Ferricytochrome c Protects Mitochondrial Cytochrome c Oxidase Against Hydrogen Peroxide-Induced Oxidative Damage

Abstract

Excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp_19,IV and Trp_48,VIIc, partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H₂O₂-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H₂O₂, with the apparent bimolecular rate constant of 5.1 ± 1.0 M⁻¹s⁻¹. Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H₂O₂-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radical(s) generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

INTRODUCTION

The mitochondrion has a central role in the production of ATP as well as the generation of reactive oxygen species (ROS). Under physiological conditions, the mitochondrion converts ~1–2% of the consumed oxygen into superoxide anion [1]. Moreover, ROS concentrations increase significantly under certain pathological events such as ischemia-reperfusion [2], muscular dystrophy [3], atherosclerosis [4], Parkinson’s disease [5], or other age-related disorders. Mitochondrial superoxide radicals are generated primarily by two ubiquinone utilizing electron transport complexes, i.e., Complex I (NADH:ubiquinone oxidoreductase), and Complex III (ubiquinone-cytochrome c reductase) [6,7]. Normally, the resulting superoxide anion is converted to H₂O₂ by superoxide dismutase with subsequent conversion to H₂O by catalase or glutathione peroxidase. However, in the presence of transition metals, H₂O₂ is converted into highly reactive hydroxyl radicals. Because the electron transfer chain components are a major source of superoxide, the local concentration of ROS near the inner membrane is potentially quite high, making the electron transfer chain itself a potential target for oxidative damage. If the resulting damage inhibits one or more of the complexes, ROS production by complexes I and III would be expected to increase in a manner analogous to that caused by electron transport inhibitors. As a result, ROS damage to the electron transfer chain could initiate a cascade of structural and functional alterations within inner mitochondrial membrane, the entire mitochondrion, and other cell components. In order to survive such a ROS attack, the mitochondrial electron transfer complexes must be equipped with efficient protective mechanisms.

Cytochrome c oxidase (CcO, EC 1.9.3.1.) is one of the mitochondrial electron transfer complexes known to be inactivated by ROS [8,9,10,11,12]. This enzyme catalyzes the transfer of electrons from ferrocytochrome c to oxygen, a reaction that proceeds through two oxy-intermediates, “peroxy-” and “ferryl-CcO”, both of which are potential free radical sources [13,14,15]. During normal electron flux, the concentration of these transient intermediates is quite low; however, during the lifetime of the mitochondrion, the potential exists for chronic free radical exposure. To avoid damage to redox-active sites, CcO is equipped with several defense mechanisms. For example, subunit III of CcO has been proposed to protect CcO from oxidative damage during enzymatic turnover [8]. An amino acid aromatic network within CcO, that facilitates radical transfer away from the redox-active, binuclear center, may also protect the enzyme from damage by radicals generated near the active site [16,17].

Cytochrome c is located in the mitochondrial inter-membrane space where its main function is to shuttle electrons from cytochrome bc₁ to CcO during respiration. In addition to its function in mitochondrial respiration, cytochrome c has a significant role in activation of a programmed cell death cascade [18]. For example, dissociation of cytochrome c from the inner mitochondrial membrane is known to be a critical step in the initiation of apoptosis [19,20,21]. Cytochrome c can also function as a cardiolipin-specific oxygenase that chemically oxidizes cardiolipin to produce CL hydroperoxides [21]. The resulting oxidized CL then releases pro-apoptotic factors into the cytosol. In addition to these roles, cytochrome c is also known to alter both the generation and elimination of H₂O₂ [22,23,24], and to regenerate dioxygen from superoxide radical anion under conditions of oxidative stress [25].

In this study, we tested the hypothesis that CcO is protected from peroxide-induced oxidative damage by its natural mitochondrial partner, cytochrome c. This hypothesis is based upon the facts that: 1) cyt c³⁺ can destroy H₂O₂ [23,24]; and 2) cyt c³⁺ binding to CcO alters the conformation of the CcO catalytic binuclear center [26,27,28]. Therefore, we have examined the extent of H₂O₂-induced changes to purified, detergent-solubilized CcO in the presence and absence of excess ferricytochrome c.

EXPERIMENTAL PROCEDURES

Materials

Sodium cholate, hydrogen peroxide, horse heart cytochrome c (≥ 95% purity), ammonium iron sulfate hexahydrate, butylated hydroxytoluene, xylenol orange, and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical were purchased from Sigma-Aldrich Co. Dodecyl maltoside was from Anatrace Inc. Bovine cardiolipin was obtained from Avanti Polar Lipids. Triton X-100 was from Roche Diagnostics. Other chemicals were analytical grade.

Methods

Purification of Cytochrome c Oxidase from bovine heart

Two forms of purified CcO, designated as A and B, were used in the present work. Preparation A involves sodium cholate solubilization of sodium deoxycholate treated Keilin-Hartree heart muscle particles followed by purification of CcO by ammonium sulfate precipitation as previously described [29]. The resulting purified enzyme was dissolved at ~100 µM in pH 7.4 buffer containing 25 mM sodium cholate and stored at −60 °C. Before it was used, the enzyme was diluted to 5–10 µM with 2 mM dodecyl maltoside, pH 7.4 buffer followed by exhaustive dialysis at 4 °C against the same buffer to remove residual sodium cholate. Preparation B involves Triton X-100 solubilization of CcO from mitochondria at neutral pH followed by purification using ion-exchange chromatography in the presence of Triton X-100. Triton X-100 was replaced with dodecyl maltoside by the second ion exchange procedure [30,31]. The resulting enzyme was concentrated to ~200 µM in dodecyl maltoside containing buffer and stored at −60 °C. The first procedure produces a form of CcO that reacts slowly with heme a₃ binding ligands; the second procedure produces a form of CcO that reacts quickly with these same heme a₃ binding ligands [32]. The data presented in this work were obtained using five different preparations of CcO with essentially the same results. All assays were done at least in triplicate using at least two different enzyme preparations.

Reaction of Cytochrome c Oxidase with H₂O₂ in the Presence or Absence of Ferricytochrome c

Dodecyl maltoside-solubilized CcO, either preparation A or B, at 10 µM in 20 mM Tris-Cl containing 2 mM dodecyl maltoside was reacted with 500 µM H₂O₂ for 30 min at room temperature in the presence or absence 0–500 µM of cyt c³⁺. In experiments that included cyt c³⁺, H₂O₂ was added 5 min after cyt c³⁺ had been mixed with CcO. The reaction was stopped by removal of both H₂O₂ and cyt c³⁺ by HiTrap Q anion-exchange column chromatography [33]. This chromatographic procedure also removes almost all phospholipids bound to CcO, except for the 3–4 tightly bound cardiolipin [33]. The resulting CcO was subsequently analyzed for changes in activity, modification of subunits and peroxidation of bound cardiolipin. It made no difference in these experiments, whether oxidized cyt c was used directly as purchased, or fully oxidized by treatment with potassium ferricyanide followed by removal of potassium ferricyanide by dialysis for 15 hours at 4 °C. In either case, cyt c³⁺ concentration was determined using ε_410nm = 106.1 mM⁻¹ cm⁻¹ [34], while the concentration of H₂O₂ was calculated using ε_240nm = 40 M⁻¹cm⁻¹ [35]. The concentration of the “peroxy” and/or “ferryl” forms of CcO were determined from the difference spectra of H₂O₂-treated minus oxidized CcO using either ΔΔε_434-412nm = 65 mM⁻¹cm⁻¹ [36] for the mixture of both forms, or ΔΔε_607–630 = 11 mM⁻¹cm⁻¹ for P-form, and ΔΔε_580–630 = 5.3 mM⁻¹cm⁻¹ for F-form [37]. Cyanide-inhibited cyt c was prepared by reacting of 500 µM cyt c³⁺ with 10 mM KCN for 1 hour at RT. The formation of the cyanide-cyt c³⁺ complex was confirmed by spectral changes at 695 nm. All visible spectra were collected using an SLM Aminco 3000 diode array spectrophotometer.

Determination of Cytochrome c Oxidase Activity

CcO activity was measured spectrophotometrically by following the pseudo-first order rate of oxidation of 25 µM ferrocytochrome c by 1.75 nM CcO at pH 7.0 in 25 mM phosphate buffer containing 2 mM dodecyl maltoside [33].

Subunit Analysis by High-Performance Liquid Chromatography

Quantitative RP-HPLC analysis of CcO subunit content was done using gradient elution from a Vydac C₁₈ reverse-phase column (5 µm, 0.46 × 25 cm, 300-Ǻ pore size) and a Waters/Millipore liquid chromatography system [9,16,38]. The gradient was made from mixtures of solvent A (0.2% TFA in water) and solvent B (0.2% TFA in acetonitrile). The column was equilibrated with solvent A and subunits eluted at 1 mL/min with a linear gradient from 25 to 50 % solvent B in 50 min, followed by a linear gradient from 50 to 85 % solvent B in 17.5 min. Elution was monitored at 214 nm after loading 0.2 – 2.0 nmol of unmodified or H₂O₂-modified CcO. Percent yield of each subunit was based upon quantitative integration of peak areas as compared with unmodified, purified CcO [9,16,38]. Fractions (0.5 mL) were collected for analysis of unmodified and modified subunits by mass spectrometry.

Analysis of Cardiolipin Oxidation

The extent of conjugated diene generation within cardiolipin after exposure of CcO to H₂O₂ was determined spectrally after (Bligh and Dyer) extraction of all phospholipids from CcO using chloroform/methanol/water [39] and subsequent purification of cardiolipin by normal phase HPLC [40]. Conjugated diene formation was monitored by the increase in absorbance at 234 nm [41].

Mass Spectrometry

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were acquired on an Applied Biosystems Voyager-DE-STR operated in either the linear or reflectron mode. HPLC-electrospray ionization tandem mass spectra were obtained using a Finnigan LCQ in conjunction with a Michrom BioResources MAGIC 2002 micro HPLC connected to a home-built microspray interface. Mass spectrometry analysis of modified and unmodified CcO subunits was done as described previously [9,16,17].

EPR Spectroscopy

EPR spectra were recorded with a Varian E-6 spectrometer. Acquisition conditions of the spectra were: modulation amplitude 10 G, frequency 9.225 GHz, microwave power 200 µW and temperature 109 K. Two series of H₂O₂-treated samples were analyzed by EPR spectroscopy to determine the effect of cyt c³⁺ upon H₂O₂-induced radical generation within CcO. The first was a mixture of CcO and a variable amount of ferricytochrome c; the second was only cyt c³⁺. In CcO-containing samples, 50 µM CcO, solubilized in 1 mM dodecyl maltoside pH 7.4 buffer containing 20 mM Tris-Cl and 4 mM K₂SO₄, was incubated with ferricytochrome c at room temperature (RT) for 5 minutes, followed by the addition of 300 µM H₂O₂. After 10 sec, the samples were rapidly frozen by immersion in a methanol-dry ice bath and then transferred into liquid nitrogen. Time-dependence of the radical signal was obtained by warming the samples to room temperature. After incubation of certain time the samples were frozen and used for EPR measurement. The concentration of radical within CcO was determined by the double integration of the EPR signal and comparison of the resulting area with that of DPPH standard. DPPH was solubilized in dimethyl sulfoxide and concentration calculated from the absorbance spectrum using an extinction coefficient of ε_525nm = 11.9 mM⁻¹cm⁻¹ [42].

Rate of H₂O₂ Decomposition by Ferricytochrome c and Cytochrome c Oxidase

The H₂O₂ concentration was measured at discrete time intervals after mixing with either cyt c³⁺, CcO or mixture of both proteins using the ferrous ion oxidation-xylenol orange assay (FOX2) [43]. The reaction conditions were 20 mM Tris-Cl buffer at pH 7.4, 2 mM dodecyl maltoside and 500 µM H₂O₂ mixed with either: 1) 500 µM cyt c³⁺; 2) 10 µM CcO; or 3) the mixture of 500 µM cyt c³⁺ and 10 µM CcO. Each reaction was initiated by the addition of H₂O₂, stopped at 5 min intervals by removal of H₂O₂ using a Microcon, Ultracel YM-3 membrane filter, with 3000 Da cutoff. Peroxide concentration in the filtrate was determined by absorbance at 560 nm 30 min after addition of the FOX2 reagent (1/9 v/v) using a standard calibration curve.

RESULTS

Ferricytochrome c Prevents H₂O₂-induced Oxidative Damage to CcO

The reaction of H₂O₂ with detergent-solubilized CcO is known to cause a time- and concentration-dependent inactivation of the enzyme [16]. The electron-transport activity of H₂O₂-treated CcO decreases 50–80% after reaction with 500 µM H₂O₂ for 30 min. at RT (inset to Figure 1). However, significant protection from H₂O₂-induced inactivation occurs when at least a 20-fold molar excess of cyt c³⁺ is added to the reaction mixture, e.g., only 5 % inactivation occurs if 200 µM cyt c³⁺ is present (Figure 1). Excess cyt c³⁺ also prevents two other H₂O₂-induced effects: 1) oxidation of Trp_48,IV and Trp_19,VIIc and 2) partial dissociation of subunits VIa and VIIa (Figures 2 & 3). When excess cyt c³⁺ is not included, ~20% of IV ~50% of VIIc elute with altered elution times due to oxidation of a single tryptophan (Trp_48,IV and Trp_19,VIIc) within each subunit [16]. The RP-HPLC elution peaks corresponding to the two oxidized subunits are absent if excess cyt c³⁺ is present during H₂O₂ exposure, i.e., > 90% of VIIc and nearly 100% of subunit IV remain unmodified (Figure 2 & 3). H₂O₂-induced oxidation of Trp_48,IV and Trp_19,VIIc without cyt c³⁺ and the absence of tryptophan oxidation in presence of cyt c³⁺ was also monitored by mass spectrometry as described previously [16,17]. In addition, excess cyt c³⁺ prevents dissociation of subunits VIIa and VIa, which normally accompanies H₂O₂-induced oxidative damage to CcO [16]. Only ~15% of subunit VIIa dissociates from CcO when it is exposed to H₂O₂ in the presence of at least a 20-fold molar excess of cyt c³⁺, rather than >40% dissociation of this subunit in its absence. Dissociation of subunit VIa is considerably reduced as well (Figure 3, panel B).

Figure 1. Ferricytochrome c protection of CcO from inactivation by H₂O₂.

Main panel: Electron transfer activity of CcO after its exposure to H₂O₂ in the presence of different concentrations of cyt c³⁺. Cytochrome c oxidase (10 µM) was reacted with 500 µM H₂O₂ for 30 min in the presence of 0–500 µM ferricytochrome c. The reaction was stopped by removal of H₂O₂ using anion-exchange chromatography, and the remaining CcO activity determined spectrophotometrically. Data were fitted to a single-exponential rise to a maximum (solid line). Inset panel: Inactivation of CcO by H₂O₂ in the absence of cyt c³⁺. Cytochrome c oxidase (10 µM) was reacted with each concentration of H₂O₂ for 30 min., excess H₂O₂ removed by anion-exchange chromatography, and the remaining CcO activity determined spectrophotometrically. Data were fitted to a single-exponential decay (solid line). Assays were performed in triplicate using three different CcO preparations. The standard deviation in experiments was estimated to be ± 5.0%.

Figure 2. Cytochrome c oxidase nuclear-encoded subunit composition after exposure to H₂O₂ in the presence or absence of ferricytochrome c.

Cytochrome c oxidase (10 µM) was reacted with 500 µM H₂O₂ in the presence of either 0, 300, or 500 µM cyt c³⁺ (top, middle and lower chromatograms). After 30 min., H₂O₂ and cyt c³⁺ were removed by HiTrap Q anion exchange chromatography and the nuclear-encoded subunit composition quantified by RP-HPLC analysis of 0.5 nmol of CcO [27]. Upper panel: RP-HPLC elution profile for all 10 of the nuclear-encoded subunits. Lower panel: Expanded profile between 33 to 49 min to better illustrate the elution of the H₂O₂ modified subunits (IV* and VIIc*). The data represents typical RP-HPLC profiles of three experiments using three different CcO preparations.

Figure 3. Ferricytochrome c protection of CcO subunits from H₂O₂-induced modifications.

Panel A: Protection of CcO subunits IV (filled triangles) and VIIc (open triangles) from H₂O₂-induced oxidations by increasing cyt c³⁺ concentration. Panel B: Protection of CcO subunits VIa (filled circles), and VIIa (open circles) from H₂O₂-induced dissociation by increasing cyt c³⁺ concentration. The percent modification of each subunit was calculated from the areas of the relevant RP-HPLC elution peaks. Differences between chromatograms were normalized on the assumption that the area under the RP-HPLC peak corresponding to subunit Va remained constant. Solid lines are single-exponential fits to the data. The results are presented as an average of three measurements using three different preparations of CcO. The deviation in experiments was estimated to be ± 4.4%.

Ferricytochrome c Protects Cardiolipin from Hydrogen Peroxide-Induced Peroxidation

Inclusion of cyt c³⁺ also protects cardiolipin that is bound to CcO from H₂O₂-induced peroxidation. If either pure or protein-bound cardiolipin is exposed to peroxides, a significant portion of the non-conjugated double bonds is converted into conjugated dienes with characteristic absorbance at 234 nm (Figure 4, thick line). However, if CcO is exposed to high concentrations of H₂O₂ in the presence of excess cyt c³⁺, the double bonds are well protected from oxidation as evident from the significantly reduced absorbance at 234 nm (Figure 4, dashed line).

Figure 4. Excess cyt c³⁺ protects CcO-bound cardiolipin from H₂O₂-induced peroxidation.

Peroxidized cardiolipin has a characteristic absorbance maximum at 234nm due to the presence of conjugated dienes; therefore, the extent of peroxidation can be determined from the UV spectrum of cardiolipin. The UV spectrum of HPLC-purified CL before (dotted line) and after exposure of CcO to 500 µM H₂O₂ in either the presence (dashed line), or absence (thick solid line) of excess cyt c³⁺. The UV spectrum was acquired online using a diode-array UV HPLC detector. In each case, cardiolipin was extracted from 2 nmole of CcO, dissolved in ethanol, and purified by HPLC. Nearly identical results were obtained for three different CcO preparations.

Hydrogen Peroxide-induced Spectral Changes for Cytochrome c Oxidase and Ferricytochrome c

Reaction of H₂O₂ with CcO results in the formation of two oxy-intermediates, the “peroxy-” or P-form” and the “ferryl-” or F-form, both of which are detected by visible spectroscopy [36]. Inclusion of cyt c³⁺ does not alter either the relative amounts or rates at which the two intermediates are generated. Reaction of 1.2 µM CcO with 500 µM H₂O₂, either in the presence or absence of 8 µM cyt c³⁺, produces a mixture that contains 0.36–0.48 µM P-form, and 0.68–0.76 µM F-form, i.e., 35 – 40% and 60%–65%, respectively (Figure 5). The pseudo-first order rate constants for the formation of the oxy-intermediates are also nearly identical in the presence or the absence of 100 µM cyt c³⁺ i.e., 0.041 s⁻¹ and 0.039 s⁻¹, respectively (data not shown).

Figure 5. Generation of the H₂O₂-induced CcO oxy-intermediates is unaffected by the presence of excess cyt c³⁺.

CcO P- and F-oxy-intermediates have a characteristic CcO_P+F – CcO_oxid difference spectrum with a minimum at 412 nm and maxima at 434, 580 and 607 nm. The difference spectrum for CcO was generated in silico by subtracting the digitized visible spectrum of 1.2 µM oxidized CcO in 20 mM Tris-Cl buffer, pH 7.4, with 2 mM dodecyl maltoside, from the digitized spectrum acquired after its reaction with 500 µM H₂O₂ for 30 min in absence and presence of cyt c³⁺. Upper Panel: H₂O₂-induced difference spectrum of CcO acquired in the absence of ferricytochrome c. Lower Panel: H₂O₂-induced difference spectral changes of cytochrome c oxidase in presence of 8 µM ferricytochrome c. In this case, the spectrum of ferricytochrome c was recorded, set to zero and was used as a reference (thin line). CcO was then added to make the solution 1.2 µM in CcO and the difference spectrum generated as described above. Concentrations of P- and F- forms were calculated using ΔΔε_607–630 = 11 mM⁻¹cm⁻¹ for the P-form, and ΔΔε_580–630 = 5.3 mM⁻¹cm⁻¹ for the F-form [32]. In both A and B forms of CcO hydrogen peroxide produces essentially the same amount of intermediates.

Hydrogen peroxide also reacts with cyt c³⁺, but the spectral effects are minimal under the conditions used. For example, maximum absorbance in the Soret region decreases by <5% when 500 µM cyt c³⁺ is exposed to 500 µM of H₂O₂ for 30 min (Figure 6, main panel). Such exposure slightly perturbs the charge-transfer bond between the heme iron and Met₈₀ sulfur [44] since very small absorbance changes also occur at 695 nm (Figure 6, inset). However, H₂O₂ does not induce any major structural changes in cyt c³⁺ as evidenced by the very small changes in the molar ellipticity at 417 nm (3–5%) and no significant alteration in either the far-UV or aromatic regions of the spectrum (data not shown).

Figure 6. H₂O₂-induced bleaching of the visible spectrum of cyt c³⁺.

Main Panel: Time-dependence of H₂O₂ –induced changes in the Soret difference spectrum of ferricytochrome c. Ferricytochrome c (500 µM) in 20 mM Tris-Cl pH 7.4 buffer containing 2 mM dodecyl maltoside was reacted with 500 µM H₂O₂ at room temperature. After 0, 10, 20 and 30 min, 10 µL aliquots were diluted 100-fold with reaction buffer and the absolute spectra were recorded. The absorption maxima in absolute spectra were 0.530, 0.527, 0.512, and 0.502, respectively. The difference spectrum was generated by subtracting the digitized visible spectrum of cyt c³⁺ before addition of H₂O₂ from the digitized spectrum acquired after its reaction with H₂O₂. Maximum absorbance in the Soret region decreased by only 0.6%, 3.5%, and 5.3% after 10, 20 and 30 min reaction of cyt c³⁺ with H₂O₂, dotted, dashed, and solid line, respectively.

Inset Panel: H₂O₂-induced changes in the far visible spectrum of a mixture of cyt c³⁺ and CcO. Difference spectra were recorded for 500 µM cyt c³⁺ and 10 µM CcO versus 10 µM CcO before (thick line) and after 30 min reaction with 500 µM H₂O₂ (thin line). Measurements were done in triplicate with the nearly identical results.

H₂O₂-Induced Free Radical Generation within Cytochrome c Oxidase: Effect of Ferricytochrome c

Exposure of CcO to H₂O₂ generates a free radical EPR signal that is characterized by the g = 2.006 with a peak-to-trough width of 12 G (Figure 7, panel A). At 109 K this signal is saturated at a microwave power above 4 mW and no hyperfine structure is detected using amplitude modulation smaller than 10 G. A maximum yield of radical occurs 10 sec after addition of H₂O₂ and corresponds to ~14% of the CcO population. Inclusion of cyt c³⁺ suppresses the radical signal in a concentration- and time-dependent manner (Figure 7, panel B). This free radical is generated as H₂O₂ reacts with the catalytic binuclear center of CcO since the EPR signal is missing if the samples containing only ferricytochrome c and hydrogen peroxide, or if the catalytic site of CcO is first blocked with cyanide.

Figure 7. Ferricytochrome c suppression of the H₂O₂-induced free radical EPR signal of CcO.

Upper Panel: Difference EPR spectra for CcO in either the absence (solid line), or presence of 100 µM (dashed line), or 800 µM f cyt c³⁺ (dotted line). All samples were frozen within 10 seconds after addition of 500 µM H₂O₂. Each difference spectrum was calculated by subtracting the oxidized CcO spectrum from that of the H₂O₂ treated enzyme. Lower Panel: Dependence of the CcO radical yield upon the cyt c³⁺ concentration for samples reacted with H₂O₂ for either 10, or 100 seconds. Solid lines through each set of data are non-linear regression fits of the data to a two-term exponential decay. The reaction was initiated by addition of 500 µM H₂O₂ to 48.3 µM CcO solubilized in 20 mM Tris-Cl buffer, pH 7.4, containing 2 mM dodecyl maltoside and 4 mM K₂SO₄. Refer to Experimental Procedures for EPR experimental details. The results are presented as an average of three experiments employing two different preparations of CcO.

Decomposition of Hydrogen Peroxide by Ferricytochrome c and Cytochrome c Oxidase

Cyt c³⁺ exhibits peroxidase/catalase-type activity, which would be expected to decrease the H₂O₂ concentration [24,58]. To determine if this activity is sufficient to affect oxidative damage to CcO, the peroxidase/catalase-like activities of cyt c³⁺, CcO, and a mixture of both proteins were determined by monitoring the catalytic destruction of H₂O₂. Both proteins catalyze a single exponential decrease in the H₂O₂ concentration with time (Figure 8). The activity of CcO is more than ten-times greater than cyt c³⁺, with corresponding second order rate constants of 63.2 ± 2.5 M⁻¹s⁻¹ compared to a rate of 5.1 ± 1.0 M⁻¹s⁻¹ for cyt c³⁺. Using these values, a pseudo-first order rate of (3.2 ± 0.6) ×10⁻³ s⁻¹ is predicted for a mixture of 10 µM CcO and 500 µM cyt c³⁺, which is within experimental error of the value experimentally determined, i.e., (3.8 ± 0.7) × 10⁻³ s⁻¹. The peroxidase/catalase-like activities of cyt c³⁺ and CcO are, however, completely inhibited if proteins are first reacted with cyanide (Figure 8). Presumably, cyanide reacts with a five-ligand form of cyt c³⁺ to produce a cyano – Fe³⁺ complex, which prevents subsequent reaction of the iron with H₂O₂.

Figure 8. Decomposition of H₂O₂ by cyt c³⁺, CcO, and a mixture of cyt c and CcO.

The peroxidase activity of 500 µM cyt c³⁺ (unfilled circles), 500 µM cyt c³⁺ that previously had been reacted with 10 mM KCN for 1 hour at RT (black-filled squares), 10 µM CcO (black-filled circles), and a mixture of 500 µM cyt c³⁺ and 10 µM CcO (grey-filled circles) were quantified by following the destruction of 500 µM H₂O₂ as a function of time using the FOX2 assay. The solid lines are non-linear regression fits of the cyt c³⁺ and CcO data to single exponentials, with second order rate constants of 5.1 M⁻¹s⁻¹ and 63.2 M⁻¹s⁻¹, respectively (the rate constant for KCN reacted cyt c³⁺ was essentially zero). The dot-dash line is the theoretical first order rate for a mixture of 500 µM cyt c³⁺ and 10 µM CcO using the two second order rate constants. All reactions were done in 20 mM Tris-Cl, pH 7.2 buffer, containing 2 mM dodecyl maltoside. The provided data are mean values of three independent experiments using three different CcO preparations and the error bars correspond to standard deviations of the measurements.

DISCUSSION

H₂O₂-induced modifications to CcO are largely prevented by high concentrations of cyt c³⁺. In contrast to results obtained in the absence of cyt c³⁺, exposure of CcO to H₂O₂ in the presence of excess cyt c³⁺ leaves the electron-transport activity of CcO unaffected. Additionally, Trp_19,VIIc and Trp_48,IV both remain unmodified; dissociation of subunit VIIa is significantly reduced; peroxidation of cardiolipin is prevented; and the observed free radical concentration within CcO is reduced. The only H₂O₂-induced alteration not prevented by excess cyt c³⁺ is the dissociation of subunit VIa, which still occurs up to ~30%. The protective effects of cyt c³⁺ against time- and concentration-dependent H₂O₂-induced modification of CcO are the consequence of two quite different effects: (1) cyt c³⁺ catalyzed decomposition of H₂O₂, and (2) cyt c³⁺-induced quenching of a peroxide-induced free radical within CcO.

Cyt c³⁺ catalyzed decomposition of H₂O₂

The mechanism for cyt c³⁺ catalyzed decomposition of H₂O₂ appears similar to either that of a classical peroxidase or catalase. Such activity would seem to be unlikely since cytochrome c contains six coordinate iron structure. However, the Met₈₀ ligand bond is relatively weak and can be displaced by rather mild perturbants, e.g., changes in ionic strength or pH, allowing external ligands, such as cyanide, azide, imidazole and peroxide, to bind to the iron of cyt c³⁺ [24,45,46,47,48,49,50,51]. A small portion of cyt c³⁺ must be present in an “open” conformation in which the 6^th axial coordination bond with Met₈₀ is broken [52] to permit such binding. Since equilibrium between the “closed” and “open” cyt c³⁺ favors the “closed” 6-coordinate heme species H₂O₂ is expected to react slowly with cyt c³⁺. Consistent with an “open” conformation hypothesis is the complete inhibition of peroxidase/catalase activity when cyt c³⁺ is first reacted with cyanide (Figure 8). Cyanide forms a strong coordination bond with any heme having an open 6^th ligand position; therefore, cyanide inhibition clearly demonstrates that catalytic destruction of H₂O₂ by cyt c³⁺ requires a direct interaction of H₂O₂ with the iron of cyt c³⁺.

A peroxidase type mechanism raises the issue of an absence of reducing equivalents to complete the catalytic cycle. The small percentage of cyt c³⁺ in the “open” conformation would be expected to react with peroxide to form the typical peroxidase intermediate, compound I, containing ferryl oxygen, Fe(IV)═O, and a radical porphyrin cation, R•⁺. It is not obvious how the 2-electron reduction of compound I to regenerate cyt c³⁺ and water would occur, but a second H₂O₂ molecule may be the source of reducing equivalents, resulting in the disproportion of two peroxide molecules, i.e., a catalase-type reaction. Such a mechanism has been used to explain the H₂O₂-induced reduction of ferryl-oxygen compounds in both met-myoglobin [53,54] and cytochrome P450 [55]. Alternatively, the side chains of aromatic acids of protein itself may donate two electrons, i. e a peroxidase-type reaction.

Regardless of the mechanism for the peroxidase/catalase-like activity of cyt c³⁺, there is no doubt that cyt c³⁺ catalyzes the slow decomposition of H₂O₂ with the second order rate of 5.1 ± 1.0 M⁻¹s⁻¹ at pH 7.4. This rate is in good agreement with previously reported values of 4–11 M⁻¹s⁻¹ [56,57,58]. Although the peroxidase activity of cyt c³⁺ is ~6–7 orders of magnitude less than that of the 5-coordinate Fe structure in peroxidases, it is sufficient to significantly decrease the H₂O₂ concentration at high cyt c³⁺ concentration.

Cyt c³⁺-Induced Quenching of a CcO free radical

The peroxidase/catalase activity of cyt c³⁺ satisfactorily explains a diminished effect of H₂O₂ on CcO due to partial destruction of H₂O₂, but it cannot explain the rapid decrease in the H₂O₂-induced free radical content of CcO. Inclusion of a 2–16 fold molar excess of cyt c³⁺ decreases the amount of the EPR-detectable radical by 30–45% 10 s after addition of peroxide. The slow peroxidase/catalase-like activity of cyt c³⁺ cannot appreciably alter the H₂O₂ concentration during such a short time interval (refer to Fig. 7).

The origin of the EPR-detectable free radical signal is unknown, but stoichiometric amounts of radicals are generated in proximity to the heme a₃-Cu_B site of CcO coincident with the formation of the peroxy- and ferryl-oxygen mixture. With both intermediates, the iron of heme a₃ is converted to an oxo-ferryl state with simultaneous generation of a radical cation, often assigned to Y₂₄₄ (e.g. Fe_a3^IV═O Cu_B^II Y₂₄₄•⁺). The Y₂₄₄ radical is not responsible for the EPR signal since electronic coupling of the radical to the iron and/or copper adjacent iron-copper binuclear center makes Y₂₄₄•⁺ EPR-silent. However, radical migration from Y₂₄₄ to a site distant from the binuclear center would make it EPR visible and would explain the EPR signal. We previously postulated such radical migration away from Y₂₄₄ along an “aromatic wire” to explain H₂O₂-induced oxidative damage to CcO-tryptophans located far from the binuclear center [16,17]. The yield of EPR detectable radical(s) in these distant site(s) is sub-stoichiometric with a maximum yield of only ~14%, but any radicals remaining near the binuclear center would go undetected. Alternatively, the steady state level of radicals may be less than stoichiometric because H₂O₂ acts as a 2-electron reductant to destroy the Y₂₄₄•⁺ radical cation before it can migrate away from the binuclear center (refer to prior discussion of peroxidase/catalase mechanism.

Three feasible mechanisms could explain the quenching of the EPR-detectable free radicals by cyt c³⁺, but none of them is entirely satisfactory. First, cyt c³⁺ may form a complex with CcO that sufficiently alters the conformation of near the binuclear center [26,27,28] to suppress a radical migration away from the catalytic center (Y₂₄₄) to the secondary distant site(s). Consistent with this explanation is the decreased yield of the observed radical together with the inhibition of the modifications of the Trp_19,VIIc, Trp_48,IV and cardiolipin in the presence of ferricytochrome c. This mechanism, however, is not completely acceptable since the cyt c³⁺-induced CcO conformation would be expected to exhibit altered H₂O₂ reaction kinetics, which does not occur (generation of the oxy-intermediates and CcO peroxidase activity are nearly identical with, or without cyt c³⁺; refer to Figures 5 & 7).

Second, cyt c³⁺ may participate in protein to protein free radical transfer in which case a protein of cyt c³⁺ would serve as an electron donor to a radical that had migrated to the surface of CcO, e.g., Trp_19,VIIc or Trp_48,IV. We were unable to detect any such alterations to cyt c³⁺ although detection of such sub-stoichiometric amounts of radicals in a 20–50 fold excess of cyt c would be difficult.

Third, cyt c³⁺ could function as a shuttle of electrons to decrease free radical content. However, in absence of electron donors in our experimental conditions this mechanism is unlikely. Moreover, if such electron transfer is occurred it would have also affect the rate of formation of P and F intermediates during the reaction of oxidized enzyme with H₂O₂. Yet both the rates and amount of oxy-intermediates are the same in the presence and the absence of cytochrome c (Fig. 6).

We conclude that cyt c³⁺ assists in two ways to shield CcO against oxidative damage. First, cyt c³⁺ protects CcO by an external mechanism: catalytic removal of H₂O₂ by cyt c³⁺. Although the rate of peroxidase activity of cyt c³⁺ is lower than for a specialized catalase or peroxidase, its location and high concentration in the mitochondrion (up to 0.7 mM [59]) potentially make cyt c³⁺ a significant contributor in the catalytic destruction of peroxide. Moreover, according to Kagan et al., interaction of cyt c³⁺ with mitochondrial cardiolipin significantly increases its peroxidase activity, which can become as high as ~200 M⁻¹s⁻¹ [21,60].

Second, cyt c³⁺ may also protect CcO from oxidative damage by suppression or elimination of detrimental free radical(s) that are generated as part of normal CcO turnover. In contrast to the purified enzyme, CcO within the mitochondrion is surrounded by cyt c on the matrix side of the inner membrane and, therefore, is well protected against H₂O₂-induced oxidative damage. In fact, CcO in vivo is relatively resistant to H₂O₂–induced oxidative damage. For example, with heterozygous Mn-superoxide dismutase knockout mice, mitochondrial H₂O₂ increases by 40–50% with significantly reduced complex I and complex V activities, but almost no detectable change in CcO electron-transport activity [61]. Furthermore, increasing the H₂O₂ concentration within isolated rat heart mitochondria, (i.e., conditions that are similar to those with knockout mice), does not lead to diminished CcO activity [62]. Purified CcO, however, is susceptible to hydrogen peroxide because it is separated from these defensive mechanisms. Altogether the present study suggests that cyt c³⁺ may have an important role in protecting the cytochrome oxidase and consequently mitochondrion as well against oxidative impairment.

ABBREVIATIONS

ROS

reactive oxygen species

CcO

bovine heart cytochrome c oxidase

P-and F-form

peroxy- and ferryl cytochrome c oxidase oxy-intermediates

cyt c³⁺

ferricytochrome c

CL

cardiolipin (diphosphatidylglycerol)

DM

dodecyl maltoside

RT

room temperature

FOX2

ferrous ion oxidation-xylenol orange assay

HPLC

high-performance liquid chromatography

ESI/MS

electrospray ionization mass spectrometry

MALDI-TOF/MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Trp_48,IV

tryptophan 48 within CcO subunit IV

Trp_19,VIIc

tryptophan 19 within CcO subunit VIIc