for Lipid Bilayers - American Chemical Society

Article pubs.acs.org/JPCB

Not Only Oxidation of Cardiolipin Affects the Affinity of Cytochrome c for Lipid Bilayers Cintia Kawai,†,‡ Juliana C. Ferreira,§ Mauricio S. Baptista,*,‡ and Iseli L. Nantes*,† †

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-580 Santo André-SP, Brazil Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo-SP, Brazil § Departamento Biofísica, Universidade Federal de São Paulo, 04021-001 São Paulo-SP, Brazil ‡

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, KD1 and KD2, 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 KD1 and KD2 values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the KD1 value 1.5-fold, increased the KD2 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.

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INTRODUCTION The interaction of cytochrome c (cyt c) with lipid bilayers is of great interest because cyt c participates in the respiratory chain, in the cellular redox balance,1−4 and in the induction of the intrinsic pathway of apoptosis.5−7 In the inner mitochondrial membrane (IMM), lipid oxidation may affect binding and activity of cyt c with repercussions in cell respiration and apoptosis.8−11 The detachment of cyt c from the IMM for participation in apoptosis implicates the existence of reversible lipid−protein interactions.5,6,12,13 Both electrostatic and hydrophobic interactions are important factors in the association of cyt c with negatively charged phospholipid membranes.13−18 Besides intrinsic lipid−protein interactions, whole-membrane properties also affect the binding and release of cyt c with the IMM. In this regard, theoretical calculations19−21 and experimental results21−23 have shown that proteins can affect the cooperative interactions among phospholipids, which favor the formation of lipid rearrangements.20−22 The cooperativity can be understood in terms of the reorganization of negatively charged lipids on a membrane surface in the presence of a positively charged protein, such as cyt c.20−23 Cyt c−membrane binding isotherms can be modeled using Hill plots, and this reveals the presence of two binding constants KD1 and KD2 that represent two © XXXX American Chemical Society

distinct states in the membranes; in addition, there is a cooperativity coefficient n.22 The effect of oxidative reactions on the cyt c−membrane interactions has been understood in terms of the effect that oxidation has on noncovalent interactions between individual phospholipids (mainly cardiolipin, CL) and cyt c. For example, there is strong evidence that oxidation of CL, which is present in the IMM and serves as an important docking site for cyt c in the IMM, decreases the affinity of cyt c for the IMM.8−10 This effect is considered to be the main factor contributing to the release of cyt c from mitochondria during redox imbalanceinduced apoptosis.5−8 There are also results indicating that lipid oxidation affects the intrinsic interactions between zwitterionic phospholipids and cyt c;24 however, the role of wholemembrane interactions has not been clearly investigated. It is important to note that lipid oxidation may change the organization of the lipid domains with repercussions for the affinity of specific proteins to these membranes.25−31 In general, oxidation of unsaturated phospholipids adds polar groups to their acyl chains, and this affects the bulk properties of the Received: May 7, 2014 Revised: August 25, 2014

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dx.doi.org/10.1021/jp504518g | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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membranes30−35 as well as the types of membrane domains that can be formed by self-assembling of the lipids within the membranes.26−28,31,32 Because cyt c−membrane interaction depends on lipid organization,25 which may be affected by the state of the whole phospholipid pool, we aim to understand the possible role of oxidized zwitterionic phosphatidylcholines in affecting the cyt c−membrane interactions. In order to obtain the binding isotherms, we took advantage of a classical method, i.e., the transfer of energy from vesicles containing lipid-bound pyrene molecules to cyt c,13,14,24 and we treated the experimental data using a cooperative model of binding.22 Two oxidized lipids were employed: PazePC (1-O-palmitoyl-2azelaoyl-sn-glycero-3-phosphocholine) and POPCOX (a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-containing hydroperoxides) (Scheme 1). These molecules mimic species

phase, with several physical consequences to the membrane.24,38,38 During these experiments, we observed that the tertiary structure of the cyt c molecule was affected by the presence of the oxidized lipid molecules, and we report those changes as well.

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MATERIAL AND METHODS

Chemicals. Horse heart cyt c (type III), HEPES, and sodium phosphate were acquired from Sigma Chemical Co. (St Louis, MO, USA). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), tetraoleoyl cardiolipin (TOCL), 1-O-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), and TopFluor fluorescent cardiolipin (CLtf) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 2-(10-(1Pyrene)decanoyl)phosphatidylcholine (PPDPC) was supplied by Molecular Probes (Eugene, OR, USA). Ammonium ́ hydroxide was acquired from QEEL Indústrias Quimicas S.A. (São Paulo, Brazil). Stock solutions of native cyt c were prepared with deionized water, and the concentration was calculated using molar absorptivity of ε409 = 1.06 × 105 M−1 cm−1.39 Preparation and Analysis of PPDPC-Containing Liposomes. Lipids were first dissolved in chloroform, which was evaporated under a flow of N2. The lipid film was kept under reduced pressure for at least 2 h, after which it was hydrated by addition of 10 mM HEPES buffer pH 7.4 at room temperature. Unilamellar liposomes were obtained by extrusion of hydrated lipid dispersions in an Avanti Mini-Extruder, acquired from Avanti Polar Lipids, Inc. Samples were subjected to 11 passes through two polycarbonate filters (100 nm pore size, Nucleopore, Pleasanton, CA, USA) installed in tandem. For fluorescence measurements, small unilamellar POPC/DOPE/ TOCL (49/30/20 mol%) vesicles containing PPDPC (1 mol %) were used, and minimal exposure of the lipids to light was ensured during the procedure. For preparing vesicles with oxidized lipids, 20 mol% of PazePC or POPCOX was mixed with POPC (29%), PPDPC (1%), DOPE (30%), and TOCL (20%). Liposome solutions were diluted with the buffer to the final lipid concentration of 25 μM. Steady-State Fluorescence Measurements. For the binding measurements using PPDPC, fluorescence emission spectra were recorded with an F-2500 fluorescence spectrometer (Hitachi, Tokyo, Japan) using excitation at 344 nm, emission at 398 nm, cuvettes with a 1 cm path length, and both emission and excitation bandpasses set at 5 nm. Two milliliters of sample solution was placed in a four-window quartz cuvette in a thermostatted cell holder kept at 30 °C. The production of each titration curve was repeated at least twice. Determination of the Dissociation Constants. Binding constants of the interaction of cyt c with liposomes were determined using the fluorescence quenching data (relative fluorescence intensity as a function of cyt c concentration) of the pyrene group from the PPDPC present in the membrane. Equation 1 was used to calculate the fraction of occupied binding sites (Y).22

Scheme 1. Molecular Structures of the Oxidized Lipids Used in This Studya

a

A, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC); B, 1-palmitoyl-2-(10-hydroperoxyl-8Z-octadecenoyl)-sn-glycero-3phosphocholine; and C, 1-palmitoyl-2-(9-hydroperoxyl-10Z-octadecenoyl)-sn-glycero-3-phosphocholine. Throughout this paper, a mixture of B and C is called POPCOX.

that are formed during conditions of cell redox imbalance, can trigger the release of cyt c from mitochondria and consequently initiate or amplify apoptosis. Although far from reproducing all the complexity of the IMM, bilayers of the PC/PE/CL proved to be a reliable model to reproduce the interaction of the cytochrome with the lipid fraction of the membranes. The previously demonstrated pH-dependent interaction of cyt c with PC/PE/CL liposomes 16 was reproduced for the interaction of cyt c with cyt c-depleted mitoplasts,36 the real IMM. In the present study, two oxidized phospholipids, PazePC and POPCOX (Scheme 1), were used in the composition of POPC/DOPE/TOCL (20/30/50 mol%) liposomes to mimic IMM exposed to oxidative stress.27,29 POPCOX bears a peroxide group that can react with heme iron and can increase the average area per lipid in membranes, leading to the lateral expansion and a liquid ordered−liquid disordered phase separation.27,28,31,37 PazePC bears a nonreactive carboxylic group and causes exposition of the carbon chain to the aqueous

Y=

n[LCn] n([L] + [LCn])

(1)

To obtain the dissociation constants, we fit the data with the model given as eq 2. This model is based on the Hill model, B



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mM) in water was reacted with 150 μL of 1% thiobarbituric acid (TBA, prepared in 50 mM NaOH). Also, 15 μL of 10 M NaOH and 75 μL of 20% H3PO4 were added to the sample, followed by further incubation for 20 min at 85 °C. The MDA−TBA complex was extracted with 300 μL of n-butanol, and the absorbance was measured at 532 nm. Reactive species of the TBA concentration were calculated from ε532nm = 1.56 × 105 M−1 cm−1.42 After 3 h of irradiation, lipid samples did not present any measurable amount of reactive species of TBA, indicating that the lipid oxidation did not progress to the formation of short-chain intermediates. Percentage of Lipid Hydroperoxides Produced by Photochemical Oxidation. The concentrations of phospholipid and hydroperoxide were both used to obtain the percentage of hydroperoxides present in the lipid mixture after the photooxidative reaction. Each photochemical synthesis gave a specific percentage (in comparison with the total concentration of phospholipids) of hydroperoxide, which averaged around 42%. The specific percentage value of hydroperoxide determined by this method was used to prepare the proper lipid mixture containing 20% of POPCOX. UV−Vis Absorption Spectrometry. Electronic absorption (EA) measurements of cyt c were conducted in a photodiode spectrophotometer (Termo Fisher Scientific Instruments, Inc., Waltham, MA, USA), using a quartz cuvette with a 1 cm path length and a slit set at 0.95 nm. Circular Dichroism (CD) and Magnetic Circular Dichroism (MCD) Measurements and Analysis. The CD measurements were taken on a Jasco J-850 spectropolarimeter (Easton, MD, USA) using quartz cuvettes with a 0.1 cm optical path bandwidth, a 1.0 nm slit, a scanning speed of 200 nm/min, a response 0.25 s, and 10 accumulations. The reactions were incubated for 10 min. Preparation and Analysis of Giant Unilamellar Vesicles (GUVs). GUVs of POPC/DOPE/TOCL containing 0.5 mol% of fluorescent cardiolipin (CLtf) were grown using the electroformation method.27,31 Briefly, a 40 μM concentration of a 2.5 mM solution of mixed lipid in chloroform was spread on the surfaces of two conductive glasses (coated with fluor tin oxide). These were then placed with their conductive sides facing each other and separated by a 2-mm-thick Teflon frame. This electroswelling chamber was filled with 0.2 M sucrose, 2.5 mM HEPES, pH 7.4 solution and connected to an alternating power generator at 1 V with a 10 Hz frequency for 3 h at 25 °C. The vesicle solution was removed from the chamber and diluted ∼6 times into 0.2 M glucose, 2.5 mM HEPES, pH 7.4 solution. This created a sugar asymmetry between the interior and the exterior of the vesicles. The vesicle solution was placed in an observation chamber. Due to the differences in the density and the refractive index between the sucrose and glucose solutions, the vesicles were stabilized by gravity at the bottom of the chamber and had better contrast when observed with phase-contrast microscopy. Observation of the GUVs was performed using an inverted microscope, the Zeiss Observe A1 (Carl Zeiss, Jena, Germany), equipped with a 40× objective. Images were taken with an AxioCam R3 digital camera (Carl Zeiss). For these measurements, the vesicle solution was placed in a special chamber, consisting of an 8-mm-thick Teflon frame between two glass plates, through which observation was possible. MALDI-TOF MS. For analysis of the whole cyt c, samples were mixed in a ratio of 1:5 (v/v) with a saturated solution (5 mg/mL) of sinapinic acid. Approximately 0.5 μL of the mixture

and the computations were performed by the program Origin v.8.5 (OriginLab Corp., Northampton, MA, USA). ⎛ Y ⎞ ⎟ = n log[C ] − log K log⎜ D ⎝1 − Y ⎠

(2)

Here, Y is the percentage of saturated binding sites, L is the number of lipid sites at which cyt c can bind, and C represents the cyt c concentration. Strictly speaking, n is the stoichiometry of the complexation in the limit of infinite cooperativity. However, in actual binding titrations, n represents the degree of cooperativity.22 This model allows the calculation of the two dissociation constants KD1 and KD2 and a cooperative coefficient n.22 KD1 is related to the intrinsic cyt c−lipid interactions, while KD2 is a constant that is related to the binding at sites created after the reorganization of the negatively charged lipids induced by cyt c.19−22 Preparation and Purification of Photodamaged POPC (POPCOX). Lipid-containing hydroperoxide was obtained by the reaction of POPC with singlet oxygen O2 (1Δg), which was generated by photosensitization of methylene blue (MB+).31 Twenty milligrams of POPC was solubilized in 2.0 mL of ethanol in the presence of 50 μM methylene blue. The sample was irradiated in a quartz cuvette using a halogen light bulb (300 W, 120 V, Ecolume) at a distance of 20 cm. The chamber was cooled by circulating water at 7 °C and was covered with plates of glass. After 3 h of irradiation, ethanol was evaporated under a flow of N2. The lipid film was resuspended in 1 mL of chloroform and passed through a silica-gel column (10 cm × 0.5 cm) pre-equilibrated with chloroform in order to remove MB+. The lipid was released from the silica by methanol. The fractions containing the phospholipids were collected, and the solvent was evaporated with N2 gas. This oxidized lipid mixture was then usedafter the determination of the concentrations of phosphate and hydroperoxide (see below)to prepare lipid films and to make vesicles containing known amounts of oxidized phospholipids. Singlet oxygen adds to the double bond of the lipids, producing two isomer derivatives of POPC. In these lipids, the hydroperoxide and the double bond are in different positions, i.e., 1-palmitoyl-2-(9-hydroperoxyl-10Zoctadecenoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2(10-hydroperoxyl-8Z-octadecenoyl)-sn-glycero-3-phosphocholine (Scheme 1). We call the mixture of these two isomers POPCOX. Phospholipid Dosage. Total phospholipid content is determined by phosphate analysis using the method of Bartlett.40 Concentrations were determined using a calibration curve obtained with KH2PO4 standard solutions varying from 0 to 100 nmol. Lipid Hydroperoxide Assay. Oxidative damage to the lipids was quantified by the content in the lipid of hydroperoxide and malonyldialdehyde (MDA).The lipid hydroperoxide (LOOH) measurement was accomplished by oxidation of Fe2+ in the presence of xylenol orange, as described by Gay and Gebicki,41 with some modifications. An aliquot of the oxidized lipid was mixed with 710 μL of chloroform:methanol (1:1.8 v/v) containing 4 mM butylated hydroxytoluene, 41 μL of HClO4 (2 M titrated), 30 μL of xylenol orange (12.6 mM prepared in water), and 20 μL of (NH4)2Fe(SO4)2 (18.7 mM freshly prepared in water) and incubated during 30 min at room temperature (25 °C). The LOOH concentration was calculated using ε560nm = 3 × 104 M−1 cm−1. To determine the MDA content, 150 μL of lipid (1 C



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was spotted onto a MALDI target and analyzed by MALDITOF MS. A sample obtained from tryptic digestion was dried by vacuum centrifugation and dissolved in 10 μL of water. An equal volume of the peptide solution was mixed with a saturated solution of α-cyano-4-hydroxycinnamic acid (5 mg/ mL in 50% acetonitrile/0.1% aqueous trifluoroacetic acid; 1:1, v/v), and 1−5 μL of the 8 pmol peptide solution was loaded onto a MALDI target and analyzed by MALDI-TOF MS. The analyses were performed in the linear, positive-ion mode using a mass spectrometer (Bruker Daltonics, Leipzig, Germany) with an acceleration voltage of 25 kV. The resulting spectra were analyzed by the flexAnalysis software program (Bruker Daltonics). The instrument was calibrated using an external calibration mixture containing cyt c and myoglobin as the standards (Protein Standard I from Bruker Daltonics). Isolation of Tuna Cyt c. Tuna cyt c was extracted from tuna heart according to the method described by Kawai et al.36 Briefly, 50 g of tuna heart muscle was homogenized at high speed in a Potter-Elvejhem tissue grinder for 4 min at 4 °C with a 100 mL ice-cold solution of 0.3% Al2(SO4)·18H2O, 6.2 mM benzamidine, 5.5 mM N-ethylmaleimide, 0.1 mM phenylmethanesulfonyl fluoride, and 5 mM EDTA. The pH was set to 4.5 with 2 M acetic acid or 2 M NH2OH, and the mixture was incubated for 30 min at 4 °C. The solution was spun at 8000g for 20 min at 4 °C, and the pH of the supernatant was set at 8.5. The solution was then filtered using Whatman no. 50 filter paper. The filtrate was dialyzed against deionized water in Spectrapor tubing (6000−8000 molecular weight cutoff) with two changes of 20 L of water per liter of filtrate per day. The cyt c was isolated via ion-exchange chromatography. Approximately 10 g of CM-32 (carboxymethylcellulose) resin, preequilibrated with 20 mM sodium phosphate (pH 8.0), was added to the dialyzed solution and maintained for 30 min, with occasional stirring at 4 °C. The solution was spun at 580g for 5 min at 4 °C, and the supernatant was discarded. The resin was washed with three column volume equivalents of phosphate buffer, and then the cyt c was displaced from the resin by 500 mM NaCl in the same buffer. Fractions containing cyt c were dialyzed against deionized water in Spectrapor tubing (6000− 8000 molecular weight cutoff) with two changes of 4 L of water.

Figure 1. Effect of cytochrome c on POPC/DOPE/TOCL/PPDPC liposomes, with and without oxidized phosphatidylcholine (PazePC and POPCOX). (A) Binding of cyt c on POPC/DOPE/TOCL/ PPDPC (49/30/20/1 mol%) liposomes in the absence of oxidized POPC (black circles), and the same liposomes containing 20 mol% of PazePC (gray circles) and containing 20 mol% of POPCOX (gray triangles) instead of POPC. (B) Percentage of fractional superficial saturation on POPC/DOPE/TOCL, PazePC/POPC/DOPE/TOCL, and POPCOX/POPC/DOPE/TOCL as a function of log cyt c concentration. The experiments were performed using 25 μM liposomes containing 1 mol% of PPDPC in HEPES, 10 mM 7.4, 25 °C, with excitation at 344 nm and emission at 398 nm (slits ex/em 5/5 nm). The solid lines represent the fitted curves. Inset: fluorescence (left panels) and optic (right panels) microscopy images of POPC/ DOPE/TOCL GUV containing 0.5 mol% of TopFluor CL in the absence (control) and presence of cyt c 0.25 μM (+0.25 μM cyt c) and 0.5 μM (+0.5 μM cyt c), using a Carl Zeiss FS02 filter cube.

positive cooperativity may be assigned to the ability of cyt c to promote clustering, also referred to as the demixing of negatively charged phospholipids favorable for the binding of additional cyt c molecules.44,45 Binding of cyt c to the IMM model was evidenced by images of giant unilamellar vesicles (GUVs) composed of POPC/DOPE/TOCL (49/30/20 mol %) and containing 0.5% of fluorescent cardiolipin (CLtf); see inset in Figure 1A. The GUVs were composed of monounsaturated lipids, POPC, DOPE, and TOCL, susceptible only to attack by singlet oxygen, to avoid the oxidation of the lipids caused by the applied electric field.46 The images obtained in the absence of cyt c reveal that CL is clustered in particular domains rather than homogeneously distributed in the bilayer (mixed with PC and PE). The addition of cyt c to the GUV suspension led to two main effects: quenching of the fluorescence by Förster energy transfer and changes in the lipid domains.13,14,23 The lipid domains can be observed in both the fluorescence and phase-contrast modes in the absence and presence of cyt c. The addition of 250 nM cyt c intensified the fluorescence in the lipid domains, suggesting an increase in CL aggregation. Therefore, it is more appropriate to assign cooperative binding to changes in the lipid organization than to a demixing. The formation of lipid domains in lipid bilayers composed of different phospholipids, as well as the lipid reorganization by the binding of proteins, has been calculated and characterized by several methods19−21 and experimental results.21−23 Increasing the cyt c concentration to 500 nM suppressed the CLtf fluorescence. This effect is consistent with the binding of additional cyt c on the CL domains expanded by the previously added cyt c.

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RESULTS Effect of Oxidized Phosphatidylcholines on the Lipid Organization with Repercussions on the Cyt c Binding Affinity. Cyt c binding to a model of IMM lipid fraction exposed to oxidative stress was assessed by fluorescence quenching, i.e., resonance energy transfer from the pyrene of PPDPC incorporated into POPC/DOPE/TOCL (20/30/50 mol%) liposomes to the cyt c heme group.43 PazePC and POPCOX (Scheme 1) were used as models of unreactive and reactive products of the oxidation of biological membrane, respectively. Figure 1A shows the effect of the native cyt c concentration on the fluorescence quenching of POPC/ DOPE/TOCL (49/30/20 mol%) liposomes containing 1 mol % of PPDPC, in the absence or presence of 20 mol% of PazePC and POPCOX. In all conditions, a plateau of fluorescence quenching was observed at cyt c concentrations higher than 200 nM. Analysis of the data shown in Figure 1A using eq 1 provides plots of the fraction of saturated binding sites (Y) as a function of the log of the cyt c concentration. All isotherms show a sigmoid profile (Figure 1B) that is consistent with a lipid reorganization leading to positive cooperativity.22 The D



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site A, which is then followed by the occurrence of lipid extended interaction, in which the acyl chain of a phospholipid is inserted into a hydrophobic channel of the cyt c structure. However, the lipid extended interaction does not preclude the detachment of cyt c from the acidic membranes by the increase in ionic strength. Therefore, to investigate whether the presence of oxidized lipid affected the binding mechanism, the membrane-bound hemeprotein was challenged by an increase in the ionic strength in the absence and presence of oxidized lipids. Figure 3 shows the dissociation of cyt c from the

The binding isotherms shown in Figure 1B were treated using the Hill-type cooperative model (eq 2).22 This model allows the quantification of the cooperativity coefficient (n) and the two dissociation constants, i.e., KD1 and KD2 that represent the dissociation constants at low and high concentrations of cyt c, respectively (Figure 2).

Figure 2. (A)Effect of oxidized phospholipid in the binding of cyt c to POPC/DOPE/TOCL/PPDPC liposomes. The Hill plot obtained for the binding of cyt c to liposomes POPC/DOPE/TOCL (black circles), PazePC/POPC/DOPE/TOCL (gray circles), and POPCOX/ POPC/DOPE/TOCL (gray triangles), as a function of the log of the cyt c concentration. The experiments were performed using 25 μM liposomes containing 1 mol% of PPDPC in 10 mM HEPES, pH 7.4, 25 °C. The solid line represents the fit of the data.

Figure 3. Binding and release of cytochrome c to POPC/DOPE/ TOCL/PPDPC liposomes with and without oxidized phosphatidylcholine (PazePC and POPCOX). Black circles show the binding and release of cyt c to POPC/DOPE/TOCL/PPDPC (49/30/20/1 mol %) in the absence of oxidized PC. Gray circles show the same liposomes containing 20 mol% of PazePC, and gray triangles show those containing 20 mol% of POPCOX instead of POPC. The experiments were performed using HEPES, 10 mM, pH 7.4, at 25 °C, with excitation at 344 nm and emission at 398 nm (slits ex/em 5/5 nm).

The dissociation constants obtained for the binding of cyt c with these membranes are shown in Table 1. Table 1. Dissociation Constants of the Cyt c Bound to POPC/DOPE/TOCL/PPDPC Liposomes Containing PazePC or POPCOX POPC/DOPE/TOCL PazePC/POPC/DOPE/ TOCL POPCOX/ POPC/DOPE/ TOCL

KD1 (nM)

KD2 (nM)

n

86.4 ± 8.6 105.4 ± 22.7

1.04 ± 0.13 4.5 ± 0.5

1.85 ± 0.02 1.94 ± 0.08

60.4 ± 19

10.9 ± 5.9

IMM mimic induced by the increase in the ionic strength provided by the addition of sodium chloride (panel B) or calcium chloride (panel C). Pyrene fluorescence from both POPC/DOPE/TOCL and PazePC/POPC/DOPE/TOCL liposomes quenched by cyt c, was completely restored by the increase in the ionic strength. This result is consistent with the electrostatic interaction between cyt c and the liposomes being the main binding force.12,43 However, only a partial cyt c release was observed by increasing the ionic strength (11 mM CaCl and 120 mM NaCl) in POPCOX/POPC/DOPE/TOCL vesicles (Figure 3B,C, triangle symbol). The incapacity of ions to detach cyt c from membranes is consistent with two types of hydrophobic interaction: drastic changes of cyt c structure that lead to the protein being buried inside the bilayer, or the formation of a hydrophobic anchor by the appending of a lipid-derived chain in the protein structure. The effect of PazePC and POPCOX on the cyt c structure was investigated by UV−visible EA, CD, and MCD, as depicted in Figure 4. Secondary and Tertiary Structure Changes of Cyt c Bound to Oxidized Membranes. Figure 4A shows the EA (upper panel) and the corresponding CD (lower panel) spectra of cyt c in buffered water (light gray lines), and associated with the IMM model in the absence (black lines) and in the presence (dotted lines) of PazePC and POPCOX (gray lines). In all conditions, the Soret and the Q-bands at the respective spectral regions of 400 and 500 nm were present.47−49 The

1.79 ± 0.01

For the IMM model without oxidized lipids, KD1 and KD2 were respectively 86.4 nM and 1.04 nM, and the cooperativity index (n) was 1.85 (Table 1). The KD2 value was nearly 100fold smaller than the KD1 value, and this is attributed to the CL reorganization promoted by the binding of cyt c to the interface. The presence of PazePC or POPCOX did not promote significant changes in KD1 and n. However, there was significant loss of affinity at high concentrations of protein, i.e., an increase of 4.5 and 10 times for KD2 in the presence of PazePC and POPCOX, respectively. The decrease in the affinity observed for the oxidized IMM model at large concentrations of cyt c is in accord with what has been reported in the literature.9,10 The partial release of cyt c from the IMM occurs in response to oxidative stress that leads to IMM permeabilization, ΔΨ loss, and alkalization of the intermembrane space to a pH of 7.4.36 Peculiar Effects of POPCOX on the Interaction of Cyt c with the IMM Model. The interaction of cyt c with negatively charged membranes at pH 7.4 involves electrostatic binding at E



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with the replacement of Met80 by another strong field ligand rather than with the conversion of heme iron to a pentacoordinated form.50,51 The corresponding CD spectra (lower panel of Figure 4A) corroborated the replacement of Met80 by another amino acid, as indicated by the decrease in the CD negative band at 24 330 cm−1. The association with the IMM model without oxidized lipids did not promote a decrease in the negative CD band at 24 330 cm−1, but instead, it promoted a slight red shift to 24 312 cm−1 accompanied by a decrease in the negative CD band. These spectral changes are also consistent with changes in the heme coordination sphere. The cyt c MCD spectra obtained with progressive increases in the magnetic field from 0 to 0.995 T (positive and negative modes) were similar for the different media (Figure 4B, lower panel). The MCD results reinforced the hypothesis that the association of cyt c with the IMM model in the presence or absence of oxidized lipids did not promote drastic structural changes enough for the burying of the hydrophilic hemeprotein in the bilayer. Therefore, the more probable cause of cyt c binding resistant to the increase of ionic strength when POPCOX was present is the formation of a hydrophobic anchor in the protein structure. This proposal was corroborated by the MALDI-TOF analysis of cyt c that was incubated with the IMM model in the presence of POPCOX. The MALDI-TOF MS analysis of the whole protein incubated with the IMM model in the presence of POPCOX revealed the presence of a population of cyt c with a mass addition of 496.6 mass units (12 807.306 peak, Figure 5A). This result is consistent with the addition of four dehydrated nonenal molecules (molecular weight 142) to the cyt c structure. The MALDI-TOF analysis of the corresponding tryptic fragments of cyt c that was incubated with the IMM model in the presence of POPCOX revealed an increase in mass compatible with a nonenal addition in the fragments 73−79, 74−79, 80−86, and 80−87; see Table 2. We also detected a fragment that had a mass compatible with the addition of two nonenal molecules in the fragment 73−79. Considering the detection of a cyt c population with a mass addition corresponding to four dehydrated nonenal molecules, we expected to detect the presence of one more modified tryptic fragment that had not ionized. Interestingly, cyt c was incubated with the IMM model suspension containing POPCOX at pH 7.4. Therefore, it is expected that cyt c predominantly interacted with the lipid interface via site A12,16the region where the nonenal addition was detected. Considering that we are studying the interaction and reaction of cyt c with a membrane model system, it was important to investigate the occurrence of detectable nonenal adducts in biological systems. Interestingly, the MALDI-TOF MS analysis of whole cyt c isolated from tuna heart revealed the presence of a population of cyt c with a mass addition of 124 Da mass units (12 172 Da peak, Figure 5B); this is equivalent to a native cyt c with one nonenal adduct.

Figure 4. Changes in the cytochrome c EA and CD spectra promoted by the interaction with POPC/DOPE/TOCL in the presence or absence of oxidized lipids. (A) Upper panel, EA spectra of samples containing 4.0 μM cyt c in the absence of liposome (thin gray line), in the presence of control vesicles POPC/DOPE/TOCL after 20 min (thick black line), and in the presence of oxidized lipid, PazePC/ POPC/DOPE/TOCL (dotted line) or POPC/POPCOX/POPC/ DOPE/TOCL (gray line) after 20 min. These experiments were performed using 500 μM liposome, 10 mM HEPES, pH 7.4, at 25 °C. Lower panel, CD spectra using the conditions described for the upper panel. (B) Upper panel, MCD spectra of cyt c (Fe3+) in buffer (diamonds line) or associated with POPC/DOPE/TOCL (thick black line) in the absence of oxidized lipids, and cyt c associated with liposomes in the presence of 20 mol% of oxidized lipid PazePC (light gray line) or POPCOX (dark gray line). Lower panel, effect of magnetic field intensity titration on 25 125 cm−1 (positive intensity) and 24 272 cm−1 (negative intensity) of cyt c MCD spectra associated or not associated with oxidized membranes. The experiments were performed using 1.5 mM liposome, 10 mM HEPES, pH 7.4, at 25 °C.

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changes observed in the cyt c EA spectra in the presence of the IMM model containing or not containing oxidized lipids are not suggestive of drastic structural changes in the protein. The association with different lipids promoted changes in the intensity of the cyt c spectra. Additionally, as has been previously described,3 the association of cyt c with POPC/ DOPE/TOCL liposomes promoted loss of the charge transfer (CT) band at 1439 cm−1 (695 nm), which is consistent with loss of Met80 as the sixth axial ligand for heme iron. However, considering that no significant blue shift of the Soret band was observed, the loss of the CT band at 1439 cm−1 is consistent

DISCUSSION The effects of oxidative stress on the biological membranes have been extensively studied by using membrane models with different lipid oxidation derivatives.52−54 These studies identified some effects of the products of lipid oxidation such as (i) increase of membrane permeability, (ii) diminution of the lipid order, (iii) formation of non-bilayer phase with consequent increase of flip-flop, (iv) decrease of the temperature of phase transition, (v) changes in the bilayer hydration, F



Article

Figure 5. MALDI-TOF mass analysis of modified horse heart and tuna cyt c. A. Horse heart cyt c. Horse heart cyt c was incubated with POPCOX/ POPC/DOPE/TOCL liposomes in 2 mM HEPES at pH 7.4 at room temperature for 15 min. The positive-ion MALDI-TOF spectrum of modified cyt c was acquired by a Bruker Daltonics MALDI-TOF mass spectrometer in reflectron-detector mode. B. Tuna heart cyt c. The protein was isolated from tuna heart according to the methodology described in the Materials and Methods section, and the mass spectra of the protein without additional treatments was acquired by a Bruker Daltonics MALDI-TOF mass spectrometer in reflectron-detector mode. Tuna cyt c was isolated from tuna heart mitochondria, and it was bound to the mitochondrial inner membrane before the purification.

Table 2. Identifiable Tryptic Fragments of Native and Modified Horse Heart Cyt c Detected by MALDI measd mass native cyt c

average/isotopic

calcd mass

error (m/z)

residue start

residue end

measd mass modified cyt ca

peptide sequence

1350.788 1634.474 1598.849 1168.515 1296.596 1433.671 1470.804 2081.155 779.242 801.235b 806.295 1623.791 678.196 907.368 964.333 604.358 634.485

mono mono mono mono mono mono mono mono mono mono mono mono mono mono mono mono mono

1349.719 1633.812 1597.774 1168.415 1295.710 1433.769 1470.690 2081.019 779.441 779.242 806.481 1623.786 678.323 906.536 964.528 604.338 634.39

1.069 0.662 1.075 0.1 0.886 −0.098 0.114 0.1360 −0.199 21.99 −0.186 0.005 −0.127 0.832 −0.195 0.020 0.095

89 9 39 28 28 26 40 56 80 80 73 61 74 80 92 56 9

99 22 53 38 39 38 53 72 86 86 79 73 79 87 99 60 13

1350.629 1633.637 1598.620 1168.113 1296.755 1433.692 1470.609 2081.162 779.325 923.397* 929.355* and 1052.891** 1623.804 801.262* 1029.908* 964.341 604.301 634.812

TEREDLIAYLK IFVQKCAQCHTVEK KTGQAPGFSYTDANK TGPNLHGLFGR TGPNLHGLFGRK HKTGPNLHGLFGR TGQAPGFSYTDANK GITWKEETLMEYLENPK MIFAGIK MIFAGIK + sodium KYIPGTK EETLMEYLENPKK YIPGTK MIFAGIKK EDLIAYLK GITWK IFVQK

a

One (*) and two (**) asterisks indicate mass of the peptide fragment compatible with single nonenal and double nonenal, respectively. Data were obtained as described in the Materials and Methods section. The residue number is based on the sequence of the mature protein. bAlso identified in modified cyt c and compatible with the fragment (mass 779.242) with the addition of sodium (theoretical mass 22.99).

with nonenal in the absence of a lipid bilayer has also been demonstrated.56 Therefore, we realize that cyt c interacted with the IMM model containing POPCOX and the significant changes were observed for KD2 values. On the other hand, cyt c was able to react with POPCOX hydroperoxide group. This reaction generates free radicals that propagate the lipid oxidation and produce lipid-derived aldehydes, such as nonenal, which, in turn, react with cyt c lysine residues facing the lipid interface (site A). The IMM model used in the present study contains TOCL, which was not expected to be attacked by the free radicals produced by the reaction of cyt c with POPCOX. However, it was also demonstrated that the reaction of cyt c with lipid peroxides generates singlet molecular oxygen that is able to attack TOCL.57 Therefore, the possibility that TOCL contributes to the generation of nonenal is not discarded. The reaction mechanism of cyt c with POPCOX is currently under investigation, and it is not the focus of the present study, which is concerned with the effect of oxidized lipids on the affinity of cyt c to the IMM.

(vi) decrease of bilayer width, and (vii) changes in the lateral phase organization.52−54 These effects are related to the reorientation of lipid derivatives to the water phase and consequently, the more moderate effects have been observed for hydroxy- or hydroperoxydieonylphosphatidylcholines than phosphatidylcholines with oxidized and cut chains with either aldehyde or carboxylic group. Regarding the latter group, it was reported that replacement of 3 mol% of POPC by PazePC was enough to promote significant increase of the miscibility transition pressure, changes in the surface potential profiles and surface pressure−area isotherm. Although more moderate hydrophobic mismatch and lipid order changes have been assigned to lipid-derived hydroperoxides than for the lipidderived carboxylic products, in the presence of cyt c, the reactivity of the lipid-derived product should be considered.55,56 We have previously demonstrated that cyt c reacts with lipidderived peroxides,43 and more recently, we have shown the formation of a hydrophobic anchor as a result of the reaction of cyt c with cholesterol carboxyaldehyde.57 In addition, the formation of nonenal−cyt c adducts after incubation of cyt c G



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Article

CONCLUSIONS This study of the cyt c interaction with POPC/DOPE/TOCL liposomes in the absence and presence of PC-derived oxidized products led to a number of conclusions. In the absence of oxidized lipids, a sigmoid binding profile was observed, and this allowed the calculation of two dissociation constants (KD1 and KD2). The sigmoid binding profile indicates that there are positive cooperative interactions between cyt c and the POPC/ DOPE/TOCL liposome membranes. Also, an increase in the cyt c concentration favors phospholipid rearrangement and formation of high-affinity binding sites for cyt c in the membranes (KD2). The cartoon shown in Figure 6 summarizes

membranes are likely to play a major role in the events preceding the detachment of cyt c from mitochondria during apoptosis. The presence of the lipid hydroperoxide also affects the structure of the protein by hydrophobic interaction and by chemical reaction.

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AUTHOR INFORMATION

Corresponding Authors

*(M.S.B.) Phone: 55-11-3091-8952. E-mail: [email protected]. *(I.L.N.) Phone: 55-11-4996-8384. E-mail: [email protected]. br. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Paolo Di Mascio and Sayuri Miyamoto (Institute of Chemistry, University of São Paulo) for their advice on the preparation of phospholipid hydroperoxide and the mass spectrometry analysis. The authors acknowledge funding from the following Brazilian institutions: FAPESP (Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo; Proc. 2012/074567, 2012/50680-5, 2012/12663-1), CNPq (Conselho Nacional para o Desenvolvimento Cientı ́fico e Tecnológico), CAPES (Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı ́vel Superior), UFABC/Núcleo de Bioquı ́mica e Biotecnologia (NBB) and Central Experimental Multiusuários (CEM), PRONEX/FINEP (Programa de Apoio aos Núcleos de Excelência), PRPUSP (Pro-Reitoria de Pesquisa da Universidade de São Paulo), Instituto do Milênio-Redoxoma (Proc. 420011/2005-6), INCT Redoxoma (FAPESP/CNPq/CAPES; Proc. 573530/2008-4), NAP Redoxoma (PRPUSP; Proc. 2011.1.9352.1.8), and CEPID Redoxoma (FAPESP; Proc. 2013/07937-8)].

Figure 6. Interaction of oxidized phosphatidycholine with cyt c. (a) At pH 7.4, cyt c interacts electrostatically with the negatively charged cardiolipin headgroup via site A and a lipid extended interaction occurs. (b) PazePC exposes terminal carboxylic group at the interface and can interact with site A. (c) POPCox can reacts with cyt c heme iron, triggering lipid oxidation and the formation of lipid aldehydes derivatives. (d) Nonenal, one of the expected products of lipid peroxidation, reacts with a lysine residue of cyt c site A and produces a hydrophobic anchor that impairs salt-induced detachment of the protein from the membrane. The divergent Lys73 and Lys72 of site A are represented in blue and are expected to be positively charged at pH 7.4 (red plus signal), and the convergent low-pKa lysine residues of site L are represented in light green and are expected to be deprotonated. Deprotonated site L at pH 7.4 is not expected to contribute to the electrostatic interaction of cyt c with the membrane. Cardiolipin structure is presented as a sketch for clarity.

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ABBREVIATIONS Cyt c, cytochrome c; PazePC, 1-O-hexadecyl-2-azelaoyl-snglycero-3-phosphocholine; POPCOX, hydroperoxide isomers derived from POPC; IMM, inner mitochondrial membrane; CL, cardiolipin; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; GUV, giant unilamellar vesicles; TOCL, tetraoleoyl cardiolipin; CLtf, fluorescent TopFluor cardiolipin; PPDPC, 2-(10-(1-pyrene)decanoyl)phosphatidylcholine; EA, electronic absorption; CD, circular dichroism; MCD, magnetic circular dichroism

some effects of oxidized PCs in the IMM model: (a) At pH 7.4, it is expected that cyt c binds on the membrane via electrostatic interaction of site A (blue) with CL that is accompanied by a lipid-extended interaction. (b) The exposure of the negatively charged carboxylic group of PazePC at the membrane surface might compete with CL for the binding of cyt c when the protein is in relatively high concentrations. The hydrophobic mismatch promoted by PazePC produces also membrane defects that contributed for an increase of KD2 value. (c) The presence of POPCOX promoted defects in the bilayer and led to a substantial decrease in the affinity of cyt c for the CLcontaining liposomes (10-fold increase in the KD2 value). (d) Cyt c is able to react with the POPC-OOH and the consequent lipid peroxidation produces nonenal that in turn reacts with a lysine residue of site A and forms a hydrophobic anchor in the cyt c structure. Therefore, the most important finding presented here is that oxidation of not only CL is important for explaining the release of cyt c from mitochondria, but also the oxidation of the whole lipid pool, which affects lipid−lipid and lipid−protein interactions, is also important. Cooperative properties of the

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