Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by

Article pubs.acs.org/JPCB

Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes Lee Serpas, Bridget Milorey, Leah A. Pandiscia, Anthony W. Addison, and Reinhard Schweitzer-Stenner* Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States

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S Supporting Information *

ABSTRACT: Visible circular dichroism, absorption, and fluorescence spectroscopy were used to probe the binding of horse heart ferrocytochrome c to anionic cardiolipin (CL) head groups on the surface of 1,1′,2,2′-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in an aerobic environment. We found that ferrocytochrome c undergoes a conformational transition upon binding that leads to complete oxidation of the protein at intermediate and high CL concentrations. At low lipid concentrations, the protein maintains a structure that is only slightly different from its native one, whereas an ensemble of misligated predominantly hexacoordinated low-spin states become increasingly populated at high lipid concentrations. A minor fraction of conformations with either high- or quantum-mixed-spin states were detected at a CL to protein ratio of 200 (the largest one investigated). The population of the non-native state is less pronounced than that found for cytochrome c−CL interactions initiated with oxidized cytochrome c. Under anaerobic conditions, the protein maintains its reduced state but still undergoes some conformational change upon binding to CL head groups on the liposome surface. Our data suggest that CL-containing liposomes function as catalysts by reducing the activation barrier for a Fe2+ → O2 electron transfer. Adding NaCl to the existing cytochrome−liposome mixtures under aerobic conditions inhibits protein autoxidation of ferrocytochrome c and stabilizes the reduced state of the membrane-bound protein.

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INTRODUCTION Cytochrome c is a small protein that serves as a model system for studying intramitochondrial electron transfer1 as well as protein folding.2 Recent work has shown that this protein also plays an important role in the first steps of the apoptotic pathway.3 To this end, cytochrome c must be released from the inner mitochondrial membrane, where the protein is bound to cardiolipin (CL). CL is a unique phospholipid with two phosphatidic acid groups connected with a glycerol backbone. The protein can induce its dissociation from the membrane surface by acquiring peroxidase capability to carry out oxidative modification of inner-membrane lipids,4 which facilitates its release from the intermembrane space. To explore the structural basis for this peculiar functionality, a lot of effort has been invested into studying the interactions between oxidized cytochrome c and CL-containing liposomes, a model system that approximates the composition of the inner mitochondrial membrane.5−21 The results obtained thus far show that oxidized cytochrome c can undergo conformational transitions upon binding to anionic surfaces in general and to CL-containing membranes in particular, especially if the protein density on the liposome surface is low. These changes involve the replacement of the native M80 ligand by either a lysine or histidine and a concomitant change in the protein’s redox potential.18,22,23 They resemble, to some extent, the changes observed at alkaline or acidic pH’s or in the presence of denaturing agents like urea or guanidinium chloride.24−29 The binding process involves multiple binding sites on the protein associated with rather different binding affinities.6,8,12,13,16,20,21 © 2016 American Chemical Society

The conformational distributions of ferricytochrome c on membrane surfaces are heterogeneous, with individual proteins differing in terms of their active-site ligation and their respective degrees of unfolding.14,15,21,30 Contrary to the rather extensive work on ferricytochrome c− liposome interactions, investigations on analogous interactions between the reduced protein and CL-containing membranes are rather limited. Iwase et al. utilized optical absorption spectroscopy to observe that bovine ferrocytochrome c became oxidized upon binding with micelles composed of 100% CL.31 An explanation for the protein’s oxidation was not provided. Nantes et al. used magnetic circular dichroism (MCD) spectra of the Soret band region to probe changes in the heme environment.32 Their data also seem to indicate that ferrocytochrome c becomes oxidized upon binding to CL-containing liposomes. However, the authors dismissed this possibility on the basis of the observation that in the absence of any reductant the addition of NaCl led to a recovery of the MCD signal diagnostic of a ferrous protein. They instead invoked the generation of a protein conformation with a ferrous high-spin state, in which the Fe− M80 linkage is broken. This assignment is in line with the observation of Droghetti et al. that ferrous cytochrome c can switch into a pentacoordinate high-spin (pcHS) state upon binding sodium dodecyl sulfate micelles and phospholipid Received: June 4, 2016 Revised: October 5, 2016 Published: November 9, 2016 12219

DOI: 10.1021/acs.jpcb.6b05620 J. Phys. Chem. B 2016, 120, 12219−12231

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The Journal of Physical Chemistry B

vacuum desiccator overnight. The lipid film was rehydrated with 25 mM HEPES buffer (pH 7.4) to obtain the desired liposome concentration. The mixture was sonicated in an ice bath at 100 W for 1.5 h and then centrifuged for 50 min at 12 000 rpm to precipitate impurities. The supernatant was decanted and left to stabilize in the dark overnight. All liposome solutions were stored under nitrogen to prevent any oxidation. Preparation of Cytochrome c−Liposome Binding Experiments. The liposomes were first diluted to the desired concentration using 25 mM HEPES buffer (pH 7.4). A cytochrome c concentration of 5 μM was used in all experiments, with the exception of those probing the charge-transfer region, for which a concentration of 50 μM was necessary. Each liposome−protein mixture was made individually for the desired lipid/protein ratios. Experiments involving NaCl were prepared by adding aliquots of NaCl solution to the liposome−protein mixtures. Electronic Circular Dichroism (CD) Spectroscopy. Spectra were recorded using a Jasco J810 spectropolarimeter, which was purged with nitrogen gas. Cytochrome c/liposome solutions were monitored using a 0.2 cm path length quartz cell (International Crystal Laboratories, Gardfield, NJ). Near-UV− visible spectra were recorded from 350−800 nm, with a scanning speed of 500 nm/min, a data pitch of 0.5 nm, a bandwidth of 5 nm, and a response time of 1 s. Five spectra were recorded per sample at 20 °C, using a Peltier solid-state heating and cooling module to control the temperature. The relatively fast scanning speed produces smoothed spectra with some amplitudes slightly less pronounced than those obtained with a lower scanning speed. By choosing these parameters for recording the CD spectra, we sacrificed spectral resolution to a limited extent for the sake of improved signal-to-noise. For the data analysis a good signal-to-noise is more important than recording the exact spectrum with high resolution. All spectra were backgroundcorrected using the Jasco spectral analysis program. Absorption Spectroscopy. Absorption measurements were performed at room temperature using 2 mm path length quartz cuvettes with a Perkin Elmer Lambda 35 UV/Vis spectrometer. Spectra were recorded between 600 and 750 nm or 470 and 750 nm, with an excitation slit width of 2.5 nm. Both the protein and liposome concentrations were increased by an order of magnitude for these experiments (50 μM for cytochrome and up to 2000 μM for CL). Fluorescence Spectroscopy. Fluorescence measurements were performed at room temperature using a 1 cm path length ICL quartz cell on a Perkin Elmer LS55 Luminescence Spectrometer. Unpolarized emission spectra were recorded between 300 and 500 nm, with a 293 nm excitation wavelength and a scanning speed of 200 nm/min. Excitation and emission slit widths of 5 and 2.5 nm, respectively, were used. Polarized fluorescence measurements were obtained at 340 nm, with 293 nm excitation and an integration time of 10 s. Excitation and emission slit widths were as above. All spectra were baselinecorrected using the program MULTIFIT.39 Light-Scattering Experiments. Particle radii measurements were obtained at room temperature in a 10 mm path length quartz cuvette using a Horiba Lb-500 Dynamic LightScattering Particle Size Analyzer (Edison, NJ). Oxygen-Free Experimentation. Small volumes (1−5 mL) of each reagent used were purged with nitrogen for 20 min. The cytochrome c−liposome solutions were made under a nitrogen atmosphere and transferred to a 0.2 cm path length ICL quartz cell, which was sealed and used for all spectroscopic experiments.

vesicles.33 Kalanxhi and Wallace investigated the binding of a variety of cytochrome c derivatives to liposomes formed with a mixture of L-α-phosphatidylcholine (PC), L-α-phosphatidylethanolamine (PE), and 1,3bis(sn-3′-phospatidyl)-sn-glycerol (CL) in a 5:4:3 ratio.34 For horse heart cytochrome c, their binding data indicate a binding affinity of ca. 105 M−1, similar to that earlier reported for ferricytochrome c binding to liposomes with a comparable fraction of CL.20 This observed binding process was at least partially inhibited in the presence of NaCl. The authors proposed a model that assumes that electrostatic binding is followed by the insertion of a CL alkyl chain into the hydrophobic channel of the protein to produce the high-spin state reported by Nantes et al.32 A very different set of experiments has been carried out by Vos and co-workers.35 They first allowed mostly oxidized cytochrome c to bind to liposomes formed with ca. 30% CL/70% PC and thereafter reduced it to ferrocytochrome c. Subsequently, they allowed the protein to react with CO, which yielded a hexacoordinated low-spin (hcLS) protein with CO as the distal ligand of the heme iron. In this experiment, reduction most likely produced a pcHS state that easily binds CO as a ligand. In the current study, we investigate whether the binding of ferrocytochrome c to CL-containing liposomes induces changes in the protein structure and concomitantly changes in its oxidation state. The average CL content of these liposomes resembles that of the inner mitochondrial membrane in its resting state.36 Thus, we address the contradiction between the binding scenarios proposed by Iwase et al.31 and Nantes et al.32 Following the strategy of earlier binding studies, we examine the binding of ferrocytochrome c to 20% 1,1′,2,2′-tetraoleoyl cardiolipin/80% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) over a broad range of CL concentrations to probe the different degrees of membrane occupation. For cytochrome c to carry out its primary role as an electron transporter, a tightly regulated equilibrium between its two redox states must exist in both the intermembrane space as well as on the inner-membrane surface of the mitochondria.37 Understanding how reduced cytochrome c interacts with CL-containing liposomes may provide insights into how this equilibrium is controlled in the mitochondria. Results obtained for the oxidized protein do not necessarily apply to the reduced state, as the conformation of the latter is known to be much more stable in solution than that of the former.38 We probed the binding of ferrocytochrome c to the above liposomes under aerobic and anaerobic conditions to assess the stability of the protein’s redox state.

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MATERIALS AND METHODS Preparation of Ferrocytochrome c Solutions. Equine cytochrome c (Sigma-Aldrich Co., St Louis, MO) with no further purification was dissolved in 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer (pH 7.4). Sodium dithionite was added to the protein solution to reduce any ferric cytochrome c present in the sample. The protein solution was then adjusted to pH 7.0 and passed through a Sephadex G10 column (GE Healthcare) to remove any remaining reducing agents (or adventitious peroxide) and impurities. The sample was then re-adjusted back to pH 7.0. Preparation of Liposomes. TOCL and DOPC (Avanti Polar Lipids, Birmingham, AL) were dissolved in a 2:1 chloroform/methanol mixture. The protocol described by Hanske et al. for liposome preparation was adopted here.15 The solvent was removed by rotary evaporation at room temperature, and the remaining lipid film was allowed to sit in a 12220


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The Journal of Physical Chemistry B Oxygen was introduced into the solutions by uncapping the sample cuvette.

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RESULTS Binding under Aerobic Conditions in the Absence of NaCl. We measured the Soret band CD and absorption spectra of 5 μM ferrocytochrome c at neutral pH (7.1) in the presence of different amounts of a 20% TOCL/80% DOPC lipid mixture. Earlier investigations have shown that the TOCL head groups are completely ionized under these experimental conditions.40 Light-scattering experiments showed that the lipids formed small unilamellar vesicles, with an average radius of 20 nm, for all CL concentrations above 35 μM, in agreement with earlier studies.19 Fusion into larger vesicles was observed at lower lipid concentrations.20 This is likely caused by the high protein density on the liposome surface.41−43 Figures 1 and S1 show the CD and absorption spectra of the Soret band region of the protein, measured with the indicated CL concentrations. The latter were calculated on the basis of the somewhat simplifying assumption that approximately half of the CLs can be found in the outer lipid layer of the formed liposome. For this reason, the respective CL concentrations might be slightly higher than those calculated.44 The red profile in Figure 1 represents the protein in the absence of liposomes. This rather complex CD signal is similar to the Soret band CD spectra of horse heart ferrocytochrome c observed earlier, but it does not display the small, sharp couplet generally observed at 417 nm.45 As this couplet is close to the positive dichroism region of the corresponding ferricytochrome spectrum, the observed spectrum could indicate a mixture of reduced and oxidized proteins. This notion is confirmed by the corresponding Q-band spectrum shown in Figure S2. If one corrects for the baseline, the Q0/Qv ratio is approximately 1.57, which is somewhat lower than that observed in the spectrum of ferrocytochrome c in the presence of sodium dithionite (1.97; shown in Figure S2 for comparison).45 To assess whether the ferro/ferricytochrome mixture changes over time on the time scale of our experiments, we measured the spectrum of a liposome-free 50 μM cytochrome c solution between 470 and 650 nm as a function of time over 2.5 h right after the protein had run over a Sephadex G10 column. As shown in Figure S2, the Qband spectrum did not change significantly over this time period. We used the molar absorptivities of ferrocytochrome c and ferricytochrome c at the Q0 and Qv positions of the reduced protein and determined that ca. 50% of the proteins in the sample are in the oxidized state. This result was reproduced by a measurement with 20 μM cytochrome c, thus demonstrating that it does not depend on protein concentration. Thus, the Q-band analysis suggests that the protocol adopted to create sodium dithionite-free samples of reduced cytochrome c actually produced a nearly equimolar mixture of reduced and oxidized proteins. Even though this would potentially complicate the interpretation of the planned binding experiments with CLcontaining liposomes, we refrained from adding sodium dithionite to completely reduce the sample because that could have provided an unwanted nonbiological stabilization of the reduced protein. The spectra depicted in Figures 1 and S1 seem to indicate a three-phase change in the Soret band CD spectrum of cytochrome c upon exposure to CL-containing liposomes. To facilitate comparison of the CD spectra some of them were shifted along the ordinate so that all spectra coalesce at 360 nm, thus compensating for the occurrence of baseline variations in the spectra due to light scattering by the liposomes in the sample.

Figure 1. Soret band CD and absorption spectra (upper panel) and tryptophan (W59) fluorescence (lower panel) as measured for selected CL concentrations of 20% TOCL/80% DOPC mixtures (red: ferrocytochrome c in solution, orange: [CL] = 37.5 μM, yellow: [CL] = 65 μM, green: [CL] = 150 μM, cyan: [CL] = 200 μM). Each solution was prepared in an aerobic environment, with a final ferrocytochrome concentration of 5 μM. The CL concentrations are numerically identical to the lipid/protein ratios.

Upon addition of liposomes, the CD signal changes significantly. With an increasing CL concentration, it transitions into a pronounced couplet, which is generally diagnostic of a native, oxidized state of cytochrome c.46 The CD spectra obtained for CL concentrations between 15 and 75 μM are nearly isodichroic at 417 nm, which indicates dominant populations of only two states of the protein in this concentration range. As the spectrum of the mixture of reduced and oxidized proteins in solution does not share this isodichroic point, the two states must be assigned to different conformations of liposome-bound proteins. Upon increasing the CL concentration above 75 μM, a second spectral change from a couplet to a positive Cotton effect band is observed, reflecting the protein’s transition from a native-like 12221


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The Journal of Physical Chemistry B oxidized state to a misligated, partially unfolded oxidized state.47 Concomitantly, the absorption spectrum exhibits a blueshift from the 413.5 nm position observed for the protein mixture in solution to 409 nm for the native oxidized state and a subsequent blueshift to 408.5 nm, which suggests that the non-native conformation still exhibits an hcLS state.48 However, a significant broadening toward higher energies with increasing lipid concentrations is also clearly visible, which might reflect some population of hexacoordinated high-spin (hcHS), pcHS,33,48 or quantum-mixed-spin states (QMS) (vide infra).49 Altogether, these spectroscopic data suggest that most of the proteins that were reduced in solution were oxidized upon binding to the liposome surface. This notion is further corroborated by the corresponding Q-band absorption spectra in Figure S3, which visually depicts the transition from the reduced to the oxidized state with increasing CL concentration. The W59 fluorescence of cytochrome c was used to determine whether or not the protein was unfolding as a result of its interaction with liposomes. The spectra in Figures 1 and S1 show the high-intensity, F, and low-intensity, P, bands identified by Pandiscia and Schweitzer-Stenner.20 The former results from tryptophan fluorescence. The origin of the phosphorescence-like P-band has not yet been identified. The gap between the two bands could be due to the inner-filter effect produced by Soret band absorption. Interestingly, the F-band intensity, which is generally very weak in the folded protein owing to a resonance energy transfer to the nearby heme group, increases even in the concentration regime for which we observed solely the transition between two redox states of the folded protein. Above a CL concentration of 100 μM, the fluorescence intensity increases concurrently with the conversion of the CD couplet into a positive Cotton band, suggesting a greater distance between the heme iron and W59 and thus a partial unfolding of the protein. To further characterize the state of the membrane-bound proteins, we measured the optical absorption between 600 and 750 nm for cytochrome−lipid mixtures with CL concentrations of 500, 1000, and 1500 μM. The corresponding protein concentration was increased to 50 μM so that the above lipid concentrations correspond to the total lipid/protein ratios of 50, 100, and 150 used for obtaining the spectra in Figures 1 and S1. We confined our measurements to these three lipid concentrations to minimize the material cost of the experiment. For the sake of comparison, we also measured the spectra of the prepared reduced protein sample and a fully oxidized protein sample in an aqueous, lipid-free solution at neutral pH. For the native oxidized protein, this spectral region exhibits a weak charge-transfer band at 695 nm, which is diagnostic of Fe−M80 ligation. As the exact wavelength position of this band depends on the solution conditions,50 we adopt the terminology of Soffer et al.,51 terming it charge-transfer band 1 (CT1) in the following. To subject the experimentally obtained spectra to a spectral analysis, we plotted them on a wavenumber scale. Next, the spectra were analyzed with our spectral decomposition program, MULTIFIT, using the same wavenumber and Gaussian bandwidths to fit the spectra observed with different TOCL concentrations. The baseline, which reflects the low-energy side of the Q0-band absorption, was fit to a superposition of a quadratic and hyperbolic function. The use of the same set of bandwidths for all fits enables a consistent treatment of the baseline. Figure 2 shows the baseline-corrected spectra and the results of our spectral analysis. We normalized the integrated intensity of the observed CT1 bands of the protein−liposome mixtures with regard to the corresponding integrated CT1 intensity of the fully oxidized native protein in

Figure 2. Optical absorption in the region between 600 and 750 nm of ferrocytochrome c in solution, ferricytochrome c in solution, and the indicated mixtures of ferrocytochrome c and 20% TOCL/80% DOPC; 50 μM ferrocytochrome c, solid brown; 50 μM ferricytochrome c, solid blue; [CL] = 50 μM, solid green; [CL] = 100 μM, solid black; [CL] = 150 μM, dashed red. The protein concentration was 50 μM. All spectra were baseline-corrected, as described in the text. The inset shows the peak intensity of the 695 nm (CT1) band as a function of (total) CL concentration.

solution. The result is plotted in the inset. To our surprise, the CT1 band of the protein in solution exhibits only 25% of the intensity of the fully oxidized protein. This seems to suggest that a much lower fraction of oxidized proteins exist in the initial mixture than the value obtained from our Q-band analysis. However, one has to take into account that the CT1 band might not always be the best tool for estimating the total fraction of native oxidized proteins. As shown in earlier studies, its integrated intensity depends significantly on solution parameters like anion concentration, temperature, and pH.50,52 At the highest CL concentration employed, the CT1 intensity reaches ca. 60% of the intensity displayed in the spectrum of the native protein. Therefore, our data provide additional and conclusive evidence for the notion that the redox state of the protein changes upon binding of the reduced protein and for the occurrence of a substantial fraction of a native-like liposomebound ferricytochrome c at all lipid concentrations investigated. It should be mentioned that the absorption spectrum of the sample with a lipid/protein ratio of 150 displays a weak band at 625 nm (16 077 cm−1). Soffer et al. termed this band chargetransfer band 2 (CT2).53 A band at this position is generally assigned to an hcHS complex of the heme iron (Fe3+), which results from a heme → iron charge-transfer transition.41,49,54,55 It could also be indicative of a ferric QM state,56 which has been identified as the resting state of horseradish peroxidase57 and other class III peroxidases.58 Pandiscia and Schweitzer-Stenner recently discovered the CT2 band in the absorption spectra of ferricytochrome c in the presence of high liposome concentrations with a high CL content (50 and 100%).21 The observed intensity of the CT2 band is significantly less than that of the corresponding CT1 band (cf. dashed red spectrum in Figure 3). Keeping in mind that the intrinsic intensity (oscillator strength) of the CT2 band is generally much higher than that of the CT1 band,59 the population of the corresponding hcHS state must be low under our experimental conditions. Spectroscopic Response Data Representing Binding under Aerobic Conditions. To visualize the changes probed 12222


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Figure 3. Upper panel, left: ΔΔε = Δε417 − Δε405 derived from Figure 1 plotted as a function of the CL concentration of cytochrome c−20% TOCL/ 80% DOPC liposome mixtures. Upper panel, right: integrated intensity of the main sub-band at 340 nm derived from the W59 fluorescence band decomposition plotted as a function of CL concentration. Lower panel: polarized fluorescence, IVV (right) and IVH (left), measured at 340 nm plotted as function of CL concentration. The solid lines result from a global fit described in the text. The error bars for the ΔΔε values are within the size of the used symbols.

between 0 and 75 μM, which reflects the conversion of unbound ferrocytochrome c to bound ferricytochrome c. The integrated fluorescence intensity measured with high CL concentrations (200 μM) is less (700) than that observed for ferricytochrome c at the same lipid concentration (ca. 1800), which might indicate a lower population of partially unfolded proteins. To perform a global analysis of the data in Figure 3, we utilized a modified version of the binding model that Pandiscia and Schweitzer-Stenner recently applied to dose−response data obtained for oxidized cytochrome c.21 For the present case, we have to consider the coexistence of ferrocytochrome c and ferricytochrome c. Hence, the reaction scheme used for our analysis is written as

by the CD and fluorescence spectra, Figure 3 shows a plot of ΔΔε = Δε417 − Δε405 and the W59 fluorescence intensity response as a function of the CL concentration. We chose to plot the difference between two dichroism values measured at two different wavelengths rather than the dichroism value measured at a single wavelength to compensate for the aforementioned baseline variations. In the Supporting Information, we show that in the presence of multiple conformers this observable can still be described as a linear combination of ΔΔε values even in the presence of the slight spectral smoothing caused by our relatively high scanning speed. Hence, the latter does not impede our data analysis. The fluorescence intensity response data were obtained by using the program MULTIFIT to decompose the fluorescence (F) spectra into three Gaussian sub-bands (at 340, 350, and 365 nm), for which the wavelength positions were kept constant (not shown).20 The integrated intensity of the most intense sub-band at 340 nm was then used to construct the spectral response graphs depicted in Figure 3. Details of this procedure are described in our earlier papers.20 In addition, Figure 3 displays the polarized fluorescence response detected at 340 nm. The spectral response data clearly reflect the observation documented in Figures 1 and 2, indicating at least two phases of cytochrome c−liposome interactions. With regard to ΔΔε, the most pronounced changes occur at low CL concentrations,

The superscript 2+/3+ indicates that we made no a priori assumption about the oxidation state that the protein adopts upon binding. The subscript p indicates the complex [cyt2+−CL] as a precursor of what we expect to be the dominant conformations, nf′ and f′, emerging from the binding of the reduced protein. We label these conformations with a prime 12223


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corresponding parameters describing the binding of reduced and oxidized proteins are identical. Only in a second step were these constraints selectively relaxed. For the final fit, all corresponding sf values of the conformations emerging from the oxidized and reduced proteins were still identical. For the fit to the fluorescence data, all snf′ values were set to zero, as done earlier for the corresponding snf values. A final global fitting of the data solely required different values for the intrinsic equilibrium constants K0λ, Δεnf, and Δεnf′ (snf for the CD response) as well as for the corresponding equilibrium constants related to the nf ⇔ f equilibrium. The thermodynamic parameters obtained are listed in Table 1, together with their statistical errors, which we

symbol to indicate that they might be slightly different from the corresponding conformations, nf and f, that result from the binding of the oxidized protein. The Q-band spectra in Figure S2 suggest that p is still a reduced state. All states of bound proteins in the above reaction scheme should be considered as representing ensembles of slightly different conformational substates,15,21,60 which are difficult to distinguish by spectroscopic means. An equilibrium between p and nf′ is indicated by the isodichroic point described in Figure 1. It reflects multiple lines of evidence for the coexistence of compact and extended conformations of oxidized cytochrome c on the surface of CLcontaining liposomes.30 The spectral response isotherm for R1 can be written as

Table 1. List of Thermodynamic Parameter Values Used to Fit Equation 1 to the Dose−Response Data in Figure 3a

S([CL]) = (s0 + {(sp + snf ′K C1 + sf ′K C1K C2)K red(1 − χox ) + (snf + sf K C2′)χox K ox }[CL])

K0red [M−1] KC1,lowb KC1,high KC2,lowc KC2,high kmod,1 kmod,2 n1 n2

/(1 + {(1 + K C1 + K C1K C2)K red(1 − χox ) + (1 + K C2′)χox K ox }[CL])

(1)

sj (j = p, nf′, f′, nf, f) are the spectral amplitudes associated with the considered states. [CL] is the concentration of unoccupied CLs, which can be calculated by combining the mass action law and the law of mass conservation, as described in our earlier paper.21 χox is the mole fraction of oxidized proteins in the absence of lipids, estimated from the Q0-band intensities in Figure S2 (i.e., 0.5, vide supra). Equation 1 treats each CL headgroup as an individual binding site. This of course is an oversimplification, as the binding of the protein can trigger a phase separation by lipid demixing, which involves the formation of CL-rich domains.43,61 The binding to such domains thus involves multiple CL head groups. However, an explicit consideration of such effects would just lead to a re-scaling of [CL] and thus of the equilibrium constants Kox and Kred, without any general change in the Langmuir-type isotherm described by eq 1. The equilibrium constants KC1, KC2, and KC2′ are assumed to increase with an increase in CL concentration, as detailed in the Supporting Information and in an earlier publication.21 Their concentration dependence can be described by sigmoidal Hilltype functions, which asymptopically approach the parameters KCj,low (KCj,low′) and KCj,high (KCj,high′) (j = 1, 2) at very low and very high CL concentrations, respectively. The parameters kmod,1 and kmod,2 determine the inflection point of the Hill functions. The binding constants Kox and Kred used in the fit can be written as K λ = K 0λ·fvdW ([CL])

50 ± 5 0 2.7 ± 0.7 0 0.5 ± 0.2 0.07 ± 0.01 0.032 ± 0.005 5.0 ± 1.0 5.0 ± 0.9

a

Some of the parameters are defined in the Supporting Information. Set to 0 and never modified. cValue was so small that the difference from zero was not statistically significant. b

obtained from a very conservative estimation procedure described in ref 21. Spectroscopic parameters are listed in Table S1. For our global fit, we calculated a reduced χ2 of 2.0. In view of the many constraints imposed on the fits, we consider the final result, with the obtained reduced χ2-value as satisfactory. Figure S4 depicts the mole fractions of the individual conformations as a function of lipid concentration. The samples’ “contamination” by conformations associated with the binding of ferricytochrome c (nf and f) is rather limited. The reason can be inferred from the concentration dependence of the effective binding constant (Keffred = Kred · KC1′ · KC2′ and Keffox = Kox · KC2), as shown in Figure S5. Owing to the rather large KC1,high constant (Table 1), Keff′red becomes substantially larger than Keff′ox at high lipid concentration, which stabilizes nf′ and f′ relative to nf and f. The curves in Figure S4 reveal that occupation of the f′ state is still modest at high lipid concentration, at which it reaches a mole fraction of 0.2. The presence of a significant fraction of nf and nf′ at 200 μM (ca. 0.58) is quantitatively consistent with the rather significant intensity of the CT1 band that we observed with the corresponding lipid/protein ratio at 2000 μM (cf. Figure 2). We also determined the fluorescence anisotropy for the 340 nm fluorescence emission as a function of the CL concentration (Figure S6). The anisotropy, rs, exhibits a broad maximum around a CL concentration of 50 μM, with an anisotropy value of ca. 0.27. At high lipid concentration, rs approaches a value of 0.15. Interestingly, the maximum coincides with the maximum of the population of conformer p, providing additional evidence for its existence. The values observed for intermediate and high lipid concentrations very closely resemble those observed for the ferricytochrome c−TOCL/DOPC mixtures at all lipid concentrations.20 Binding under Anaerobic Conditions in the Absence of NaCl. The data presented thus far suggest that reduced cytochrome c is oxidized upon binding to the liposome surface.

(2)

that is, the product of an intrinsic binding constant, K0λ, and a CL concentration-dependent van der Waals gas type correction term, f vdW([CL]), as derived by Heimburg and Marsh.61 The subscript λ = ox,red indicates the oxidation state of the binding protein. The van der Waals term becomes important at low lipid concentrations, when the occupation of the membrane surface by cytochrome c is high. A detailed account of this binding model and the underlying assumptions is given in ref 21. The solid lines in Figure 3 result from the fit of our model. The agreement with the experimental data is very satisfactory. All spectral response data were fitted using the same equilibrium constants. The following constraints were imposed on the fitting procedure. First, we used the parameter values obtained in earlier work for the binding of ferricytochrome c (i.e., Kox, KC2,low′, KC2,high′, kmod,ox, snf, and sf).21 We further assumed that 12224


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from using the Q-band intensities for determining the fraction of oxidized and reduced proteins because we are not certain that the Q-band spectra of reduced proteins in solution and on liposomes are identical. Taken together, the results of our experiments under anaerobic conditions show that O2 functions as an oxidant of reduced liposome-bound cytochrome c under aerobic conditions. To construct a spectral response curve, we calculated ΔΔε′ = Δε430 − Δε405 and plotted this as a function of the CL concentration (Figure 5). Our data indicate a linear decrease with

At high lipid concentrations, the oxidized protein undergoes a structural change and concomitant replacement of the M80 ligand, as observed earlier.21 An electron transfer from peroxidized lipids can be ruled out because, contrary to bovine CL or 1,1′,2,2′-tetralinoleoyl cardiolipin, TOCL is not oxidizable.22,62 To check whether or not O2 serves as an oxidant for liposome-bound cytochrome c, we measured the Soret CD and absorption band spectra of the protein under anaerobic conditions (N2 atmosphere) as a function of CL concentration. The results are depicted in Figures 4 and S7. The data reveal that

Figure 5. ΔΔε′ = Δε430 − Δε405 derived from Figure 4 plotted as a function of the CL concentration of cytochrome c−20% TOCL/80% DOPC liposome mixtures. The solid line results from a fit described in the text.

increasing [CL]. Compared with the dichroism response data in Figure 3, the changes are less pronounced and the midpoint lies at higher CL concentrations, indicating a lower affinity of the binding process. We fit the data to a model that resembles R1 but in which the nf′ and f′ states are now reduced and nf as well as f are omitted. The solid line is the result of this fit. The respective fitting parameters are listed in Table S2. As this analysis is based only on a single set of spectral response data, the uncertainties, particularly of K0 and KC2,high, are rather large. What one can say with certainty is that both kmod values are lower for the anaerobic case compared to those for the aerobic case. As a consequence, the overall binding affinity for anaerobic binding is lower than it is for aerobic binding. Whether or not the respective intrinsic binding constants, K0, are different cannot be determined on the basis of the existing data. When we opened our sealed cuvette to allow for the influx of O2, a conversion to the oxidized form occurred on a time scale of minutes, revealing that O2 is the oxidant of ferrocytochrome c at low as well as at high lipid concentrations. Figure S8 shows the Soret CD signal of ferrocytochrome c−liposome mixtures with CL concentrations of 35 and 100 μM measured at different times after the samples’ exposure to O2. The data reveal the conversion of the modified reduced state into a native-like, oxidized state at lower and a partially unfolded, non-native oxidized state at higher CL concentrations, as expected. These data provide independent confirmation of the notion that the reduced state cannot be maintained for liposome-bound cytochrome c under aerobic conditions. One might suspect that the observed rapid oxidation of membrane-bound cytochrome c is caused by the accumulation of

Figure 4. Selected Soret band CD and absorption spectra of native ferrocytochrome c (green) and selected mixtures of ferrocytochrome c and 20% TOCL/80% DOPC liposomes measured under anaerobic conditions (red: ferrocytochrome c in solution, orange: [CL] = 37.5 μM, yellow: 65 μM, green: 150 μM, cyan: 200 μM). The inset in the lower panel shows the corresponding Q-band spectra, all of which are indicative of a reduced, native-like state of the protein.

the typical (+)(−)(+) shape of a ferrocytochrome CD signal converts into a very broad positive Cotton effect signal with increasing CL concentration. This signal is reminiscent neither of the couplet nor of the Cotton band that we observed under aerobic conditions. The peak intensity of the corresponding absorption spectrum decreases slightly. The corresponding shift in the peak position is rather modest (from 412.5 to 410 nm) and the resulting band measured at [CL] = 200 μM lies well below the peak wavelength of native ferricytochrome c in solution.21 The data thus suggest that the protein undergoes structural changes upon binding, while staying predominantly in the reduced state. That notion is unambiguously confirmed by the optical spectrum in the Q-band region, shown in the inset of Figure 4. It clearly displays the Q0 − Qv band separation with a Q0/Qv ratio of ca. 1.75, which is diagnostic of a (mostly) hexacoordinated reduced state of the heme iron. Here, we refrain 12225


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ferrocytochrome c to PC/PE/CL membranes.32 Figure 6 shows the CD spectra of ferrocytochrome c−liposome mixtures

O2 in the hydrophobic interior of the liposome’s lipid membrane.63,64 To test whether the oxidation of ferrocytochrome c could actually be caused by a high concentration of oxygen molecules at the liposome surface, a homogenous mixture of aqueous ferrocytochrome c was exposed to a nearly pure O2 atmosphere. Figure S9 shows the absorption spectrum of the ferrocytochrome c sample before and after oxygen gas exposure. It does not exhibit any significant differences that would indicate a change in the protein’s oxidation state. It is therefore unlikely that the rapid oxidation of cytochrome c on the liposome surface is caused by a higher local concentration of O2. As pointed out earlier,21 the characteristics of the cytochrome c binding explored by our experimental protocol are clearly distinct from the A-site binding that Kinnunen and co-workers deduced from binding studies, where they added different amounts of oxidized cytochrome c to a batch of CL-containing liposomes.6,8 However, one might wonder whether C- and L-site binding might play a role in our experiments. Generally, the neutral pH value chosen for our studies is not favorable for these interactions, which were reported to be dominant at a more acidic pH.6,12,65 However, our pH value of 7.1 is rather close to the effective pKa value that Kawai et al. reported for the protonation process that initiates L-site binding.12 Therefore, we measured the absorption spectra of four representative lipid−protein mixtures, that is, 25, 50, 100, and 150 at pH’s 7.4 and 6.5. The former value lies clearly outside of the L-site binding region,12 and it is actually identical to the pH value that Nantes et al. used for their study.32 At the latter value, L-site binding (and possibly also some C-site binding) should be dominant.8,65 The observed spectra of the pH 7.4 sample in Figure S10 are very similar to those observed at pH 7.0 (Figures 1 and S3), with both the B- and Q-band indicating liposome-induced oxidation with an increasing lipid/protein ratio. The Q-band spectra seem to indicate that the conversion to the oxidized protein is actually completed at slightly lower CL concentrations. The corresponding spectra of the pH 6.5 sample are also indicative of protein oxidation, but in addition, we observed a more substantial blueshift of the Soret band at higher lipid/protein ratios (higher CL concentrations), which indicates the formation of a hcHS protein. At low CL concentrations, we observed an increase in the sample’s turbidity, consistent with what Nantes and coworkers described as characteristics of L-site binding.12 Therefore, we can conclude that the observed protein oxidation upon binding of cytochrome c to TOCL-containing liposomes does not seem to depend on the specific binding process, whereas the latter seems to have an influence on the ligation state of the oxidized non-native protein. A more thorough analysis of the pHdependence of both ferricytochrome c and ferrocytochrome c binding to TOCL-containing liposomes is currently underway in our laboratory. Binding Under Aerobic Conditions in the Presence of NaCl. Our results show that in the absence of salt, the reduced state of cytochrome c is not maintained following binding to a TOCL-containing membrane surface. Under physiological conditions, however, ca. 100 mM NaCl is present, so we wondered whether the presence of Na+ and Cl− ions could at least partially inhibit the oxidation of ferrocytochrome c. It is well established that NaCl inhibits the high-affinity A-state binding of ferricytochrome c to CL-containing liposomes.6 At high CL concentrations the addition of NaCl shifts the equilibrium between different conformations of ferricytochrome c toward more compact, native-like conformations.14,15,21 Nantes et al. reported that Ca2+ and Na+ ions inhibit the binding of

Figure 6. Soret band CD spectra as measured for selected CL concentrations of 20% TOCL/80% DOPC liposomes (red: ferrocytochrome c in solution, orange: [CL] = 37.5 μM, yellow: [CL] = 65 μM, green: [CL] = 150 μM, cyan: [CL] = 200 μM) in the presence of 150 mM NaCl. All solutions have a final protein concentration of 5 μM. CL concentrations in micromolar units are numerically equivalent to the lipid/protein ratios.

measured after the addition of 150 mM NaCl at different lipid concentrations. The ΔΔε dose−response in Figure 7 shows a small but clearly discernible change in this spectroscopic parameter, indicating that the protein still interacts with the added liposomes. However, all spectra, including those recorded at high lipid concentrations, differ clearly from the spectra of the ferri/ferro mixtures obtained in the absence of salt (Figure 1). We assign the spectral changes to structural alterations in the protein, which is still in a reduced hcLS state. This notion is corroborated by the observation that the Soret band shows a negligible blueshift in the presence of the liposomes and NaCl (cf. Figures 6 and S11) and is proven by the re-emergence of the Q0 − Qv splitting (Figure S12). The Q0/Qv = 1.4 ratio might indicate the existence of a minor fraction of oxidized protein, but in general terms, the data allow us to confirm the observation of Nantes et al.32 that the addition of NaCl restores the reduced state. However, contrary to observations by these authors, our data suggest that cytochrome c binding to the liposome surface is not (completely) inhibited. To check the validity of this interpretation of our CD data, we also measured the W59 fluorescence in the presence of NaCl and plotted the integrated intensity of the S1 sub-band (340 nm) as a function of CL concentration in Figure 7. Surprisingly, we obtained a nearly 12226


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membrane surface composed of 20% TOCL and 80% DOPC leads to structural conversions and heme-iron oxidation. We identified three representative liposome-bound states, denoted p, nf′, and f′. The protein is clearly oxidized in the nf′ and f′ states. These two states are very similar, but not identical, to states nf and f, which we inferred from the response data of ferricytochrome binding to the surface of the above liposomes. Conformations f and f″ exhibit nearly identical spectroscopic parameters (sf′ and sf); nf′ and nf do not show any detectable fluorescence, but the corresponding Δε values differ substantially (−30 and −12 M−1 cm−1, respectively). Even more important is the difference between the KC2,high values that describe the equilibrium between nf(nf′) and f(f′). This constant is clearly lower for the nf′ ⇄ f′ equilibrium (0.48) than it is for nf ⇄ f′ (1.45).21 Under normal circumstances one would expect that the ensemble of liposome-bound cytochrome c exhibits the same equilibrium distribution irrespective of the history of its formation. This is apparently not the case. One might wonder whether this discrepancy reflects a nonequilibrium situation similar to that observed by Kinnunen and co-workers for their Asite binding.8 We have to admit that our present thermodynamic modeling of the ferrocytochrome c response data and the ferricytochrome c data in ref 21 is still not comprehensive enough to account for ferricytochrome and ferrocytochrome binding to CL-containing liposomes in a fully consistent manner. Under anaerobic conditions, the protein binds to the added liposomes but remains in the reduced state. The addition of NaCl under aerobic conditions returns most of the protein to its reduced state and inhibits binding at high CL concentrations. Proposed Binding Mechanism. We propose the following overall reaction scheme to explain our data

Figure 7. Upper panel: ΔΔε response indicating ferrocytochrome c binding to 20% TOCL/80% DOPC liposome mixtures measured in the presence of 150 mM NaCl. Lower panel: Fluorescence response indicating ferrocytochrome c binding to 20% TOCL/80% DOPC liposome mixtures measured in the absence (●) and presence (△) of 150 mM NaCl. The solid and dashed lines have been inserted for visual purposes only and do not represent the result of any fit performed on the data.

Before we discuss this scheme in detail we have to recall that these aerobic experiments were performed far from equilibrium, as the kinetics of an electron exchange with O2 is slow, owing to the limited permeability of the native protein for O2. Reoxidation of the protein in solution did not occur on the time scale of our experiments with 5 μM cytochrome c, but this apparently changed upon the binding of the protein to the liposome surface. Upon binding to the liposome surface, the reduced protein adopts predominantly the p conformation, with a modified hexacoordinated iron(II) conformation, in which the heme group might be more accessible to O2. The existence of a reduced state of cytochrome c that might be more amenable to oxidation by O2 has been proposed by Cassatt and Marini on the basis of ferrocytochrome c oxidation by various ferricyanide derivatives.66 As a consequence, p converts into an ensemble of oxidized nf3+′ conformations, which are compact14,15 and, to a major extent, native-like, with M80 as axial ligands. Oxidized state nf′3+ converts into the highly fluorescing f3+′ state. The equilibrium is shifted from p toward nf′ and f′, with increasing CL concentration. A minor fraction of the f′ population comprises a partially unfolded conformation, in which the heme iron is either in an hcHS or a QM state. A similar transition has been inferred from optical absorption data of ferricytochrome c bound to liposomes with 50 or 100% CL content.21 As indicated above, we can rule out lipid peroxidates as the source of protein oxidation on liposomes for two reasons: TOCL

linear increase in the fluorescence intensity with increasing lipid concentration. Compared with the dose−response obtained in the absence of NaCl, there is no discernible difference at low lipid concentrations, at which the p and to a lesser extent the nf′ conformations dominate (Figure S4). Only in the regime of the f′/f conformation at high CL concentrations did we observe a fluorescence reduction that is not as pronounced as that observed for ferricytochrome c under this condition. A thorough understanding of the apparently nontrivial influence of NaCl on the binding, conformation, and redox state of cytochrome c requires further systematic studies, which are out of the scope of this paper. Here, we just emphasize the reestablishment of the reduced state of cytochrome c as an important and somewhat surprising observation.

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DISCUSSION Summary of Results. In this paper we show that under aerobic conditions the binding of ferrocytochrome c to a 12227


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The Journal of Physical Chemistry B is not considered amenable to peroxidation22,62 and the peroxidation mechanism investigated by Musatov et al. (for bovine CL, which is polyunsaturated) does not require the presence of O2.67 Moreover, as shown by these authors, peroxidized CL leads to the destruction of the heme group by a Fenton-type reaction, similar to that observed for horseradish peroxidase.68 This has not been observed in our experiments. If the protein−liposome system is kept under anaerobic conditions, the conversion to the oxidized state is prevented, which evidences the involvement of O2.66 The CD spectra taken at very high lipid concentrations exhibit a positive Cotton band that is much broader than that in the corresponding spectrum of oxidized cytochrome c. Another striking difference concerns the noncoincidence between the CD and absorption maxima. In nf3+′, the noncoincidence, λΔεmax − λεmax, between the peak positions of the CD and absorption bands is positive, that is, the CD peak lies on the lower energy side of the absorption band. As revealed by Figure 4, the situation is reversed for nf2+′. Previous work has shown that such a noncoincidence reflects a splitting of the B-band caused by the removal of the twofold degeneracy of the excited B-state (Eu symmetry in D4h). This is due to a quadratic Stark effect produced by a very strong internal electric field and by vibronic perturbations of the excited state..69 In the native conformation, a transition from the oxidized to reduced state brings about a switch of the positions of Bx and By, owing to an orientational change in the electric-field vector in the heme plane.69 This can lead to a sign switch of the noncoincidence effect. From our data, we infer that this appears to occur for the partially unfolded nf′ state as well. Influence of NaCl on Ferrocytochrome c Binding. The interpretation of the influence of NaCl on aerobic ferrocytochrome c binding is not straightforward. In principle, one could expect that the addition of this salt would partially inhibit protein binding and stabilize the more folded, native-like nf′3+ state on the membrane surface.14,18,21 Both effects would have led to a reduced fluorescence and a recovery of the B-band CD couplet of the native and native-like states of the oxidized protein. Contrary to this expectation, we observed that the protein was converted back to its reduced state, in agreement with the observations of Nantes et al.32 This observation led these authors to conclude that, in contradiction to the report of Iwase et al.,31 ferrocytochrome c does not get oxidized upon binding to CLcontaining liposomes. However, our data unambiguously show that oxidation does indeed occur. Anion binding to cytochrome c in solution is known to stabilize the oxidized state47 so that ion− protein interactions cannot be invoked to explain our observations. Therefore, we hypothesize that the binding of both Na+ and Cl− to the membrane (Na+ to phospholipid head groups and CO groups, Cl− to the choline group of DOPC)70−72 can change the fluidity of the membrane in a manner that would allow for a deeper penetration of the protein into the more hydrophobic part of the outer layer. This could drive water out of the heme pocket, decrease the dielectric constant of the heme environment, and increase the redox potential, thus stabilizing the reduced state by moving its redox potential above that of the O2/O2−/H2O2 system (0.281 V at neutral pH).73 This scenario might also facilitate the proposed insertion of a CL tail into the protein interior.10,18,34 The W59 fluorescence data clearly suggest that some structural rearrangements and unfolding of ferrocytochrome c occur upon binding even when the oxidation state is maintained. A direct comparison of fluorescence data obtained under aerobic and anaerobic

conditions has to take into account that quenching owing to the Förster mechanism should be weaker in the reduced state due to a smaller overlap between the fluorescence emission and the Soret band absorption. We can certainly state, however, that the fluorescence intensity is reduced at high CL concentrations, in the presence of NaCl, which indicates a reduced fraction of the nf2+′ state. The rather linear slope of both the dichroism and fluorescence response to the addition of CL-containing liposomes (Figure 7) also indicates a lower overall binding affinity and thus some inhibition of cytochrome c binding. This results from Na+ and Cl− binding to the liposome and protein surfaces, respectively. We would like to emphasize, however, that reduced binding cannot account for the obtained preponderance of the reduced state. Comparison with the Literature. With regard to cytochrome c oxidation on CL-containing liposomes, our data strongly support the conclusions that Iwase et al. drew from their optical spectroscopy data for cytochrome c−CL mixtures (i.e., vesicles with 100% CL content).31 Nantes et al. had argued against this possibility on the basis of their observation of NaCl recovering the MCD signal of the ferrous protein.32 Our data suggest that NaCl does indeed restore the reduced state of the protein. A concrete mechanism still needs to be identified. Kalanxhi and Wallace investigated the binding of horse heart cytochrome c to liposomes formed with PC/PE/CL liposomes (5:4:3) by monitoring the absorbance changes in the Q-band region.34 The authors did not report any changes in the oxidation state and fit their binding isotherms with a single sigmoidal function. The description of their experimental protocol seems to suggest that they performed their experiments under anaerobic conditions, which would explain the absence of any protein oxidation. Kapetanaki et al. allowed oxidized horse heart cytochrome c to react with liposomes formed with 28% CL/72% PC and then subjected the mixture to reduction with sodium dithionite.35 They found that the treated protein could bind CO with a very high affinity. The authors performed their experiments in the region of low CL concentration. On the basis of the result of this study, one can expect that these authors’ protocol produced ferrous nf′ and f′ conformations. The latter should be susceptible to a replacement of the non-native ligand (histidine or lysine) by CO. This stabilizes the partially unfolded f states and increases their population. Hence, the results of Kapetanaki are generally consistent with those of our analysis of both ferricytochrome and ferrocytochrome binding to CL-containing liposomes. In a more recent study, Bradley et al. used MCD spectra covering the near-IR region of the cytochrome c spectrum to investigate the influence of the binding of cytochrome c to CLcontaining liposomes on the ligation state of horse heart cytochrome c in the oxidized and reduced states.74 Their results are at variance with those reported in this study and our earlier studies in many respects. For the binding of the oxidized state, they report the occurrence of two coexisting low-spin states, none of which has M80 as its distal heme ligand. They assign these two spin states to the H/K and H/OH− ligated heme groups. The authors performed their experiments with a rather low CL/protein ratio of 30. Their observation is contradicted by the appearance of the 695 nm band in the optical spectra of ferricytochrome c at similar CL/protein ratios, which unambiguously showed the presence of native-like states of liposomebound proteins.21 Moreover, the existence of a B-band couplet (Figure 1) indicates that the M80 ligand is maintained. Ligation with either K or OH− would move F82 away from the heme and 12228


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The Journal of Physical Chemistry B thus eliminate the negative maximum of the observed couplet.75 For the ferrous protein, the data of Bradley et al. suggest a conversion into a pcHS state. This confirms an earlier observation, by Droghetti et al.,33 of the formation of pcHS states of partially unfolded cytochrome c upon the protein’s reaction with sodium dodecyl sulfate micelles and dioleoylphosphatidylglycerol vesicles. There is no indication of the formation of a ferrous pcHS state in the spectra of anaerobic ferrocytochrome c−lipid mixtures. Its presence would cause a redshift of the Soret band, whereas our data do not indicate any significant shift at low CL concentrations and exhibit a blueshift at high CL concentrations (Figure 4). We wonder whether these serious discrepancies can be explained by the different protocols used to prepare the sample. Bradley et al. subjected their proteins to 100% ethanolic CL, whereas we used a TOCL/DOPC mixture to form small unilamelar vesicles. In line with our earlier work,20,21 this study shows compelling evidence for the notion that the binding of cytochrome c to CLcontaining liposomes does not trigger universal unfolding. Depending on the initial redox state, a substantial fraction stays in a native-like state, in which the Fe−M80 ligation is maintained. Alvarez-Paggi et al. provided experimental and computational evidence for the notion that upon binding to electrostatic surfaces the protein can undergo conformational changes while maintaining its ligation state.76 Recently, Mandal et al. carried out a very thorough experimental characterization of a ferricytochrome c−25% TOCL/75% DOPC mixture for a lipid/protein ratio of 40:1, which would correspond to a very low CL/protein ratio.77 The authors found that under these conditions the protein resides on the membrane surface without significant penetration and protein unfolding. That is, what we would expect on the basis of our own results. Interestingly, however, the protein nevertheless exhibited a measurable peroxidase activity. This discrepancy between structure and function certainly needs further investigation. Summary and Conclusions. Our visible CD and absorption experiments reveal that under aerobic conditions ferrocytochrome c is oxidized upon binding to CL-containing liposomes. We surmise that this reaction is facilitated by a more open structure of the protein on the liposome surface, which allows for easier access of molecular oxygen to the active site. As shown earlier, the newly oxidized protein can adopt predominantly two different types of conformations: the first one being native-like, with an intact M80-Fe3+ linkage, and the other one being partially unfolded, with a non-native, yet-to-be-identified distal ligand. However, in the present case the population of the partially unfolded state is much lower than that earlier observed for ferricytochrome c−20% TOCL/80% DOPC mixtures at the same high CL concentrations. Currently, it is unclear why the equilibrium between nf3+′ and f3+′ is different from that between nf and f′.21 Moreover, we observed a (not yet quantified) minor subfraction of a partially unfolded high-spin conformation emerging at high CL concentrations, in agreement with the findings of Pandiscia and Schweitzer-Stenner.21 In the presence of NaCl, the reduced state is to a major extent restored at all CL concentrations. This observation might suggest a change in the local heme environment that leads to a lower effective dielectric constant. Our results reveal, again, the importance of NaCl in maintaining the functionality of the electron-transport chain in the mitochondria; a process that heavily depends on the presence of both intact ferrocytochrome c and ferricytochrome c.37 As shown earlier, NaCl lowers the fraction of the hcLS nf3+′ and practically eliminates an nf3+′ subfraction, which exhibits a

pentacoordinate heme iron. In the present study, we show that NaCl can maintain the reduced state of the protein even when it is bound to a CL- containing surface. This ensures that the protein can carry its electron from the cytochrome c reductase to the cytochrome c oxidase in the mitochondrial respiratory chain.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b05620. Additional fitting parameters and the mole fractions of the involved species of the protein−liposome mixtures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 1-215-8952268. ORCID

Anthony W. Addison: 0000-0002-0706-7377 Reinhard Schweitzer-Stenner: 0000-0001-5616-0722 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge partial financial support of this work by the Department of Chemistry of Drexel University. REFERENCES

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Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by

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