Not Only Oxidation of Cardiolipin Affects the Affinity of Cytochrome c for Lipid Bilayers

Abstract

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

Cardiolipin Structure and Oxidation Are Affected by Ca2+ at the Interface of Lipid Bilayers

“…Different features observed in the FTIR spectra at pH 2 and 6.5 warrant that CL was not totally protonated at pH 6.5. Even in liposomes with mixed lipids, in the absence of proteins, cardiolipin forms domains (Kawai et al, 2014b) and is reasonable to consider that singly and doubly unprotonated CL molecules may be present in these lipid bilayers. In Figure 2, the organization of CL as vesicles, at pH 6.5 is indicated by the sharp band peaking at 1,732 cm −1 with a shoulder at 1,718 cm −1 that is assigned to the C=O ester group.…”

Photodynamic Efficiency: From Molecular Photochemistry to Cell Death

“…Considering that one of the main events of apoptosis is the detachment of cytochrome c from mitochondria, Kawai et al demonstrated that lipid oxidation and reorganization within the membrane change the binding of cytochrome c to liposomes that mimic the inner mitochondrial membrane (IMM). That is, in the presence of 1-palmitoyl-2-azelaoyl- sn -glycero-3-phosphocholine (PazePC), which bears a nonreactive carboxylic group, the dissociation constant between cytochrome c and IMM was increased, while in the presence of a mixture of two hydroperoxide isomers derived from 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), which have a reactive peroxide group, this same dissociation constant was lowered [ 59 ].…”

Mitochondrial Cristae Architecture and Functions: Lessons from Minimal Model Systems

“…Relatively few studies regarding the possible compartmentalization of inner membrane mimicking only-lipid systems have been performed. In some of these, experimental strategies based on epi-, confocal and two photon excitation fluorescence microscopy techniques have been employed to study the lateral structure of membranes using giant vesicles as model systems [ 80 , 100 , 139 , 140 , 141 ].The most common approach relies in the partition properties of particular fluorescent probes into the possible coexisting lipid domains ( Figure 10 : for experiments A, B, C and E) where the fluorescence intensity images obtained will be limited to information about the lipid domain’s shape and size. In the case of Laurdan labeled membrane ( Figure 10 : experiment D), local physical properties of the possible lipid domains were used to associate their presence with equilibrium thermodynamic phases.…”

“…In the case of Laurdan labeled membrane ( Figure 10 : experiment D), local physical properties of the possible lipid domains were used to associate their presence with equilibrium thermodynamic phases. In the experiments presented in Figure 10 E,F, phase contrast imaging allows to follow the GUV morphology [ 140 , 142 ]. As it can be seen in the left panel of Figure 10 , for some IMM-mimicking lipid compositions, the fluorescent probe used is homogeneously distributed throughout the membrane, and no mesoscopic domains are observed.…”