Coexistence of Native-Like and Non-Native Cytochrome c on Anionic Liposomes with Different Cardiolipin Content

Abstract

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.

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.…”

“…WT Cc undergoes CL-dependent conformational changes that allow H 2 O 2 to access the heme crevice (74). Hence, we addressed whether the affinity differences between WT and Y48pCMF Cc for CL could affect their peroxidase activity.…”

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

“…In particular, the rupture of the heme-Fe-M80 coordination and the tertiary rearrangement of cyt c, involving the 40's Ω loop and the M80-containing Ω loop, have been reported to occur in CL-bound cyt c [7,20,21]. The difficulties encountered in the study of the cyt c/CL binding process are correlated with several factors, such as the ionic strength of the solution, the membrane curvature, the CL/ cyt c molar ratio at which the binding reaction is followed, which significantly affect the results obtained [7,17,19,45,46]. Moreover, the finding that CL-bound cyt c is present in solution as a heterogeneous ensemble of forms characterized by different heme coordination represents a further complication [15,16,28].…”

“…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].…”