Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I′ band of the protein and the CH₂ and CD₂ stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Changes of the phospholipid bilayer structure under the adsorption of ferricytochrome c on its surface]

Changes in the mobility of phospholipid molecules in liposomes membranes under adsorption ferricytochrome c on its surface were studied by means of NMR and EPR spectroscopy. It is found that the interaction of cytochrome molecules with vesicles causes the broadening of 1H-NMR signals of hydrophobic as well as polar groups in cardiolipin and phosphatidylcholine in the presence of lauric or phosphatidic acid. This broadening of 1H-NMR signals in hydrophobic groups may be caused by decrease in the rate of lateral diffusion of phospholipid molecules. The changes in the correlation time of hydrophobic spin-proub in liposomes containing phosphatydiloholine and cardiolipin with the increase of ferricytochrome c concentration were also observed. These changes suggest that the formation of protein-phospholipid clusters results in the impair of the regular structure of phospholipid bilayer.     

1H-n.m.r. evaluation of the ferricytochrome c-cardiolipin interaction. Effect of superoxide radicals.

The interaction between ferricytochrome c and cardiolipin was investigated by 1H n.m.r. at 270 MHz. From the phospholipid-induced changes of the protein spectral features it is concluded that the first 2 equivalents of cardiolipin cause a conformational change at the lower part of the solvent-exposed haem edge, involving a rearrangement of the hydrogen-bond interactions of propionate 6, thus partly accounting for the lowered redox potential of cytochrome c in the presence of cardiolipin. The increased value for the pK of the alkaline isomerization of ferricytochrome c shows that cardiolipin stabilizes the native structure of the protein, indicating that the oxidized form assumes ferrocytochrome c-like properties. Peroxidation of cardiolipin by superoxide radical ions drastically decreases the protein binding to this phospholipid. The implications of this finding, and the likelihood of the ternary cytochrome c-cardiolipin-cytochrome c oxidase complex, for the binding of cytochrome c to cytochrome c oxidase in vivo, are discussed in relation to peroxidative damage following ischaemia and reperfusion.     

Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.     

Calorimetric studies of cytochrome oxidase-phospholipid interactions

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.     

Cholesterol effects on the interaction of cardiolipin with anti-cardiolipin antibody

(1) Human antibodies to cardiolipin, phosphatidic acid and phosphatidylserine were assessed by binding to nitrocellulose paper and subsequent reaction with an enzyme-linked or radioactively labelled second antibody to human IgG. (2) The addition of cholesterol to constant amounts of cardiolipin impregnated in the nitrocellulose paper resulted in a profound fall in antibody binding beginning at a 0.5 to 1 molar ratio of cholesterol to cardiolipin and stabilizing at about 15% of the original level. (3) Antibody binding to phosphatidic acid and phosphatidylserine also showed extensive cholesterol-induced inhibition beginning at a slightly lower molar ratio of cholesterol to phospholipid. (4) The structural array of neither the cardiolipin alone impregnated in nitrocellulose nor the phospholipid together with cholesterol is known. It is possible that the specific cardiolipin phase structure required for human antibody recognition was disrupted by cholesterol.     

Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids

Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.     

Apocytochrome c interaction with phospholipid membranes studied by Fourier-transform infrared spectroscopy.

Apocytochrome c, the heme-free precursor of cytochrome c, has been used extensively as a model to study molecular aspects of posttranslational translocation of proteins across membranes. In this report, we have used Fourier-transform infrared spectroscopy to gain further insight into the mechanism of apocytochrome c interaction with membrane phospholipids. Association of apocytochrome c with model membranes containing the acidic lipid dimyristoylphosphatidylglycerol (DMPG) as a single component results in a drastic perturbation of phospholipid structure, at the level of both the acyl chains and the interfacial carbonyl groups. However, in a binary mixture of DMPG with acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54), the perturbing effect of the protein on the acidic phospholipid is greatly attenuated. In such a membrane with mixed lipids, the physical properties of the DMPG and DMPC components are affected in a similar fashion, indicating that apocytochrome c does not induce any significant segregation or lateral-phase separation of acidic and zwitterionic lipids. Analysis of the apocytochrome c spectrum in the amide I region reveals that binding to phospholipids causes considerable changes in the secondary structure of the protein, the final conformation of which depends on the lipid to protein ratio. In the presence of a large excess of DMPG, apocytochrome c undergoes a transition from an essentially unordered conformation in solution to an alpha-helical structure. However, in complexes of lower lipid to protein ratios (less than or equal to approximately 40:1), infrared spectra are indicative of an extended, intermolecularly hydrogen-bonded beta-sheet structure. The latter is suggestive of an extensive aggregation of the membrane-associated protein.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.     

Phospholipids as ionophores.

The ionophoretic capabilities of phospholipids have been examined by direct measurement in a Pressman cell of the phospholipid-mediated translocation of cations across an organic phase separating two aqueous phases. Cardiolipin and phosphatidic acid were the most active inonophores among the phospholipids tested, with activities comparable to that of X537A in respect to the translocation of divalent cations. Cardiolipin translocates both divalent and monovalent cations at approximately equal rates. The ionophoretic activity of cardiolipin could be modulated by other phospholipids (inhibition), by butacaine (stimulation), by complexation with cytochrome c (inhibition), and by ruthenium red and lanthanum (inhibition). The rate of translocation of cations mediated by cardiolipin was independent of pH over a wide pH range (5.4 to 8.3). The same general pattern of properties observed for cardiolipin applied to phosphatidic acid except for stimulation by butacaine. Complexation of phospholipid mixtures, such as asolectin or mitochondrial lipid, with reduced cytochrome c, enhanced the ionophoretic capability of these phospholipids by 1 order of magnitude. The complex thus formed has the properties of a polyionophore. The possible physiological significance of this enormous ionophoretic potential of phospholipids is examined.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Δ2-4)CYP1B1 and (Δ2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Δ2-4)CYP1B1 than the (Δ2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.     

Cytochrome c specifically induces non-bilayer structures in cardiolipin-containing model membranes

(1) The effect of cytochrome c addition on the phospholipid structure of liposomes composed of cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylcholine or phosphatidylethanolamine in a pure form or in mixtures was investigated by 31P-NMR and freeze-fracture techniques. (2) Cytochrome c specifically induces the hexagonal Hii phase and possibly an inverted micellar structure of part of the phospholipids in cardiolipin-containing model membranes. (3) These results are compared with the effect of Ca2+ on cardiolipin and are discussed in relation to the structure and function of the inner mitochondrial membrane.     

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.     

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN*

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (<r.m.s. deviation>_bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (<r.m.s. deviation>_bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser-Raman investigation of lysozyme-phospholipid interactions

The interaction of lysozyme with mixed 1,2-dipalmitoyl-l-phosphatidic acid/1,2-dimyristoyl-l-phosphatidylcholine liposomes was investigated by laser Raman spectroscopy. Substantial changes were observed in the spectra of both the lipid and protein in the mixed liposomes over the range 10–62°C. At temperatures below 27°C, interaction with lipid appears to slightly increase the amount of helical structure in lysozyme at the expense of random conformation. At temperatures above 30°C, considerable β-sheet is irreversibly formed. Onset of β-formation appears to coincide with the formation of disordered lipid side-chains in the acidic component of the lipid.At all temperatures, the O-P-O diester stretching mode at 782 cm^?1 is much more intense in the lipid/protein mixture than in lipid alone. It is observed that the dimyristoyl phosphatidylcholine chain-disorder transition is lowered by 3°C, while that of the phosphatidic acid is lowered by 12°C, yet the post-transition conformation contains a significantly higher proportion of trans-segments in the presence of lysozyme.These results are interpreted in terms of: (1) a polar interaction between acidic phospholipid and lysozyme at temperatures below either chain-disorder transition, in which lysozyme is essentially excluded from the hydrophobic portion of the lipid and (2) an interaction at higher temperatures which involves the lipid side-chains of dipalmitoyl phosphatidic acid in the disordered state and is manifested by a substantial conformational change.