Nitric Oxide Inhibits Peroxidase Activity of Cytochrome c· Cardiolipin Complex and Blocks Cardiolipin Oxidation*

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c·CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO–, respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c·CL complex, NO chemically reacted with H2O2-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H2O2-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H2O2-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c·CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3′-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1′2,2′-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1′2,2′-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c·CL complexes.

complex III (ubiquinol:cytochrome c reductase) and complex IV (cytochrome c oxidase). The electron transporting function of cyt c is effectively accommodated by its hexa-coordinate arrangement of heme iron, whereby Met 80 and His 18 represent the two axial ligands (1). The hexacoordinate organization of heme-iron, however, hinders binding and chemical interactions of cyt c with small molecules such as NO, O 2 , CO, and H 2 O 2 ; therefore cyt c in solution exhibits only a marginal peroxidase activity (2)(3)(4)(5)(6). The hindrance can be eliminated, and the redox catalytic reactivity can be conferred on cyt c by converting its hexa-coordinate arrangement into a penta-coordinate form by full or partial unfolding of the protein with strong denaturing agents such as guanidine chloride or chemical modification of Met 80 (7,8). Similar structural perturbations accompanied by an exchange and a loss of axial ligands and an increase of peroxidase activity were observed when cyt c interacted with negatively charged phospholipid membranes (3-5, 9 -11). The structure of cyt c bound to the anionic membranes and its peroxidase activity strongly depend on the experimental conditions, such as membrane composition, protein-to-lipid ratio, ionic strength, and pH (1,5,(7)(8)(9)(10)(11)(12). Denatured cyt c was shown to display an increased NO binding and a significant peroxidase activity in the presence of H 2 O 2 (7,8,(13)(14)(15). Chen et al. (13) reported NO consumption by HOCl-modified cyt c. However, the studies were performed in model systems under conditions and/or treatments hardly compatible with known physiological or pathophysiological roles of cyt c.
NO was shown to play an important role in the regulation of mitochondrial respiration (16). One established mechanism of this regulatory function is reversible inhibition of cytochrome c oxidase (17). The discovery of mitochondrial nitric-oxide synthase emphasized the significance of NO in mitochondrial functions (18,19), although NO can also reach mitochondria from external sources because of its hydrophobic nature and facile diffusion in biomembranes (20,21). An increased production of NO has been demonstrated during early stages of apoptosis indicating its potential involvement in regulation of programmed cell death (22,23). One possible pathway is the interactions of NO with superoxide generated by disrupted electron transport yielding a very reactive oxidant, peroxynitrite, that may cause mitochondrial damage during apoptosis (20,21,24). However, the protective effects of NO in apoptosis also were demonstrated (25).
Our previous work has identified CL as a physiologically relevant regulator of peroxidase activity of cyt c markedly enhanced during apoptosis (5). Specific peroxidation of CL catalyzed by the peroxidase activity of CL-bound cyt c molecules contributes to mitochondrial outer membrane permeabilization and the release of proapoptotic factors from the intermembrane space of mitochondria into the cytosol, a critical step in the intrinsic pathway of apoptosis.
The cyt c⅐CL complex has very different structural and catalytic properties than cyt c in solution. Therefore its interactions with NO may differ substantially from those of isolated cyt c. We were specifically * This work was supported by National Institutes of Health Grants U19 AI068021, HL70755, and ES09648-01A2 and Pennsylvania Department of Health Grant SAP 4100027294. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  interested in the NO regulation of peroxidase activity of cyt c⅐CL complex because of its role in induction of apoptosis. Although several recent studies indicate that CL-induced unfolding of cyt c is associated with the formation of its penta-coordinate state (5,9), interactions of the cyt c⅐CL complex with NO and its effects on the peroxidase activity of the complex remain unknown. Here we report, for the first time, that CL facilitates the binding and chemical interactions of NO with cyt c and its reactive intermediates formed in the presence of H 2 O 2 . Thus, NO can act as a regulator of the H 2 O 2 -dependent peroxidase activity of the cyt c⅐CL complex.
For reduction of cyt c to its ferrous form, 10 mM ascorbate was added to the solution of cyt c (4 mM) in 25 mM phosphate buffer, pH 7.4, 100 M DTPA and incubated for 20 min. The solution was then passed through a Sephadex G-25 column (HiTrap Desalting, 1.6 ϫ 2.5 cm; Amersham Biosciences) equilibrated with the same buffer. The concentration of cyt c was determined spectrophotometrically (15).
Liposomes containing DOPC and cardiolipin were prepared by sonication of lipids suspension under N 2 and used immediately after preparation. The optical measurements were carried out on a Shimadzu UV160U spectrophotometer; EPR spectra were recorded on a JEOL-REIX spectrometer. All of the original spectra are prototypical of at least three experiments.

NO Concentration
NO flux (concentration of NO produced in unit of time) was calculated according to the following equation, NO concentration in the solution was estimated from the concentration of NO donor, assuming that NO oxidizes at a normal oxygen concentration ([O 2 ] ϭ 220 M) via bimolecular process with a rate constant k NOox ϭ 10 3 (M Ϫ1 s Ϫ1 ) (24,27). The corresponding equation is as follows, where Flux NO was computed from Equation 1.
The inhibition constant (IC 50 ) was determined as the concentration of an NO donor (or Angeli's salt) that caused a 2-fold decrease of the cyt c activity. Corresponding half-inhibiting NO concentration was estimated from IC 50 of NO donor according to Equation 2.

Low Temperature EPR Measurements
Nitrosylation of cyt c was performed in the presence of DOPC⅐TOCL liposomes with different content of CL (total lipid, 8 mM) in 25 mM sodium phosphate buffer, pH 7.4.
For cyt c nitrosylation by NO donors, ferro-cyt c (100 M) was incubated for 15 min with liposomes in the presence of 500 M NO donor (DEANOate at room temperature or PAPANONOate at 37°C) under N 2 . When Angeli's salt (750 M) was used as a donor of nitroxyl (HNO, pK a ϭ 11.4), it was incubated with ferri-cyt c (200 M) for 10 min at room temperature. The reaction was stopped by freezing the samples in liquid nitrogen. The spectra were recorded at 77 K under the following instrumental settings: center field, 3200 G; scan range, 500 G; modulation amplitude, 5 G; microwave power, 10 milliwatt; time constant, 0.1 s; scan time, 4 min; receiver gain, 5 ϫ 10 2 .

Peroxidase Activity Measurements
Etoposide-based Assay-EPR spectra of etoposide phenoxyl radical were recorded at 25°C in gas-permeable Teflon tubing (inner diameter, 0.8 mm; thickness, 0.013; Alpha Wire Corp., Elizabeth, NJ). The tubing (length, ϳ6 cm) was filled with 60 l of sample, double-folded, and placed in an open 3.0-mm-internal diameter EPR quartz tube. The spectra were recorded under the following EPR conditions: center field, 3350 G; sweep width, 50 G; microwave power, 10 milliwatt; field modulation, 0.5 G; receiver gain, 10 3 ; time constant, 0.03 s; scan time, 8 min. The time course of etoposide radical signal was obtained by repeated scanning of the field corresponding to a part of the EPR signal (sweep width, 5 G; the other instrumental conditions were the same).
To obtain a solution of etoposide phenoxyl radicals free of the radicalgenerating system, horseradish peroxidase (40 g/ml) was incubated with etoposide (200 M) and H 2 O 2 (100 M) for 4 min; then the solution was quickly passed through an Amicon Ultra PL-30 filter (Millipore Co., France). The filtrate containing etoposide phenoxyl radicals was used immediately to monitor its interactions with reactive nitrogen species.

PAGE of Cyt c⅐Cardiolipin Complexes
Cyt c-Fe(III) (30 M) was incubated with DOPC⅐TOCL liposomes in the presence of H 2 O 2 (200 M each 15 min). The peroxidase reaction was stopped by 1400 units/ml catalase. Then the loading buffer was added, and the samples were immediately loaded onto a gel. SDS-PAGE was run using 12.5% running gel and 4% stacking gel as described in Ref. 29. The gels were stained with Coomassie R-250 Brilliant Blue.

Western Blotting of Cyt c⅐CL
For blotting, the proteins were electrophoretically transferred to nitrocellulose membrane, and the membrane was incubated with anti-DMPO or anti-nitrotyrosine antibodies. Incubation with anti-DMPO antibodies was performed essentially as described in Ref. 30. In the case of anti-nitrotyrosine antibodies, nonspecific binding was blocked by the incubation in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20 (TBS-T) with 3% bovine serum albumin for 1.5 h at room temperature. The membrane was then incubated with anti-nitrotyrosine antibodies (1:5000 in TBS-T with 1% bovine serum albumin) overnight at 4°C. After washing five times with TBS-T, the membrane was incubated with the secondary antibody conjugated with alkaline phosphatase for 1 h at room temperature. After washing three times with TBS-T, the bands were visualized using chemiluminescent substrate for the detection of alkaline phosphatase, Lumi-Phos TM WB.

Detection of NO
NO was quantitated amperometrically using a NO-selective electrode (Iso-NO; 2-mm shielded sensor: WPI, Sarasota, FL). The samples were incubated at room temperature in a reaction chamber under continuous stirring. Changes in current output (pA) were recorded, and NO was quantified using a standard curve generated by the addition of NaNO 2 in nitrite-free water under reducing conditions (KI/H 2 SO 4 ).

Lipid Extraction and Two-dimensional HPTLC Analysis
Lipids were extracted from liposomes using the Folch procedure (31). The lipid extracts were separated and analyzed by HPTLC (high performance thin layer chromatography) as previously described (32). Lipid phosphorus was determined as reported in Ref. 33.

Phospholipid Hydroperoxides
Phospholipid hydroperoxides were determined by fluorescence HPLC of products formed in MP-11-catalyzed reactions with a fluorogenic substrate, Amplex Red, according to our newly developed protocol (5,34). Oxidized phospholipids were hydrolyzed by porcine pancreatic phospholipase A 2 (0.2 unit/l) in 25 mM phosphate buffer containing 1.0 mM Ca 2ϩ , 0.5 mM EDTA, and 0.5 mM SDS (pH 8.0 at room temperature for 30 min). After that, 50 M Amplex Red and MP-11 (1.0 g/l) were added, and the samples were incubated at 4°C for 40 min. The reaction was terminated by the addition of 100 l of a stop reagent (10 mM HCl, 4 mM butylated hydroxytoluene in ethanol). After centrifugation at 15,000 ϫ g for 5 min, aliquots of supernatant (5 l) were injected into Eclipse XDB-C18 column (5 m, 150 ϫ 4.6 mm). The mobile phase was composed of 25 mM NaH 2 PO 4 , pH 7.0, methanol (60:40, v/v). The flow rate was 1 ml/min. The resorufin (an Amplex Red oxidation product) fluorescence was measured at 590 nm after excitation at 560 nm. Shimadzu LC-100AT vp HPLC system equipped with a fluorescence detector (RF-10Axl) and an autosampler (SIL-10AD vp) was used. The data were processed and stored in digital form with Class-VP software.

ESI Tandem Mass Spectrometry
ESI (electrospray ionization) tandem mass spectrometry of TLCL oxidation products was performed by direct infusion into a Finnigan MAT TSQ 70 mass spectrometer with a triple-quadrupole tandem mass spectrometry analyzer (Thermo Electron Co.). Sheath flow was adjusted to 5 l/min, and the solvent consisted of chloroform:methanol (2:1, v/v). The electrospray probe was operated at a voltage differential of Ϫ3.5 kV in the negative ion mode. Mass spectra for doubly and singly charged CL were obtained by scanning in the range of 400 -950 and 1200 -1800 m/z, respectively, every 1-1.5 s and summing individual spectra. The source temperature was maintained at 70°C.

Statistical Analyses
The data are expressed as the means Ϯ S.D. of at least triplicate determinations. Changes in variables were analyzed by one-way analysis of variance for multiple comparisons. The differences were considered significant at p Ͻ 0.05.

RESULTS
Cyt c Heme Nitrosylation-We used low temperature (77 K) EPR spectroscopy to study the effects of CL on nitrosylation of cyt c. Only weak predominantly hexa-coordinate EPR signal was observed from a system in which cyt c-Fe(II) and an NO donor, DEANOate, were incubated with DOPC liposomes lacking TOCL. The spectrum of hexacoordinate NO-heme complex showed the characteristic shape with rhombic symmetry around the paramagnetic center. There were peaks at g ϭ 2.068 and 1.986 and, in between, an "inverted S shape" (35). Because of the broadness of the peaks and their overlapping, the g values must be considered only as tentative. Incubation of ferro-cyt c with DOPC liposomes containing TOCL (at 1:1 molar ratio) in the presence of DEANOate resulted in a marked increase of the amount of nitrosylated cyt c, as well as the appearance of a signal from a penta-coordinate heme-iron complex. In the presence of TOCL, a new three-line signal with a splitting of 17 G emerged at g ϭ 2.009, and a new low field peak also appeared at g ϭ ϳ2.094. Based on the results presented on Fig. 1, the percentage of penta-coordinate cyt c can be estimated by subtraction of spectrum a from spectrum b or c. The estimate shows that ϳ20% of NO-heme was penta-coordinate at cyt c:TOCL ϭ 15 (TOCL ϭ 1.5 mM, Fig. 1A, panel b), and its content increased to more than 40% at cyt c:TOCL ϭ 40 (TOCL ϭ 4 mM, Fig. 1A, panel c).
Similarly, the incubation of a nitroxyl donor, Angeli's salt (26,36), with ferri-cyt c⅐TOCL complexes caused the formation of typical EPR signals of heme-Fe(II)-nitrosylated species (Fig. 1B). In this case too, the increased amount of nitrosylated cyt c with markedly expressed signal of its penta-coordinate form was observed in the presence of TOCL (Fig.  1B, panels b-d ).
The complex of NO with ferri-cyt c is not detectable by EPR because the NO⅐ferri-porphyrin complex is EPR silent (37). However, optical absorption spectroscopy readily detected the interaction of cyt c-Fe(III) with NO (Fig. 2). The spectrum of ferri-cyt c⅐TOCL complexes showed significant changes after the addition of 100 M of DEANOate (Fig. 2), indicative of the Fe(III)-NO complex formation (15,22,37). The Soret maximum shifted from 409 to 415 nm, and a new peak at 562 nm was observed. The effect was not detectable with solubilized cyt c in the absence of DOPC⅐TOCL liposomes.
Combined, these results demonstrate that CL facilitates binding of NO with cyt c. Thus, heme of cyt c in the complex with CL is accessible for nitrosylation by NO and HNO both in its ferro-and ferri-states.
Redox Interactions of NO and H 2 O 2 with Cyt c⅐CL Complex-The nitrosylation of heme sets the stage for chemical interactions of NO with catalytically active form of the cyt c⅐CL peroxidase complex. This was assessed in EPR experiments in which nitrosylated ferro-cyt c⅐CL was exposed to H 2 O 2 whereby the addition of H 2 O 2 resulted in an almost 80% quenching of the EPR signal of nitrosylated cyt c⅐CL (Fig. 3A). The effect is not likely due to competitive binding to the complex of H 2 O 2 because the latter is a distinctly weaker ligand for ferro-cyt c than NO (38, 39), yet the effect was observed at equimolar concentrations of NO and H 2 O 2 .
Conversely, when H 2 O 2 was incubated with cyt c-Fe(III)⅐CL complexes, a typical EPR signal of protein-derived radicals of the activated peroxidase was induced (Fig. 3B) (3, 40)    ers, and high molecular weight aggregates remaining in the stacking gel (Figs. 4A and 5A). Formation of oligomeric species is predominantly due to recombination of tyrosyl radicals, i.e. via dityrosine covalent linking by formation of stable carbon-carbon bonds. Because the phenoxyl groups are retained in dityrosine, it is likely that dityrosine can be further oxidized by the peroxidase activity, resulting in the formation of other oxidation products such as trityrosine and other polymers.
To further characterize interactions of NO with reactive intermediates of cyt c⅐CL complexes, we utilized a recently developed immunospin trapping technique using antibodies against a spin trap, DMPO. The protocol has been shown to be specific for protein-derived, likely tyrosyl, radicals in different activated hemoproteins (30,41), including cyt c (13).
Using a Western blotting technique with antibodies against DMPO, we found that the antibodies readily interacted with all of the bands corresponding to different oligomeric forms of cyt c (Fig. 4B). This indicates that the presence of the immobilized DMPO nitrone adducts likely formed on tyrosyl radicals of activated cyt c. High molecular weight aggregates contained more DMPO adducts than low molecular weight aggregates. A concentration-dependent decrease of DMPO spin adducts with protein (tyrosyl) radicals was observed in the presence of an NO donor, PAPANONOate (Fig. 4B). Notably, PAPANONOate also caused a dose-dependent inhibition of the formation of high molecular weight aggregates of cyt c and increased the proportion of its monomeric form revealed by PAGE (Figs. 4A and 5A).
To more definitively characterize the involvement of tyrosyl radical intermediates, we performed Western blotting using anti-3-nitrotyrosine antibodies (13,42,43). Oligomeric forms of cyt c, in particular its high molecular mass aggregates, revealed a positive staining for 3-nitrotyrosine when PAPANONOate was added to the incubation system (particularly at high concentrations) (Fig. 5B). In a separate series of low temperature EPR experiments, we confirmed that PAPANONOate was able to quench H 2 O 2 -induced signals of protein-derived radicals, similarly to the effects of DEANOate (Fig. 3B).
Maximal Thus, protein-derived, likely tyrosyl, radicals are produced as reactive intermediates of catalytically competent peroxidase cyt c⅐CL complexes. Because NO interacts with these radical intermediates, we further studied whether NO acted as an inhibitor of peroxidase activity of cyt c⅐CL complexes.
Inhibition of Peroxidase Activity of cyt c⅐CL Complexes-We tested the peroxidase activity of cyt c-Fe(III)⅐CL complexes in the presence and absence of NO donors using two different protocols (Fig. 6). In the first one, we used EPR spectroscopy to monitor the formation of etoposide phenoxyl radicals during one-electron oxidation of etoposide (44). The addition of H 2 O 2 to cyt c⅐TOCL complexes in the presence of etoposide produced a characteristic EPR signal of etoposide phenoxyl radical (Fig.  6A) whose magnitude monotonously increased, reached a plateau after 4 min of recording, and did not decay over a subsequent 5-min period. In the absence of TOCL, the magnitude of the signal was ϳ50 times less. In contrast, preincubation of cyt c⅐CL with DEANOate caused a progressive decrease of the magnitude of the signal (IC 50 , 25 Ϯ 5 M; the corresponding half-inhibiting NO concentration, ϳ4 M). Because the lifetime of etoposide phenoxyl radicals is on the order of several min at M concentrations (45), we demonstrated, in separate experiments, that NO did not directly interact with the etoposide radicals. This was shown as a lack of any effect of DEANOate on the EPR signal of the etoposide radical detectable after its separation from the generating system, horseradish peroxidase/H 2 O 2 , by ultrafiltration/centrifugation. Second, we used cyt c⅐CL-peroxidase-catalyzed oxidation of DAB (28) and found that DEANOate caused inhibition of DAB oxidation in a dose-dependent manner with IC 50 ϭ 10 Ϯ 3 M (the corresponding half-inhibiting NO concentration, ϳ2.5 M) (Fig. 6B). These results demonstrate that NO acts as an effective inhibitor of peroxidase activity of cyt c⅐CL complex.
NO Consumption by cyt c⅐CL Peroxidase-We further assessed consumption of NO during incubation of ferri-cyt c⅐CL complexes using an  NO-selective electrode (13,37). Concentration of NO in solution as measured by NO electrode current initially increased linearly after the addition of DEANOate and then came to saturation as NO production equaled NO consumption as a result of NO interaction with oxygen and cyt c⅐CL peroxidase. An initial increase of NO concentration computed from NO electrode current in the absence of H 2 O 2 agreed very well with the initial NO production value computed from the concentration of DEANOate (0.101 M/s versus 0.105 M/s). A steady-state NO concentration value of ϳ7-8 M in the absence of H 2 O 2 also agrees with the computed NO production rate and the reported bimolecular rate of NO oxidation in the saturation oxygen conditions (k 2 ϭ 10 3 M Ϫ1 s Ϫ1 ) (27). An initial rate of increase of NO concentration and saturation NO concentration value were markedly inhibited upon the addition of cyt c⅐TOCL and H 2 O 2 to the incubation system (Fig. 7). NO consumption in the peroxidase reaction of cyt c⅐CL complex computed from the changes of slopes of initial current increases in Fig. 7 was ϳ0.03 M/s, corresponding to a bimolecular rate constant of peroxidase reaction of ϳ10 M Ϫ1 s Ϫ1 comparable with estimations of the peroxidase activity of cyt c⅐CL complex in similar conditions performed by other methods. H 2 O 2 -induced NO consumption did not take place when cyt c and DOPC liposomes were incubated with DEANOate in the absence of TOCL. At ratios of cyt c:NO Յ 3:1, no significant quenching of NO response was observed in the absence of H 2 O 2 (cyt c:CL ϭ 1:25). At high ratios of cyt c to NO (Ն10:1), a significant inhibition of NO release occurred likely because of direct binding of NO to heme iron (data not shown). Thus, peroxidase function of cyt c⅐CL complex may be viewed as a regulatory mechanism to control NO levels in mitochondria.
Inhibition of Cardiolipin Oxidation-A physiologically important peroxidase function of the cyt c⅐CL complex is its ability to induce peroxidative modifications of CL, particularly important in the execution of an apoptotic program (5). Therefore, we tested whether CL oxidation was affected by NO. Mass spectrometry assessment of TLCL oxidation by cyt c⅐TLCL complexes revealed a significant accumulation of different hydroxy-hydroperoxy-, and hydroxy/hydroperoxy-derivatives of TLCL as evidenced by the appearance of signals with m/z of 731.3, 739.3, 755.3, 755.5, 770.9, and 786.9 as compared with only one signal at m/z 723.8 for nonoxidized doubly charged ion of TLCL. In the presence of PAPANONOate, the signals of oxidized derivatives of TLCL were almost completely suppressed (Fig. 8A). We further employed our newly developed Amplex Red-based protocol with fluorescence HPLC detection of products to quantitatively assess TLCL oxidation by cyt c⅐H 2 O 2 (Fig. 8B). We found that 1 h of incubation of cyt c⅐TLCL in the presence of H 2 O 2 resulted in accumulation of (165 Ϯ 25) pmol CL-OOH/nmol TLCL. When the incubation was performed in the presence of PAPANONOate, only 30 Ϯ 4 pmol CL-OOH/nmol TLCL were formed in the reaction. This indicates that NO acted as an effective inhibitor of TLCL peroxidation catalyzed by the peroxidase activity of cyt c. No nitrated derivatives of CL were observed in mass spectrometry experiments, indicating that quenching of lipid centered radicals by NO is a relatively unimportant antioxidant mechanism compared with NO interaction with oxoferryl reactive intermediates of the peroxidase reactions and quenching of protein-centered radicals.

DISCUSSION
Numerous studies on protein unfolding/refolding employed cyt c as a favorite object (1, 7, 46 -48). These established that the native hexacoordinate state includes axial ligation of Met 80 to the heme that can be ruptured and replaced by His 26 and His 33 under destabilizing conditions (1,47). Five cooperative and hierarchic folding units of different stabilities have been revealed in horse heart cyt c such that substructures of  higher stability are dependent on the unfolding of lower stability substructures whereby destabilization of Met 80 -containing domain displays the lowest stability (46,48). Different physical treatments and chemical modifiers including strong oxidants of Met 80 (hypochlorous acid and singlet oxygen) have been shown to cause destabilization of cyt c resulting in appearance of its peroxidase activity (8,49). One can expect that Met 80 in cyt c⅐CL will be much more exposed to solvent and more prone to interaction with some other oxidants, for example, per-oxynitrite (43,50,51). So, modification of peroxidase activity of cyt c⅐CL complex by peroxynitrite may be due not only to the nitration of tyrosine residues but as well to the oxidation of Met 80 in the cyt c⅐CL complex. Further, several anionic phospholipids, particularly CL, were demonstrated to cause destabilization of cyt c (5,9,10). However, the physiological relevance of these findings remained poorly understood.
We have recently reported that the formation of cyt c⅐CL complexes occurs during apoptosis, resulting in marked enhancement of peroxi- dase activity in mitochondria (5). Notably, the peroxidase activity was directed toward oxidation of CL and accumulation of oxidized CL and stimulated the release of pro-apoptotic factors, including cyt c, from mitochondria. Shidoji et al. (52) demonstrated that CL oxidation products, particularly CL hydroperoxides, did not effectively bind cyt c. Thus, interactions of cyt c with CL play an important role at very early mitochondrial stages of apoptosis because of the selective ability of the complex to catalyze CL oxidation. This raises the question of potential regulation of peroxidase activity of cyt c⅐CL complex in mitochondria.
NO has been known to act as an effective inhibitor of heme-peroxidases via both binding to their heme as well as quenching of the catalytic reactive intermediates, peroxidase compounds I and/or II (53,54) and nitration of reactive protein radicals formed as a result of peroxidase reaction (13,40,43). Our previous studies showed that a dominant mechanism of the antioxidant effect of NO on the peroxidation reaction catalyzed by myoglobin and hemoglobin is NO reduction of oxo-ferryl reactive species (53). We also demonstrated the effectiveness of NO in inhibiting lipid peroxidation dependent on peroxidase activity of myoglobin and hemoglobin in several different types of cells (55,56). NO has been shown to serve as one electron substrate for both Compound I and Compound II for a number of peroxidases such as myeloperoxidase (MPO) (57,58), eosinophil peroxidase, lactoperoxidase (58), and horseradish peroxidase (59,60 HOCl-oxidized cyt c exhibited a higher consumption of NO compared with native protein (13). Thus, it is likely that NO may act as an important regulator of peroxidase activity of the cyt c⅐CL complex in mitochondria.
Although the presence of NO in mitochondria has been established as diffused through membrane or produced by NO synthase (18,20), its role in mitochondrial physiology is less clear. In fact, NO-dependent production of peroxynitrite has been associated with damage of mitochondrial electron transporters (20,24 (21,24). Superoxide is believed to be largely formed toward the matrix close to the location of MnSOD (20). On the other hand, NO may diffuse through the outer mitochondrial membrane. Thus, the sites of origin and the fluxes of the radicals may be spatially separated. Therefore, even at relatively high NO concentrations (ϳ1-2 M), one can expect substantial H 2 O 2 production in mitochondria, allowing for the activation of peroxidase activity of cytochrome c⅐CL complex. Our results provide a reasonable explanation for potential beneficial effects of mitochondrial NO (nitric-oxide synthase) because of its regulatory role in the control of peroxidase activity of cyt c⅐CL complexes in mitochondria.
Our measurements of optical spectra of cyt c⅐CL complexes showed that stable complexes between CL-bound ferri-cyt c and NO are formed at rather high NO concentrations of ϳ8 -10 M, whereas a strong inhibition of lipid peroxidation by cyt c⅐CL is achieved at lower (ϳ2-4 M) concentrations of NO. Thus, Fe-NO complex formation is a relatively unimportant mechanism of NO regulation of peroxidase activity of cyt c⅐CL, compared with the reduction by NO of the peroxidase reactive intermediates: heme-centered compounds I and II and protein-centered radicals generated in the presence of H 2 O 2 or organic (lipid) hydroperoxides. Although stable protein-derived radicals were observed in our EPR experiments, no measurable concentrations of compounds I and II were detected. This is in contrast to other heme proteins displaying peroxidase activity such as myoglobin, horseradish peroxidase, and myeloperoxidase, where long-lived compound I and II species are readily detectable in the presence of H 2 O 2 (53,57,59,60). Apparently, oxoferryl reactive intermediates are very short-lived in the case of cyt c⅐CL catalyzed peroxidase reaction, and their formation is immediately followed by the oxidation of protein-centered (tyrosine) residues in close vicinity of cyt c heme and formation of protein radicals (probably Tyr and Trp centered).
Recently, stimulation of myeloperoxidase by low micromolar NO concentrations was demonstrated (57). MPO exerts specific catalytic and thermodynamic properties (58,63). This peroxidase has very unusual reactivities of compounds I and II. Compound I/compound II couple of MPO has a very high redox potential of ϩ1.35 V and is able to oxidize halide anions to their respective hapohalous acids (63). Compound II/ferric MPO is much less active, and its reduction constitutes the limiting step of MPO peroxidase activity (57,63). Nitric oxide effectively reduces compound II of MPO and thus at low concentrations stimulates peroxidase activity of MPO. There are no indications so far that compounds I and II of cyt c have different reactivities; they are also short lived (they are not observed spectroscopically), possibly because of the effective tyrosine oxidation. Thus, it is unlikely that nitric oxide will have an activation effect on the peroxidase activity of cyt c as has been shown for MPO.
A demonstrated strong inhibitory effect of NO on the peroxidase activity of cyt c⅐CL complex combined with its increased amount in mitochondria suggest that NO may regulate lipid peroxidation and apoptotic activity of CL membrane-bound cyt c during apoptosis. Notably, heme-nitrosylated cyt c has been detected in the cytosol of apoptotic cells (22,23). Moreover, intramitochondrial origin of cyt c nitrosylation has been established along with its sensitivity to expression of Bcl-2 proteins and the importance for apoptosis. However, specific mechanisms involved in nitrosylation of hexa-coordinate heme of cyt c remained controversial. Our results clearly indicate that NO is capable, in the absence of H 2 O 2 or other sources of oxidizing equivalents, to effectively nitrosylate heme in cyt c⅐CL complexes. It is tempting to speculate that formation of such complexes is a prerequisite for the production of heme-nitrosylated cyt c in mitochondria and its subsequent release into the cytosol of apoptotic cells.