bonding between lysine side chains and PO2(-) or hydrophobic interactions. With regard to site 2 binding, our results are reminiscent of the two-state equilibrium between a compact C and an extended E-state proposed by Pletneva and co-workers (Hanske et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 125-230). We conjecture that the nonelectrostatically bound proteins should have higher abilities to maintain the redox potential that is required for the function as an electron transfer protein.

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Cytochrome c binding to cardiolipin receptors on the surface of TOCL/DOPC(20% : 80%) liposomes induces a conformational change, which is not reversible after the protein's dissociation at low ionic strength. Addition of 100 mM NaCl switches the protein back to its native state.