Coexistence of Native-like and Non-Native Partially Unfolded Ferricytochrome c on the Surface of Cardiolipin-Containing Liposomes

Abstract

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.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…6C). Data for the interaction of WT Cc with liposomes are consistent with the K D values reported before (68,(72)(73)(74), but they diverge from the lower K D values reported in the literature (75) and the A/C two-site binding model described by Kinnunen and coworkers (66,67). Such discrepancies are still unresolved, and further research is necessary to harmonize data from all these different binding assays in a unified and single model.…”

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

“…7, 65, 66 Both the compact and extended conformers are competent for peroxidase activity. 7, 67 However, peroxidase activity is higher for the extended conformer, suggesting that it could be more effective at CL oxidation.…”

The key role played by charge in the interaction of cytochrome c with cardiolipin

“…The models proposed to describe the CL/cyt c interaction identified some regions involved in the protein-liposome interaction [5,6,17,18,26,47]; among others, the Met80-containing 66-92 region, which comprises the cleft formed by the 67-71 and 82-85 residues and a network of positively charged residues (i.e., Lys72, Lys73, Lys86) that may facilitate the insertion of the acyl chain, is particularly noteworthy [5,6,26]. This prompted us to focus our studies on this region, which is considered crucial for the cyt c/CL binding process.…”

“…CL-bound cyt c shows altered tertiary structure, a perturbed heme crevice and the displacement of Met80 from the sixth coordination position of the heme-Fe atom [5][6][7][8][16][17][18][19]. Depending on the experimental conditions, the sixth coordination position of the heme iron remains unbound or is occupied by another side chain (likely a Lys or a His residue) [7,20,21].…”