Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine

Free Radical Biology & Medicine, Vol. 37, No. 12, pp. 1963–1985, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.08.016

Serial Review: Signaling by Toxicants Serial Review Editor: Henry Jay Forman OXIDATIVE LIPIDOMICS OF APOPTOSIS: REDOX CATALYTIC INTERACTIONS OF CYTOCHROME C WITH CARDIOLIPIN AND PHOSPHATIDYLSERINE Valerian E. Kagan,*,y,z Grigory G. Borisenko,*,§ Yulia Y. Tyurina,*,b Vladimir A. Tyurin,*,b Jianfei Jiang,* Alla I. Potapovich,* Vidisha Kini,* Andrew A. Amoscato,O,** and Yasu Fujii* *Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health; yDepartment of Pharmacology, and Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA; §Research Institute of Physico-Chemical Medicine, 119992 Moscow, Russia; b Sechenov’s Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223 Saint Petersburg, Russia; OMass Spectrometry Facility, University of Pittsburgh Center for Biotechnology and Bioengineering, Pittsburgh, PA 15219, USA; and **Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA

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(Received 17 May 2004; Revised 24 August 2004; Accepted 26 August 2004) Available online 23 September 2004

Abstract — The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c’s equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c’s redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner. D 2004 Elsevier Inc. All rights reserved. Keywords — Cardiolipin, Phosphatidylserine, Cytochrome c, Peroxidation, Peroxidase activity of cytochrome c, Apoptosis, Free radicals Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of cytochrome c with anionic phospholipids. . . . . . . . . . . . . Electrostatic and hydrophobic interactions. . . . . . . . . . . . . . . . . . . Functional consequences: antioxidant and peroxidase activities of cyt c . . . Reactivity of solubilized (water-soluble) cyt c toward hydrogen peroxide.

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This article is part of a series of reviews on bSignaling by Toxicants.Q The full list of papers may be found on the home page of the journal. Address correspondence to: V.E. Kagan, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15260, USA; Fax: +1 412 383 2123; E-mail: [email protected]. 1963

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Peroxidase activity of modified cyt c . . . . . . . . . . . . . . . . . . . . . . . . Peroxidase activity of membrane-bound (lipid-bound) cyt c . . . . . . . . . . . . . PS and CL hydroperoxides in apoptosis: suicidal nature of cyt c as a peroxidase . . Effect of protein-membrane binding on the electron transport function, antioxidant activity of cyt c, and disruption of the Met80-Fe bond . . . . . . . . . . . . . . Mitochondrial cytochrome c and its interactions with cardiolipin. . . . . . . . . . . . . . CL-bound cyt c: mitochondrial stress sensor and redox regulator of apoptosis . . . . . Two pools of cyt c and two pools of CL . . . . . . . . . . . . . . . . . . . . . . . . Oxidation of CL and its role in cyt c release . . . . . . . . . . . . . . . . . . . . . . Regulation of cyt c peroxidase activity: the role of mitochondrial nitric oxide synthase (mtNOS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytochrome c in the cytosol and its interactions with phosphatidylserine . . . . . . . . . PS/cyt c as a catalyst of PS oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . PS/cyt c complex as a signal for PS externalization . . . . . . . . . . . . . . . . . . . Recognition of PS and PSox and phagocytosis of apoptotic cells . . . . . . . . . . . . Effects of PS and PSox on phagocyte cytokine and ROS production and inflammatory response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-oxidant regulation of apoptosis: blockage of phospholipid oxidative signaling pathways. Can we selectively prevent CL and/or PS oxidation? . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

Apoptosis—ingeniously orchestrated cell death— involves a genetically predetermined chain of events that leads to disassembly of intracellular structures and macromolecules and culminates in selective clearance of unwanted or irreparably damaged cells by phagocytes. Mitochondria are key regulators in both intrinsic (initiated by endogenous death signals) and extrinsic (triggered by toxic damaging signals) pathways of apoptosis. Cytochrome c (cyt c), a relatively simple component of the mitochondrial electron transport system, turns into a dreadful soloist when it finds itself in the new cytosolic environment, where it performs a well-characterized initiation of apoptosome formation and activation of caspase-dependent apoptotic processes (reviewed in [1–3]). Although the presence of heme moiety of cyt c is essential for these functions, cyt c’s redox catalytic features are not required, and its Znsubstituted homologs are as effective as those that contain Fe [4]. Recently, two other essential functions of cyt c in apoptosis that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria [5,6] and phosphatidylserine (PS) in the plasma membrane [7]. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that

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facilitates release of cyt c (along with several other pro-apoptotic factors) into the cytosol [8–10] and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes [11,12]. Although associations between cyt c and these essential apoptotic pathways of phospholipids have been established, specific mechanisms of cyt c’s catalysis of their oxidation remain unknown. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids results in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. This new concept is compatible with a leading role for reactive oxygen species (ROS) in the execution of the apoptotic program, with cyt c as the main executioner. INTERACTIONS OF CYTOCHROME C WITH ANIONIC PHOSPHOLIPIDS

Electrostatic and hydrophobic interactions Cyt c is a peripheral membrane protein of the inner mitochondrial membrane; it is composed of a single polypeptide chain of 104 amino acid residues and a covalently attached heme group and is roughly spherical, with a diameter of 34 2. Its heme group is surrounded by tightly packed hydrophobic side chains, and there is a hydrophobic channel from the protein surface to the

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heme crevice [13]. At the opening of this hydrophobic channel on the protein surface, a highly conserved cluster of positively charged lysines is opposed by invariant Asn52, capable of forming two hydrogen bonds with the protonated phosphate [13]. Overall, at physiologic pH, cyt c is a basic protein with eight noncompensated positive charges [14]. Consequently, its interactions with membrane lipids occur primarily through binding to anionic (acidic) phospholipids [15]. As shown in Fig. 1, binding of cyt c with anionic phospholipids, such as PS and CL, remarkably changes the electrophoretic behavior of cyt c/liposome complexes, such that migration of cyt c to the cathode is no longer possible. In contrast, the electrophoretic mobility of cyt c/phospholipid liposomes is not affected in the presence of the phospholipid phosphatidylcholine (PC), which does not carry a net negative charge. Attachment of cyt c to membranes containing anionic phospholipids involves electrostatic and hydrophobic interactions and hydrogen bonding [16]. Several hypotheses to describe these interactions have been put forward: (i) cyt c binds to membrane without penetration, (ii) cyt c penetrates into phospholipid bilayer to a different extent [16], or (iii) vice versa, one of the phospholipid acyl chains penetrates into cyt c (bextended lipid anchorageQ) [17]. Based on quenching of fluorescent-pyrene-labeled liposomes by cyt c or quenching of fluorescent-Zn2+-substituted heme by brominated lipids, existence of two binding sites for anionic phospholipids on cyt c—one of which facilitates electrostatic interactions (A site), while another entails hydrophobic forces (C site)—has been proposed [14,18,19]. The most current and commonly accepted model suggests that

cyt c’s interactions with membranes induce significant conformational changes, which include disturbance of hexa-coordinated heme iron bonds in the protein, opening of heme crevice, and partial unfolding of the protein accompanied by the adjustment of the hydrophobic face of amphipathic a helices toward the hydrocarbon core of the lipid bilayer [20–22]. The model accommodates data on spectral changes of the protein upon binding to various phospholipid vesicles that are reminiscent of a well-known cyt c unfolding induced by acids or denaturing agents. Membrane-associated cyt c is likely to have a disrupted packing of core side chains and heme crevice. Enhanced fluorescence of Trp59 suggests a shift of the corresponding loop out of the heme by ~20 2, which is accompanied by accommodation of Trp59 in the hydrophobic lipid environment [22]. Conformational circular dichroism analysis demonstrated that tight binding of cyt c decreases its a-helical content (70–79%) and increases its h sheet up to 135%, indicating that not only tertiary but also secondary structure of the protein is affected by its interaction with membranes [21]. Finally, analysis of H-2H1 exchange in lipid-bound cyt c revealed that a large group of amides (28 of the 42 core amides) exchange via a highly concerted global unfolding transition that exposes all these otherwise protected protons to solvent [23]. Overall, binding to anionic phospholipids (particularly CL) causes profound changes in cyt c structure induced by electrostatic interactions mediated by the A site (primarily of conserved Lys at positions 72 and 73 with deprotonated phosphate group) and simultaneous hydrophobic interactions at the C site that facilitate hydrogen bonding of invariant Asn52 with the protonated phosphate group [14,17–19]. The electrostatic interactions with anionic phospholipids at the A site can be reversed by increased ionic strength and do not involve pronounced conformational changes in the protein [17]. In contrast, the C-site-mediated association of cyt c with anionic phospholipids is not sensitive to increasing ionic strength [17] and causes conformational changes in the protein and disruption of the S-Fe-coordination bond between Met80 and heme iron [24,25]. The latter is consistent with EPR studies showing that membranebound cyt c has an altered high spin state of heme Fe3+ [25]. The striking changes in cyt c structure incurred by its tight association with membranes are also manifested in its functional properties, including its redox behavior as discussed below.

Fig. 1. Anionic phospholipids (CL, PS) but not phosphatidylcholine bind with cyt c and affect its migration during native PAGE. Cyt c (80 AM) was incubated with small unilamellar phospholipid liposomes (4 mM, DOPC, DOPC:DOPS = 1:1 or DOPC:CL = 1:1) for 60 min; 5 Ag of cyt c was loaded onto a 6% native PAGE (pH 7.0). DOPC, 1,2dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero3-phospho-l-serine; CL, 1,1V,2,2V-tetraoleoyl cardiolipin.

Functional consequences: antioxidant and peroxidase activities of cyt c Reactivity of solubilized (water-soluble) cyt c toward hydrogen peroxide. Redox-active, heme-containing proteins are frequent suspects as inducers of oxidative

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stress because of their propensities to produce free radicals and to oxidize important biomolecules in peroxidase-like reactions, through which heme-containing peroxidases catalyze two-electron reduction of hydrogen peroxide into water (and organic peroxides into alcohols) (see Scheme 1). During this reaction, heme is activated to form compound I (a highly oxidized Fe(V) state/oxoferryl S heme with porphyrin radical cation, Fe(IV)jO Por +), which can subtract electrons from various substrates (usually through two, one-electron steps involving the formation of intermediate compound II with Fe(IV)/oxoferryl heme, Fe(IV)jO) and subsequently return to a resting state (Fe(III)) [reviewed in 26]. Solubilized (membrane-free) cyt c is a notable exception among heme-containing proteins in that it reacts very slowly with peroxides (0.2 M1 s1 at pH 7.0) [27]. All six coordination positions in heme iron of cyt c are occupied by ligands that prevent its interaction with peroxides [28,29]. As a result, the reaction rate of H2O2 with cyt c is 3 orders of magnitude lower than that with Mb, which contains penta-coordinated heme iron (102 M1 s1), and 8 orders of magnitude lower than that with the classical peroxidases, horseradish peroxidase and myeloperoxidase (~107 M1 s1). In classical peroxidases, not only is the sixth coordination bond of heme iron available for binding peroxides but also the heme environment favors cleavage of the O-O bond [26,30]. In mitochondria, cyt c oxidase, whose content is only slightly lower than that of cyt c, reacts with H2O2 with a constant rate of 10 3 M 1 s 1 [31]. Not surprisingly, cyt c K m for H2O2 is about 70 mM, while for cyt c oxidase it is ~1.2 mM [27,32]. Thus, solubilized cyt c is not a strong competitor with other hemoproteins for H2O2-dependent peroxidase reactions. These poor qualifications of cyt c as a peroxidase not only shed some doubt on the potential biological significance of this activity but also prompted speculation that cyt c functions as an antioxidant enzyme mostly by efficiently converting superoxide into molecular O2 [33]. Considering that cyt c concentration in the intermembrane space exceeds 0.5 mM, it may function as a protective "antioxidant shell" around mitochondria. The ability and contribution of cyt c into removal of H2O2 and oxidation of intramitochondrial reductants was not seriously considered. Despite solubilized cyt cTs seemingly incompatible attributes toward pro-oxidant activity, the pro-oxidant activity of cyt c has been documented in a number of studies in model systems. More than a decade ago, peroxide-induced oxidation of lipids in mitochondria was unequivocally shown to depend on the presence of cyt c [34]. Moreover, several recent reports documented that cyt c can promote peroxide-dependent oxidation of ascorbate, glutathione, NADH, phenols, peptides, and

proteins [35–37] and generate fatty acid hydroperoxide radicals [38]. Although the mechanisms of cyt ccatalyzed pro-oxidant activity are not fully understood, cyt c’s catalytic pro-oxidant role in the presence of H2O2 and organic peroxides is generally accepted as a peroxidase-like activity.1 What are the factors and conditions that convert solubilized cyt c from an antioxidant enzyme to a pro-oxidant peroxidase? In this review, we present evidence that the tightly and hydrophobically membrane-bound fraction of mitochondrial cyt c is markedly distinct in properties from its solubilized form and represents a peroxidase that may act as a mechanism for destruction of mitochondria that is triggered by overproduction of superoxide/H2O2 resulting from disrupted electron transport. We speculate that this mechanism is critical to initiating an intrinsic apoptotic program and to fulfillment of the PS-signaling apoptotic pathway. Peroxidase activity of modified cyt c. In cyt c, heme iron has two axial bonds: one with His17 on the proximal side of heme and one with Met80 on the distal side. The bond with Met80 impedes iron interactions with H2O2. However, different conditions resulting in the dissociation of the Met80 bond (e.g., pH shifts and temperature changes) significantly affect cyt c’s properties. Modifications of cyt c, leading to disruption of Fe-Met80 bond, induce peroxidase activity; oxidation of Met80 by HOCl, nitration of Tyr67 by ONOO (which promotes Fe-Met bond disruption), or carboxy-methylation of Met80 displace Met80, disrupt its interactions with iron, and facilitate reactivity of the iron [39–41]. For example, carboxy-methylation of cyt c increased the rate of its reaction with H2O2 by ~60 times, decreased K m to 25 mM (pH 7.0), and stimulated H2O2-dependent oxidation of different substrates. Acidic conditions, known to induce unfolding of the protein, further amplified peroxidase activity of carboxy-methylated cyt c: at pH 3, the reaction rate of carboxy-methylated cyt c with H2O2 exceeded the rate of native protein by three orders of magnitude [41]. However, it is not quite clear whether

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In addition to its oxidation of substrates associated with consumption of peroxides, cyt c can oxidize unsaturated lipid acyl chains, with generation of lipid radicals that subsequently form lipid peroxides upon addition of oxygen [24,34]. In these circumstances, cyt cdependent lipid peroxidation was referred to as peroxidase activity of cyt c [24]. However, the role of peroxides in the mechanism of these reactions has not been shown. In addition, peroxides were produced during these reactions; this is contrary to bclassicalQ peroxidasedependent processes during which peroxides are consumed. Thus, despite the semantic similarity of the terms peroxidase and peroxidation, we adhere to using the classical meaning of the term peroxidase activity. (Note that cyt c-induced lipid peroxidation could occur as a result of cyt cTs peroxidase activity).

Cytochrome c and apoptotic peroxidation

disruption of Fe-Met bond is required for activation of cyt c to a peroxidase by anionic phospholipids. It is possible that conformational changes and markedly increased accessibility of cyt c’s heme to H2O2 induced by CL/PS binding is sufficient for activation of its peroxidase function. Peroxidase activity of membrane-bound (lipid-bound) cyt c. The role of cyt c as a redox shuttle carrying electrons between complexes III and IV of mitochondria is entirely compatible with the occupancy of all six of its heme coordination bonds which hampers binding and activation of any small molecules (such as oxygen, H2O2, NO, etc.). Reportedly, interaction and binding of cyt c to negatively charged PS or CL in liposomes or membranes results in rupture of the Fe-Met80 bond following partial unfolding of the protein; this causes enhancement of the peroxidase activity of cyt c. Whatever the detailed mechanism is, such enhancement was directly observed in two independent series of experiments in which we assessed the peroxidase activity of cyt c toward two different substrates: 2V,7Vdichlorodihydrofluorescein (DCFH2) (evidenced by its oxidation to a fluorescent 2V,7V-dichlorofluorescein (DCF)) and etoposide (by the EPR detection of its one-electron oxidation intermediate, etoposide phenoxyl radical). We found that the rate of DCFH2 oxidation by cyt c/H2O2 in the presence of PS-containing liposomes was increased 3-fold relative to its oxidation rate with solubilized cyt c; CL-containing liposomes facilitated DCFH2 oxidation 5-fold (Fig. 2) (V. Kini et al., unpublished observations). Similarly, the magnitude of etoposide phenoxyl radical generated by cyt c/H2O2 in the presence of PS-or CL-containing liposomes was 10and 20-fold greater, respectively, than that without liposomes or with PC-containing liposomes (Osipov et al., unpublished observations). In addition, binding to liposomes facilitated the formation of protein-centered (tyrosyl) radicals upon addition of H2O2. Formation of transient protein-based radicals is a common feature of heme-containing proteins treated with oxidants. Usually, compound I formed after reaction with H2O2 triggers intramolecular electron transfer from a nearby tyrosine or tryptophan residue to the porphyrin, generating a corresponding amino acid radical. Such tyrosyl radical was detected on cyt c by the spin-trapping technique [42]. With solubilized protein, this highly unstable tyrosyl radical was barely observable by direct lowtemperature EPR spectroscopy but became prominent when cyt c was bound to PS and (especially) CL liposomes (Fig. 3). Interestingly, the relative intensities of protein-centered signals were proportionally similar to the rates of DCFH2 oxidation (cyt c:cyt c-PS:cyt cCL = 1:3:10).

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It is tempting to speculate that both PS- and CL-bound cyt c represent posttranslational modifications of the heme protein that can direct its redox activity toward oxidation of proteins and lipids to yield products that elicit proapoptotic behavior. In fact, our preliminary experiments indicate that incubation of cyt c and H2O2 with dioleoylPC (DOPC)/arachidonyloleoyl-PS (AOPS)-containing liposomes produced ~7 pmol lipid hydroperoxides per Ag Pi while essentially no oxidation was observed upon incubation of a mixture of DOPC/AOPC liposomes under the same conditions (V.A. Tyurin et al., unpublished results). Moreover, DOPS/CL liposomes also underwent a very strong oxidation in the presence of cyt c/H2O2. This emphasizes cyt c peroxidase activityTs tremendous potential for catalyzing lipid peroxidation, including selective peroxidation of PS and CL during apoptosis. In fact, incorporation of cyt c into human myeloid HL-60 cells or into cyt c null (cyt c /) mouse embryonic cells remarkably enhanced oxidation of PS [43]. Presumably, highly oxidized heme (compound I or II) and proteinbased tyrosyl radical formed in the presence of H2O2 in PS-or CL-activated peroxidase forms of cyt c are capable of subtracting an electron from unsaturated acyl chains of phospholipids followed by the addition of oxygen and formation of lipid hydroperoxide, similar to peroxidase reactions of cyclooxygenases (reviewed in [44,45]). Obviously, newly formed lipid hydroperoxides may function as substrates for cyt c peroxidase activity and further propagate lipid peroxidation, even in the absence of H2O2 (Scheme 1, inset). Determining specific features of these cyt c-catalyzed peroxidase reactions of lipid oxidation is essential. Of particular importance is the extent to which PS and/or CL that are directly involved in cyt c activation and conversion into a peroxidase can be simultaneously oxidized and whether these peroxidized molecular species of PS and CL are still capable of interacting with cyt c in ways that sustain its peroxidase activity. PS and CL hydroperoxides in apoptosis: suicidal nature of cyt c as a peroxidase. Hydroperoxide-driven activation and formation of reactive intermediates of peroxidases are usually suicidal to the protein [45,46]. Whereas any suicidal interactions can hardly support the uninterrupted shuttling function of cyt c in mitochondrial electron transport, cyt c’s peroxidase-based role in execution of apoptotic process can be regulated by its suicidal substrates. Indeed, H2O2 caused destruction of cyt c as evidenced by decreased absorbance in the Soret band of the optical spectrum of cyt c and the appearance of a new EPR signal with g = 4.3. Both of these features signal significant disturbance in the heme environment and probably account for the decomposition of the tertiary protein structure. This destructive effect of

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possess only one third of the electron transport activity of electrostatically bound cyt c [49]. Moreover, the CLS bound cyt c would not be able to convert O2 – into O2, rendering it unable to fulfill an important superoxidescavenging antioxidant function in the intermembrane space of mitochondria [33]. These considerations lead us to suggest that mitochondria contain different pools of cyt c, each with distinctively different functions that are determined by the specific type of interaction of cyt c with CL. It is also likely that molecular species of CL, which differ in their fatty acid composition, are important determinants of CL/cyt c interactions (see below). MITOCHONDRIAL CYTOCHROME C AND ITS INTERACTIONS WITH CARDIOLIPIN Fig. 2. Binding of anionic phospholipids (CL, PS) but not PC confers peroxidase activity onto cyt c as evidenced by oxidation of DCFH2 to DCF. Cyt c (2 AM) was preincubated without (1) or with small unilamellar phospholipid liposomes [100 AM, DOPC (2); DOPC:DOPS, 1:1 (3); DOPC:TOCL, 1:1 (4)] for 1 h at 25 8C in 10 mM phosphate buffer, pH 7.0, and then H2O2 (10 AM) and DCFH2 (10 AM) were added for 5 min incubation. Peroxidase activity of cyt c was evaluated by fluorescent monitoring of the formation of DCF (excitation, 502 nm; emission, 522 nm). Data shown are representative of three independent experiments.

H2O2 was more pronounced in PS-and CL-bound cyt c relative to its solubilized form. Reducing substrates, such as phenol or ascorbate, prevent protein destruction in phospholipid-bound cyt c, suggesting that the bselfdestructiveQ behavior may have a regulatory role. Indeed, if polyunsaturated phospholipids are among the major substrates for peroxidase activity of cyt c, then loss of enzyme activity may be viewed as an important factor that regulates excessive oxidation of phospholipids during controlled execution of apoptosis. Effect of protein-membrane binding on the electron transport function, antioxidant activity of cyt c, and disruption of the Met80-Fe bond. Conformational changes in the protein may be associated with a significant shift of cyt c’s redox properties [47]. For carboxymethylated cyt c, the redox potential of the cyt c 2+/cyt c 3+ couple is more negative (reducing) than that for the native protein by 500 mV [48]. If binding of cyt c to CL in mitochondria and subsequent loosening of Met80-heme bond results in changes of cyt c redox properties similar to those of the carboxy-methylated protein, then this phospholipid-bound cyt c could not effectively function as an electron acceptor from complex III. Indeed, tightly membrane-bound cyt c in mitochondria has been shown to

CL-bound cyt c: mitochondrial stress sensor and redox regulator of apoptosis In mitochondria, most of cyt c is membrane bound. Of this, at least 15% is tightly bound protein (due to both electrostatic and hydrophobic interactions); the remaining cyt c is loosely bound—likely interacting with the membrane electrostatically and readily detached from the membrane by high ionic salt solutions, e.g., KCl [16]. As mentioned previously, loosely bound and solubilized cyt c, on the one hand, and tightly bound cyt c on the other, carry out different tasks: The former participates in S electron transport and O2 – scavenging, i.e., it inhibits ROS formation and development of oxidative stress. The latter CL-bound cyt c is not a likely participant of electron transport but rather is responsible for the peroxidase activity that eliminates H2O2 formed in mitochondria and prevents H2O2 diffusion into the cytosol. However, this antioxidant function can be effectively accomplished only at the expense of oxidation of other reductants such as ascorbate, GSH, phenolic compounds, and vital mitochondrial constitutents (such as proteins and polyunsaturated phospholipids). Normally generated amounts of H2O2 can be handled by cyt c along with other antioxidant defense systems (such as catalase, GSH peroxidases, thioredoxins, and peroxiredoxins) [50–53]. However, this mechanism can significantly change during states of excessive oxidative stress. For instance, intrinsic and extrinsic apoptotic stimuli send signals to mitochondria which disrupt electron transport, yielding large-scale production of S O2 –, H2O2, and/or ONOO. The latter two species can be sources of oxidative equivalents for the peroxidase activity of CL-bound cyt c, which can readily induce extensive peroxidation of CL in the inner mitochondrial

Scheme 1. Schematic representation of cyt c functions in mitochondria: pools participating in electron transport and acting as a peroxidase activated during apoptosis are shown. Insert depicts the formation of oxoferryl Fe(IV)jO form of cyt c by H2O2 or CL hydroperoxides (CL-OOH).

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Fig. 3. EPR spectra of protein-derived radicals formed from cyt c bound to anionic phospholipids (CL or PS) upon incubation with H2O2. Cyt c 1 mM was incubated with or without small unilamellar liposomes for 1 h at 25 8C in 10 mM Hepes buffer, pH 7.0, then H2O2 (2 mM) was added. Reaction of cyt c with H2O2 was stopped after 10 s by freezing the samples in liquid nitrogen. EPR spectra from frozen samples were detected at 77 K under the following conditions: 3230 G, center field; 100 G, sweep width; 1 G, field modulation; 0.2 mW, microwave power; 0.3 s, time constant; 2 min, time scan. Liposomes were prepared either from dioleoylphosphatidylcholine (DOPC), from DOPC and dioleoylphosphatidylserine (DOPS), or from DOPC/TOCL at a ratio of 1:1 using sonication. The final phospholipid concentration was 50 mM.

membrane (Scheme 1). The consequences of CL oxidation are twofold: First, oxidized CL, in contrast to nonoxidized CL, does not tightly bind cyt c; hence, it cannot retain cyt c in the inner membrane. Rather the oxidized CL releases the cyt c into intermembrane space. Second, CL oxidation is important for the formation of the mitochondrial permeability transition pore (MPTP) [8–10]. Thus, CL-bound cyt c functions as a peroxidase, transducing and amplifying apoptotic signals that arrive at mitochondrial electron transfer complexes and produce an excess of oxidants (H2O2 or ONOO). Characteristically, CL-bound cyt c also induces its own detachment from the inner membrane and release from mitochondria through MPTP by producing oxidized CL catalyzed by cyt c’s own peroxidase activity (Scheme 1). While this chain of events elucidates features of CL oxidation, it does not reveal the pathways through which CL regulates peroxidase activity of the specific pool of cyt c that is tightly bound to the inner mitochondrial membrane. Two pools of cyt c and two pools of CL The most fully characterized function of cyt c in mitochondria is that of an electron carrier of the

mitochondrial respiration chain between complexes III (ubiquinone-cytochrome c oxidoreductase, 11 subunits) and IV (cytochrome c oxidase, 13 subunits) [54–56]. Complexes III and IV associate to form a supercomplex whose functional activity requires an inner mitochondrial membrane lipid CL [57–63]. Complexes III and IV generate an electrochemical proton gradient across the inner membrane where CL may act as a proton trap [64]. CL is the most essential component for the assembly of complexes III and IV [60]. Eight to 9 molecules of CL are tightly bound to complex III [57,65] and 25 to 30 molecules to complex IV purified from bovine heart tissue [66]. CL-enriched domains and its protein complexes may be assembled into patches and rafts [21,64]. Deficiency of CL leads to alteration in the stability of mitochondrial membranes, to increased permeability, to decreased respiratory rate, and to totally dysfunctional mitochondria [61–63]. CL is found exclusively in the inner mitochondrial membrane where it is accountable for 25% of all phospholipids [67]; it is predominantly confined to the membrane [68] but small amounts of it may be present in the outer layer of the outer membrane [69]. This translocation of CL from the inner to the outer mitochondrial membrane reportedly occurs due to the activity of phospholipid scramblase 3 [70]. There are two major pools of CL in mitochondria—loosely bound CL and CL that is tightly bound with proteins [5]. Through these pools, CL realizes the stabilization of physical properties of membranes and specific interactions with proteins and complexes [57,60,65,66]. In addition to its role in the functioning of complexes III and IV, CL is also essential for normal operation of other mitochondrial complexes (adenine nucleotide translocase, CL synthase, and tricarboxylate, pyruvate, and phosphate carriers) [71]. Within the context of this review, we will further focus on the interactions of CL with cyt c as they relate to cyt c function as a peroxidase during apoptosis. In addition to a small fraction of solubilized cyt c in mitochondria, the majority of it is membrane bound [72]. The binding of cyt c to the inner membrane can be loose or tight [16,21,73]. Tightly membrane bound cyt c is likely to owe its association with the membrane to CL. In fact, CL is necessary for cyt cTs insertion into mitochondrial membrane [74]. The majority of mitochondrial CL (about 70%) is represented by its molecular species containing linoleoyl/ oleoyl fatty acid residues [74]. There is almost a 70-fold excess of CL available for 1:1 stoichiometric binding with cyt c, yet the question of why the inner membrane contains only 5 to 15% of tightly bound mitochondrial cyt c is still unclear (Kagan et al., unpublished results). It has been shown that cyt c binds with higher affinity to

Cytochrome c and apoptotic peroxidation

the membranes containing unsaturated CL than to those containing saturated CL [75]. It is tempting to speculate that a relatively minor fraction of CL molecular species containing more unsaturated and longer-chain fatty acid residues (C20:4 and C22:6) are mostly involved in specific interactions with cyt c in the inner membrane, resulting in its tight binding. To this end, we attempted to perform MS analysis of CL molecular species. MS analysis of phospholipid mixtures does not usually provide quantitative information on their abundance. Within a given species of phospholipids, the relative intensities of CL signals are approximately proportional to the abundance [77]. However, it is recognized that all species may not ionize to the same degree, therefore making the protocol semiquantitative at best. With these limitations, we performed MS analysis of molecular species of CL (Fig. 4), and relative intensities of MS signals are shown in Table 1. We found that about 30% of CL molecular species in mouse heart mitochondria contain C20:4 and C22:6 (Table 1). This is consistent with data showing that alterations of fatty acid composition of CL affect the

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activity of electron transport complexes [78]. The hypothesis also helps to rationalize the high oxidizability of CL by peroxidase activity of cyt c and cyt cTs detachment from peroxidized CL in the membrane and release into the cytosol during apoptosis (Scheme 1). In fact, CL was reported to cause a drastic broadening of the linewidth of the downfield signals in 1H NMR spectra at 31.4 and 34.2 ppm assigned to cyt cTs heme methyl group-3 and-8, respectively [75]. Furthermore, oxidation of CL disrupted its interactions with cyt c as evidenced by resolution of the NMR spectra. Interestingly, monoand dihydroperoxides of CL were less effective in broadening the signals than nonoxidized CL, and CL trihydroperoxides induced almost no broadening of the linewidth of the signals [75]. There appears to be a critical link between changes in CL mitochondrial topography and oxidation and detachment and release of cyt c during apoptosis. CL translocation from the inner to the outer mitochondrial membrane has been demonstrated using two models— staurosporine-treated HL-60 cells and TNF-a-treated

Fig. 4. Representative mass spectrum of mouse heart cardiolipin. Lipids were analyzed by electrospray ionization tandem mass spectrometry by direct infusion into a triple-quadrupole mass spectrometer (Micromass, Inc., Manchester, England). Sheath flow was adjusted to 5 Al/min and the solvent consisted of chloroform:methanol (1:2, v/v). The electrospray probe was operated at a voltage differential of 3.5 keV in the negative-ion mode. Mass spectra for the (M-H)-CL species were obtained by scanning in the range of 1200–1800 m/z every 1.5 s and summing individual spectra. In addition, scans in the range of 400–950 m/z were also taken for any doubly charged CL species. Source temperature was maintained at 708C.

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Table 1. Fatty Acid Composition of Different Molecular Species of CL from Mouse Heart Mitochondria

osporine, as evidenced by caspase activation and PS externalization (Fig. 5).

CL Species, m/z

Fatty acid chain analysis

Oxidation of CL and its role in cyt c release

1422 1424 1426 1428 1448 1450 1452 1454 1470 1472 1474 1476 1478 1480 1496 1498 1500 1544

(C18:2)3/(C16:1)1 (C18:2)3/(C16:0)1 (C18:2)2/(C18:1)1/(C16:0)1 (C18:2)1/(C18:1)2/(C16:0)1 (C18:2)3/(C18:1)1 (C18:2)2/(C18:1)2 (C18:2)1/(C18:1)3 (C18:1)4 (C18:3)2/(C18:2)1/(C20:3)1 (C18:3)1/(C18:2)2/(C20:3)1 (C18:2)2/(C18:1)1/(C20:4)1 (C18:2)1/(C18:1)2/(C20:4)1 (C18:1)3/(C20:4)1 (C18:0)1/(C18:1)2/(C20:4)1 (C18:2)3/(C22:6)1 (C18:2)2/(C18:1)1/(C22:6)1 (C18:2)1/(C18:1)2/(C22:6)1 (C18:2)2/(C22:6)2

% of total CL 1.85 1.11 1.63 1.47 18.79 18.32 6.95 2.53 8.29 9.23 6.95 2.91 1.27 1.30 7.86 4.20 1.90 3.36

Note. Lipids were analyzed by electrospray ionization mass spectrometry by direct infusion into a triple-quadrupole mass spectrometer (see legend for Fig. 4). Tandem mass spectrometry (MS/MS analysis) of individual mitochondrial CL species was used to determine the fatty acyl chain composition. Collision-induced dissociation spectra were obtained by selecting the ion of interest and performing daughter ion scanning in Q3 at 400 m/z using Ar as the collision gas. The mass spectrometer was operated at unit resolution for MS scans and slightly below unit resolution for MS/MS scans. The percentage of each species was determined based on total ion current.

U937 cells. Importantly, CL migration occurred prior to any detectable changes in mitochondrial membrane potential, plasma membrane exposure of PS, and DNA fragmentation but after the production of ROS [79]. During reoxygenation, simultaneous release of cyt c and CL occurs in cerebral mitochondria [80]. Recent data indicate that mitochondria-mediated ROS production affects the activities of complexes III and IV of the mitochondrial respiratory chain through oxidative damage of CL, but the role of cyt c/CL interactions in this process has not been established [81]. If the hypothesis that polyunsaturated molecular species of CL tightly bound to cyt c are those that primarily undergo oxidation catalyzed by peroxidase activity of cyt c is correct, one would expect that dietary manipulations of CL fatty acid composition should affect the sensitivity of cells to apoptosis. Indeed, increased levels of docosahexaenoic acid (C22:6) in CL (up to 48 mol%) in HT-29 cells grown in the presence of this fatty acid resulted in a significantly increased sensitivity of mitochondrial membrane potential and their production of oxidants in the cells [82]. Our preliminary experiments with HL-60 cells grown in the presence of docosahexaenoic acid showed that enrichment of CL with this highly unsaturated fatty acid conferred an enhanced sensitivity of the cells to apoptosis induced by staur-

Nonoxidized, polyunsaturated CL is important in the mitochondrial bioenergetics, optimizing the activity of key mitochondrial proteins [83]. However, during apoptosis, CL migrates from the inner to the outer mitochondrial membrane where it seems to be essential for cyt c release from the mitochondria. The mechanism of release of cyt c—a critical event in propagation of apoptosis—remains unclear. It is likely that CL participates in both essential steps related to cyt c release: the detachment of cyt c from the inner membrane and the formation of a permeability transition pore [81,84,85]. Release may occur in two steps, wherein cyt c is first dissociated from CL and then is let loose into the extramitochondrial environment. The movement of cyt c from the mitochondria to the outside requires pro-

Fig. 5. Caspase-3 activity (A) and PS externalization (B) during apoptosis induced by staurosporine in docosahexaenoic acid-pretreated HL-60 cells. HL-60 cells were preincubated with or without 10 AM docosahexaenoic acid for 1 h at 37 8C, then cells were incubated in the absence or presence of 0.4 AM staurosporine for the indicated time. Caspase-3 activity was measured by using an EnzChek Caspase-3 Assay Kit (Molecular Probe); PS externalization was determined by annexin VFITC binding assay. Open squares, control; open circles, docosahexaenoic acid; closed squares, staurosporine; closed circles, docosahexaenoic acid + staurosporine. Data points represent the mean F SE, n = 3. *p b 0.05; **p b 0.01 vs staurosporine alone at the same time point.

Cytochrome c and apoptotic peroxidation

apoptotic proteins Bid and Bax to permeabilize the outer mitochondrial membrane without actually disrupting the membrane [85]. The dissociation of cyt c from CL is achieved by the breach of their electrostatic/hydrophobic interactions [75] likely caused by peroxidation and the appearance of polar hydroperoxide groups in one or more of CL’s polyunsaturated fatty acid residues [75]. The presence of hydroperoxide groups in CL is essential for the disruption of its binding with cyt c, as CL with a hydroxygroup in the fatty acid residue did not cause the dissociation of cyt c but was able to bind to cyt c to the same extent as can intact CL [86]. Peroxidase activity of cyt c/CL complex utilizes CL as its substrate, yielding CL hydroperoxides. The latter can be utilized by the complex as a source of oxidizing equivalents to further propagate CL oxidation (Scheme 1, inset). Thus, the selfpropagated accumulation of CL hydroperoxides should inevitably cause increased detachment of cyt c from CLdependent binding sites on the inner mitochondria membrane. Because CL hydroperoxides are suicidal substrates for cyt c/CL peroxidase activity destruction of cyt c occurs in the course of the reaction. This oxidative modification of cyt c can be also accountable, at least in part, for dissociation of cyt c/CL complex. If generation of CL-hydroperoxides is a prerequirement for the liberation of cyt c [87], the mechanisms responsible for CL oxidation are likely to regulate early events in apoptosis. Interestingly, cyt c was still releasable by Bax in CL-deficient yeast mitochondria [88], suggesting CL participation in retention of cyt c rather than in facilitating its release. There are several speculations about possible sources and mechanisms of CL oxidation. Mitochondria are a major source of ROS, superoxide and its dismutation product H2O2, which are generated both during physiologic respiration and during disrupted electron transport [83,86]. It has been noted that a burst of ROS damages mitochondria by causing profound loss of CL [63]. Its high degree of unsaturation and its location in the inner mitochondrial membrane near to the site of ROS production make CL susceptible to oxidation [83]. However, neither superoxide nor H2O2 are effective in direct oxidation of vulnerable substrates. Specific catalytic mechanisms of CL oxidation in mitochondria also remain unknown. Using a succinate + antimycin A system to produce ROS, Paradies et al. [83] showed that about 30% of cyt c was dissociated from the submitochondrial particles (SMPs) and that this effect was prevented by addition of SOD + catalase. Treatment with Fe-ADPascorbate, an inducer of mitochondrial membrane lipid peroxidation, also led to cyt c dissociation. The content of CL also decreased in the succinate + antimycin A-treated SMPs and addition of SOD + catalase prevented this loss. A strong linear correlation between levels of CL and

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dissociation of cyt c was also shown [81,87]. Based on protective effects of anti-oxidants against both lipid oxidation and cyt c dissociation from membranes the authors concluded that CL oxidation was responsible for the effect. It should be noted, however, that measurements of peroxidation were conducted in total lipid extracts rather than in the CL fraction. The role of CL peroxidation was confirmed by a decreased dissociation of cyt c upon addition of exogenous nonoxidized CL. A decrease in dissociation was not observed upon addition of other phospholoipids such as PC and phosphatidylethanolamine (PE) to the ROS-producing SMP system [81,87]. In another study in neuronal cells, Kirkland et al. [89] demonstrated CL peroxidation and a decrease in the CL after neuronal-growth-factor-withdrawal-induced apoptosis. Specific catalytic mechanisms of CL oxidation were not identified in this study or in a number of other studies indicating that peroxidation of CL occurs during apoptosis [76,89]. Release of cyt c into the extramitochondrial space after its dissociation from CL is linked to mitochondrial membrane permeabilization [90]. The presence of CL is also required for effective pore formation [79,84,85]. For permeabilization reactions mediated by t-Bid (caspase-8-digested Bid) and Bax, the presence of CL in the mitochondria is both sufficient and necessary [85]. Studies in cell-free systems by Kuwana et al. [85] found no discontinuities in the membrane at the ultrastructural level in t-Bid/Bax-permeabilized, CLcontaining liposomes [85], suggesting that release of mitochondrial intermembrane proteins during apoptosis is not accompanied by membrane rupture. Bax appears to cooperate with voltage-dependent, anion-channels (VDAC) to form cyt c-permeable pores in the outer membrane [91]. Bernardi et al. [92] speculated that mitochondrial membrane permeabilization involves two independent steps: the first may occur in a permeability–transition–pore-complex (PTPC)-independent manner and the second may involve physical and/or functional interactions of pro-apoptotic proteins with PTPC proteins. In conclusion, various studies indicate that CL is vital for retaining cyt c in the inner membrane of the mitochondria until the protein is required for initiating the downstream apoptotic processes. Oxidative damage to CL triggers the release of cyt c, which occurs first as dissociation from CL due to breaches in the hydrophobic and electrostatic interactions caused by peroxidation of CL acyl chains. This is then followed by the release of the soluble form of cyt c into the extramitochondrial space through a pore whose formation may also depend on the presence of (oxidized) CL (Scheme 1). Numerous studies indicate that ROS are essential for CL oxidation, but they do not characterize any specific mechanism. Our

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hypothesis identifies the CL–cyt c complex as a peroxidase whose catalytic activity is responsible for CL oxidation and, hence, for detachment and release of cyt c from mitochondria. It is therefore, important to consider possible regulatory pathways involved in control of CL oxidation by cyt c. The roles of expression and activity of different antioxidant enzymes are important but also trivial, as they regulate the cellular content of H2O2, one of the required cofactors of peroxidase reactions. The discussion that follows emphasizes the possible involvement of NO and some other reductants that can act as effective regulators of cyt c peroxidase activity and, therefore, of cyt c release and of apoptosis in general. Regulation of cyt c peroxidase activity: the role of mitochondrial nitric oxide synthase (mtNOS) As a pluripotent regulator and free radical, nitric oxide (NO) is likely involved in controlling several mitochondrial processes, including respiration, membrane phospholipid oxidation, and the execution of apoptosis. Interactions of NO with cyt c that, one way or another, participate in the same events have not been considered important. These interactions were probably neglected because of the relatively low reactivity of solubilized cyt c toward NO. Because redox properties of cyt c depend on its binding to CL in the inner membrane, the possible role of the cyt c/NO bcoupleQ in view of the protein interaction with membranes bears discussion. Inhibition of respiration by NO has been shown at different levels: in whole organisms, in single cells, and in isolated mitochondria [93–95]. In mitochondria, NO can block respiration at each major site of the electron transport chain: It directly inhibits complex I (through protein thiol nitrosylation and/or formation of iron–nitrosyl complexes with Fe-S centers) [96], complex III (through nitrosylation of cytochrome b heme) [95], cyt c (via nitrosylation of heme) [97], and complex IV (through nitrosylation of cyt c heme or binding to CuB) [98]. In addition, NO can be reduced by ubiquinol to yield nitroxyl anion and ubisemiquinone, resulting in disruption of electron flow between complexes I and III [98]. Cytochrome c oxidase (complex IV) appears to be most sensitive to the inhibitory effect of NO [99]. Inhibition of cytochrome c oxidase inevitably causes reduction of electron transporters and production of S O2 – that can either dismutate to H2O2 or react very rapidly with NO to yield a reactive and deleterious oxidant, ONOO-. H2O2 can form highly oxidized oxoferryl states of cytochrome c oxidase, which are S able to oxidize NO into reactive NO2 . Thus, the mitochondrial environment is favorable for activation

of NO and its conversion into damaging reactive nitrogen species. Surprisingly, mitochondria express their own NO synthase (mtNOS), which is capable of generating NO in amounts sufficient to inhibit respiration up to 25% [100]. If NO can depress their function and induce oxidative damage, why do mitochondria need and generate it? Interestingly, NO-related oxidative stress in mitochondria (i.e., protein tyrosine nitration and irreversible impairment of the respiration) has been detected only at relatively high NO concentrations (N1 AM) [101]. Such high levels of NO are usually observed during inflammation or ischemia/reperfusion. Among the many controversial roles of NO, its potent antioxidant function may become central under some conditions. It is possible that low concentrations of NO are indispensable for its antioxidant protective function rather than for its pro-oxidant effects in mitochondria. For instance, NO is a potent radical scavenger capable of quenching oxidation of polyunsaturated lipids in membranes [102]. Another possible antioxidant function of NO is the reduction of oxoferryl species of hemoproteins and inhibition of peroxidase activity. An enticing hypothesis is that NO acts as a regulator of peroxidase activity of CL-bound cyt c in mitochondria. As discussed above, CL-bound cyt c, through its peroxidase activity, can act as a receptor of proapoptotic signals in mitochondria and participate in execution of the apoptotic program by oxidizing CL and triggering cyt c release from mitochondria. As a good reducing substrate for a variety of peroxidases, NO can react with highly oxidized states of heme in the CL/cyt c complex and prevent oxidation of other important molecules such as CL and other membrane phospholipids. In addition, there is a good chance that NO can inhibit membrane-bound cyt c through nitrosylation of its heme: Increased accessibility of the catalytic site in CL-bound cyt c results in increased activity of the heme toward small molecules such as H2O2 and NO. Similar changes in carboxy-methylated cyt c have been shown to increase its affinity for NO at least by two orders of magnitude [103]. It is likely that one of NO’s very important functions in mitochondria is to regulate the peroxidase activity of CL-bound cyt c, and, hence, it has a regulatory role in the execution of the apoptotic program. In conclusion, CL-bound cyt c acts as a peroxidase and catalyzes CL oxidation, thus functioning as a stress sensor and redox regulator of apoptosis. It is likely that one of the important NO functions in mitochondria is to regulate the peroxidase activity of CL-bound cyt c via two different mechanisms: (1) reduction of highly oxidized states of heme in peroxidase cycle and (2) binding to heme in reduced state. Under normal

Cytochrome c and apoptotic peroxidation

conditions, NO generated by mtNOS participates— along with other reductants—in elimination of H2O2, thus protecting mitochondria against oxidative stress. Massive production of superoxide triggered by proapoptotic stimuli yields excessive amounts of H2O2 and/or ONOO-, both of which can be utilized as sources of oxidizing equivalents for peroxidase-catalyzed oxidation of CL, thus contributing to propagation of apoptosis. CYTOCHROME C IN THE CYTOSOL AND ITS INTERACTIONS WITH PHOSPHATIDYLSERINE

PS/cyt c as a catalyst of PS oxidation Through its electrostatic and hydrophobic interactions with negatively charged CL, cyt c activates itself to a peroxidase that catalyzes oxidation of CL and forces cyt c out of mitochondria into the cytosol. CL is not normally found outside of mitochondria, but activation of cyt c to a peroxidase is likely to occur in a new cytosolic environment through interactions with other anionic phospholipids. Among those, PS represents a particularly good candidate. Although the affinity of cyt c is significantly lower for PS than for CL, the former also produces stable complexes with cyt c (see Fig. 1) that confer peroxidase activity (see Fig. 2); as a result, selective oxidation of polyunsaturated PS would occur. If this happens during dispatch of the apoptotic program, cyt c released from mitochondria into the cytosol can act as a redox catalyst, causing selective oxidation of PS. Our previous work has shown that execution of apoptotic cascade in a variety of cell lines by different intrinsic pro-apoptotic stimuli resulted in selective oxidation of PS, while more abundant phospholipids such as PC and PE remained much less oxidized or nonoxidized (reviewed in [7,104,105]). Apoptosis-associated production of H2O2 was essential for PS oxidation as cells overexpressing catalase did not respond by PS oxidation to pro-apoptotic stimuli [106]. In addition, cyt c was essential for catalyzing PS oxidation as oxidation did not occur in cyt c / mouse embryonic cells. Integration of exogenous cyt c into cyt c / cells reconstituted PS sensitivity to oxidation upon addition of H2O2 [43]. In combination, these results provide strong support for our hypothesis that, in a manner similar to CL/cyt c interactions in mitochondria, formation of PS/cyt c complexes in the cytosol creates a peroxidase that can effectively and selectively oxidize PS. Because massive oxidation of PS takes place in the plasma membrane [107] and precedes its externalization on the cell surface [104,105], we suggested that oxidation of PS may be essential for egression of PS during apoptosis and for recognition of apoptotic cells by phagocytes.

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PS/cyt c complex as a signal for PS externalization Asymmetric distribution of phospholipids across membranes is a fundamental feature of living cells. Normally, PS is confined to the inner monolayer of the plasma membrane [108,109], which contains N65% of total cellular PS [110]. During apoptosis, lipid asymmetry collapses, resulting in exposure of PS on the cell surface [111]. Externalized PS acts as an important beat-meQ recognition signal for macrophage receptors [112]. PS surface exposure is one of the early events in apoptosis that may originate from the balance between inward and outward PS translocations catalytically driven by two enzymatic activities, aminophospholipid translocase (APT) and a nonspecific phospholipid scramblase (PLSCR) [108,109]. APT II, a 116-kDa protein, is responsible for localizing PS in the inner leaflet of the plasma membrane [113]. Four isoforms of PS-specific ATP II have been identified [114] and the enzyme has been isolated from several sources [115–117]. During intrinsic apoptosis, the inhibition of APT is redox dependent and sensitive to oxidation and other modifications of its SH groups [118,119]. In contrast, Fas-triggered extrinsic apoptosis in type II cells (e.g., Jurkat cells)—a process that is also accompanied by PS externalization and complete inhibition of APT—is only partly redox dependent and cannot be fully reconstituted by –S-S-reducing agents [120]. Given that PS oxidation precedes its externalization it is likely that changes in PS distribution within the plasma membrane require cyt c acting as a catalyst for PS oxidation [43,107]. It is clear that mitochondrial disruption and release of cyt c are critical for PS signaling pathways during intrinsic apoptosis but can also contribute to extrinsic apoptotic pathways. Because PS oxidation seems to be important for its externalization, it is likely that oxidized PS (PSox) interacts with APT differently than nonoxidized PS. Among many possible explanations, the following three causes of enhanced PS externalization induced by PSox may be considered: (1) APT fails to recognize PSox, (2) APT is modified (poisoned) by the oxidized substrate PSox, and (3) PSox acts as a bpseudo-scramblaseQ and facilitates transbilayer diffusion of PS (and PSox). PSox incorporated into the outer monolayer of cell membrane of different cells (Raji cells, HL-60 cells) was internalized as effectively as PS. In addition, PSox did not affect translocation of fluorescently labeled PS (1-palmitoyl-2(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)-sn-glycero-3-phosphoserine, NBD-PS) from the outer to the inner leaflet of plasma membrane [121]. These results indicate that APT may recognize PSox and is not inhibited by it. Another possibility for PSoxTs

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enhancement of PS externalization is through activation of the scrambling activity. PLSCR are thought to mediate Ca2+-dependent bidirectional transbilayer movement of plasma membrane phospholipids [122]. Recently, four human PLSCR genes—the mouse and rat orthologous genes [123,124]—have been identified, yet the cellular function of PLSCR remains unclear. PLSCRs may be involved in plasma membrane reorganization in response to cell stimulation [125,126], injury [127], and apoptosis [128,129]. PLSCR is a multiply palmitoylated 35-kDa protein [130] whose activity during cell stimulation and apoptosis is regulated by protein kinase Cy (PKCy) [131]. The enzyme is enriched in lipid rafts [132]—specialized membrane subdomains, which contain high amounts of PS [133]. The formation of rafts may be associated with PS exposure during apoptosis [11,134]. In platelets, a direct correlation between expression of PLSCR and Ca2+-ionophore-induced externalization of PS has been demonstrated [135]. However, results from several laboratories do not support involvement of PLSCR in PS exposure during apoptosis. In fact, a reverse correlation between the expression of PLSCR and the ability of cells to expose PS during apoptosis has been reported [136]. Further, the amount of protein was found to be normal in Scott syndrome patients whose blood cells were defective in both PLSCR activity and PS externalization [108]. Detergent-solubilized PLSCR isolated from Scott syndrome erythrocytes exhibited normal Ca2+-induced scrambling of phospholipids [137]. Finally and most notably, no evidence of defective platelet function or Ca2+-induced PS exposure in platelets and red blood cells from mice deficient in PLSCR1 was detected [138]. This raises the question of whether PLSCR protein is responsible for phospholipid scrambling in plasma membrane. There are, however, alternatives to protein-associated phospholipid scrambling pathways that may involve formation of transbilayer lipid boundaries that facilitate bidirectional diffusion of phospholipids across the membrane [109]. For example, phospholipid oxidation may cause the formation of transbilayer lipid pores through which other lipids may migrate bidirectionally with a diffusion rate orders of magnitude higher than the bflip-flopQ rates in nonoxidized bilayers [139]. From this, we hypothesized that PSox, formed during apoptosis, stimulates externalization of PS through increased rates of PS and/or PSox transmembrane diffusion. We confirmed this experimentally using Raji cells with very low PLSCR activity under conditions when APT was inactive (due to exhaustion of ATP): exogenously added and internalized PSox increased both the content of externalized PS on the cell surface and the number of

annexin-V-positive cells with externalized PS (after inhibition of APT) in a dose-dependent manner [121]. Thus, PSox can facilitate scrambling of PS (and PSox) and contribute to PS externalization during apoptosis. If this mechanism occurs during apoptosis, then not only PS, but also PSox, should be present on the surface of apoptotic cells where it may serve as an additional recognition signal for macrophages [140]. Using two models of oxidant (H2O2)- or nonoxidant (staurosporine)-induced apoptosis in HL-60 cells, we were able to show that PSox appears along with PS on the cell surface in a time-dependent manner. Using a newly developed protocol for assessment of aminophospholipid oxidation on the cell surface [141], we found that the majority of oxidized PSox underwent externalization during apoptosis. Thus stimulation of PS oxidation/externalization during apoptosis results in egress of not only PS but also significant amounts PSox. Therefore, it is important to determine the extent to which PSox can act or modulate recognition of apoptotic cells by macrophages. Recognition of PS and PSox and phagocytosis of apoptotic cells PS exposed on the surface of apoptotic cells is believed to be a recognizable, universal beat-meQ signal for macrophages [112]. Various macrophage receptors, including the scavenger-receptor superfamily [142], integrins [143], compliment receptors [144], and phophatidylserine receptor (PSR), may recognize PS and initiate ingestion of apoptotic cells. Although many of these receptors can bind PS directly, an assortment of bbridgingQ molecules such as h-2 glycoprotein [145], milk fat globule protein (MFG-E8) [146], protein S [147], and thrombospondin [148] facilitate interactions between macrophages and exposed PS on apoptotic cells. The PSR is one of a number of macrophage receptors involved in removal and clearance of apoptotic cells [11,149,150]. However, specific mechanisms through which PSR recognizes PS are still unknown. A recent study indicated that annexin I, which is also externalized during apoptosis, stimulates phagocytosis through PSR [151]. It is possible that annexin I is involved in PS translocation or in its aggregation, thus creating high local concentrations of PS in the outer leaflet of plasma membrane [152]. Lipid peroxidation is responsible for the generation of the apoptotic phenotype [7,10,86,104,105,153]. Many receptors implicated in phagocytosis of apoptotic cells can bind oxidized phospholipids [154,155]. In addition, bridging molecules can have higher specificity for oxidatively modified PS than for nonoxidized PS. In particular, we have shown that MFG-E8 preferentially interacts with PSox and, to a lesser degree, with

Cytochrome c and apoptotic peroxidation

nonoxidized PS and PC [140]. C-reactive protein (CRP), an acute-phase protein of immune response, binds to oxidized PC and, thus, can promote the clearance of CRP-opsonized apoptotic cells by its direct binding to Fcg receptors on the macrophage [156]. There is also accumulating evidence that cells undergoing apoptosis generate oxidatively altered cell surface moieties [157]. Scavenger receptors known to bind OxLDL [157] compete with apoptotic cells for uptake by phagocytes [158]. It is likely that both PS and PSox serve as apoptotic cell ligands that are essential for their recognition by phagocytes [119]. The presence of PSox in the outer leaflet of the plasma membrane stimulated recognition and phagocytosis of cells to a greater extent than equivalent amounts of PS, suggesting that PSox may act as an additional enhancing signal. Antioxidant prevention of PS oxidation during apoptosis was associated with diminished phagocytosis of apoptotic cells by macrophages [7,119]. Furthermore, the phagocytosing activity of macrophages shows typical threshold responses for their dependence on the PS content on the cell surface [159]. It is possible that PSox acts to amplify the macrophage response to PS and presents an additional reassurance signal—confirming that the cell is indeed apoptotic and should be phagocytosed. In a manner consistent with this interpretation, PSox decreased the threshold for macrophage digestion of cells expressing PS on their surface [7,119,159]. It is not clear which among many receptors is responsible for recognition of PSox. Both anti-PSR and anti-CD36 antibodies more effectively suppressed phagocytosis of Jurkat cells enriched with PSox than the same cells enriched with PS, indicating that not only PSR but also other receptors may be engaged in interactions with PSox [7]. It is likely that other oxidized phospholipids such as oxidized molecular species of PC can participate in stimulation and tuning of phagocytosis [156]. Effects of PS and PSox on phagocyte cytokine and ROS production and inflammatory response Apoptosis is critical for tissue homeostasis and results in fast, effective removal of dying cells, either by neighboring cells or by bprofessionalQ phagocytes such as macrophages and dendritic cells. Recognition and uptake of apoptotic cells by macrophages involve multiple receptors: not only the previously mentioned scavenger receptors and integrins but also other pattern recognition molecules, including CD91 and CD14 [160]. Triggering of these receptors by microbial agents initiates inflammation and stimulates an acquired immune response; however, interaction and recognition of apoptotic cells by these receptors has a totally opposite effect—it blocks the inflammatory response and prevents an acquired immune response. Recently, interactions with two macrophage

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receptors, PSR and CD91, were shown to be particularly important for the recognition and engulfment of apoptotic cells. In a PSR knockout mouse, defects in clearance of apoptotic cells and their accumulation in the lung caused abnormal development leading to neonatal lethality [161]. Notably, whereas stimulation of CD91 alone or its activation by necrotic cells triggered pro-inflammatory reactions, triggering of PS receptor-dependent mechanisms induced anti-inflammatory pathways. If both the CD91 and the PS receptors are engaged, the antiinflammatory responses seem to dominate [162,163]. Thus, resolution of inflammation depends not only on effectively removing apoptotic cells but also on actively suppressing the macrophage production of inflammatory mediators [164,165]. This suppression occurs through switching off production of pro-inflammatory mediators and by stimulating production of anti-inflammatory cytokines, such as TGF-h1 from recruited macrophages that have internalized apoptotic cells. Disruption of PS-dependent apoptotic signaling is associated not only with the failure to effectively clear apoptotic cells but also with triggering the inflammatory response. For example, treatment of apoptotic cells with annexin V to block recognition of PS by PSR efficiently inhibited the uptake of irradiated cells by macrophages and skewed the phagocytosis of irradiated cells toward inflammation. The anti-inflammatory activity of PS liposomes in microglia suggests that, as with peripheral macrophages, PS can modulate microglial activation toward an anti-inflammatory phenotype [166]. It is tempting to speculate that PSox may exert even a stronger anti-inflammatory potency through its ability to enhance recognition of apoptotic cells and stimulate phagocytosis. In several studies, ingestion of apoptotic cells in vitro actively suppressed production of pro-inflammatory growth factors, cytokines, chemokines (e.g., GM-CSF, MIP2, IL-1a, KC, IL-8, and TNF-a), and eicosanoids (thromboxane B2) [167]. This down-regulation of proinflammatory mediators in response to apoptotic cells has been shown in human monocyte-derived macrophages, murine macrophage cell lines (RAW264.7 and J774), microglial cells purified from neonatal rat brain, bonemarrow-derived macrophages, fibroblasts, and mammary epithelial cells [167,168]. Down-regulation of inflammatory responses by apoptotic cells is associated with secretion of TGF-h1, prostaglandin E2, and/or plateletactivating factor by macrophages following phagocytosis [169,160]. In fact, direct in vivo installation of apoptotic cells into inflammatory lesions resulted in accelerated resolution of inflammation mediated by increased production of TGF-h1 in lungs of LPS-stimulated mice [166]. Furthermore, PS-containing liposomes, but not PC-containing liposomes, induced TGF-h1, although not so dramatically as intact cells did [167].

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In microglia, nuclear factor-kB (NF-kB) and p38 mitogen-activated protein kinase (p38) are essential to the synthesis of pro-inflammatory molecules. In LPSactivated microglia, PS liposomes did not affect NF-kB activation but inhibited the phosphorylation of p38, revealing potential molecular signaling pathways evoked by PS/PSR interactions which are likely to be related to suppression of microglial cells’ pro-inflammatory activities [168]. Because activated phagocytes regulate inflammation not only through releasing pro-/anti-inflammatory signaling mediators [169] but also by generating reactive nitrogen and oxygen species such as NO and superoxide, it is important to determine whether PS-dependent signaling also impinges on this pathway. Only a few reports address this question. PS liposomes strongly reduced the LPS-induced release of NO in microglial cultures from neonatal rat brain [166,170]. These findings have been confirmed and extended by using PS-exposing apoptotic PC12 cells [171]. In addition, NO production by mouse peritoneal macrophages stimulated with LPS was downregulated by PS liposomes through the induction of TGFh [170,172–174]. In our experiments [120], apoptotic cells effectively suppressed production not only of NO but also of superoxide by RAW264.7 macrophages stimulated by zymosan. Importantly, supression by PS-containing liposomes was significantly less effective than that by apoptotic cells, suggesting that there may be other signals (in addition to externalized PS) on the apoptotic cell surface that contribute to this effect. Overall, phagocytosis of apoptotic cells not only prevents the release of toxic and immunogenic intracellular contents but also stimulates macrophages to express an anti-inflammatory phenotype and to suppress production and release of toxic reactive oxygen and nitrogen species. All these factors are likely to be critical in the resolution of inflammation. Antioxidant regulation of apoptosis: blockage of phospholipid oxidative signaling pathways. Can we selectively prevent CL and/or PS oxidation? Generation of ROS and subsequent oxidative stress are components of a final common pathway for execution of intrinsic apoptosis via mitochondrial disfunction. Mitochondrial permeability transition and cyt c release, the well-established hallmarks of apoptosis, are associated with this excessive generation of radical intermediates and oxidative stress [175–178]. This implies that enhancement of antioxidant protection should be associated with the development of resistance to apoptosis. Indeed, there are numerous reports indicating that increased levels of GSH [179,180] and over-expression of antioxidant enzymes such as Mn-superoxide dismutase, catalase, and thioredoxins [181–186] boost resistance to pro-apoptotic agents generated through intrinsic pathways. The significance of

the effects of antioxidant enhancement should be carefully considered in the context of their potential interference in the execution of the apoptotic process. A separate issue is selective inhibition of phospholipid oxidation by lipid-soluble antioxidants, including both natural (e.g., vitamin E) and pharmacologic compounds. Because oxidation of two major phospholipids, CL in mitochondria and PS in plasma membrane, is involved in apoptotic signaling, interruption of their effects by antioxidants may be linked with defective signaling functions. Antioxidants that are effective inhibitors of PS oxidation may affect PS externalization and/or clearance of apoptotic cells by macrophages and resolution of inflammation. Indeed, etoposide, a phenolic anti-cancer drug and potent lipid antioxidant [187] known to induce apoptosis through inhibition of topoisomerase II [188], prevents PS oxidation, inhibits PS externalization, and hinders phagocytosis of apoptotic cells by macrophages [119]. In Jurkat cells, vitamin E effectively inhibited cyt c-dependent PS exposure during intrinsic actinomycin D-induced apoptosis [189]. It is possible that lipid-soluble antioxidants capable of inhibiting oxidation of CL in mitochondria would effectively regulate detachment of cyt c, formation of mitochondrial permeability pores, and release of cyt c into the cytosol. To date, we know of no direct, reliable data on CL oxidation in mitochondria during apoptosis. New analytical approaches and protocols must be developed to address this important aspect of regulation of intrinsic apoptosis by lipid-soluble antioxidants. It is likely that ROS production is relatively less important during execution of the extrinsic apoptotic pathway, particularly in type I cells, in which mitochondrial involvement is minimal [190–194]. Integration of vitamin E into cells during Fas-triggered apoptosis proved consistent with this approach, as it had no effect on PS externalization or on recognition of these cells by macrophages [120]. Antioxidants are commonly regarded effective antiinflammatory agents [195–197]. However, natural and pharmacologically used antioxidants can affect key mechanisms of oxidative apoptotic signaling through their interaction in phospholipid-dependent pathways. Therefore, therapeutic strategies based on the use of antioxidants must consider the potential effects of antioxidants on death receptor and drug-induced apoptosis. CONCLUDING REMARKS

The primary life-supporting function of cyt c is control of cellular energetic metabolism as a mobile shuttle in the mitochondrial electron transport chain (Scheme 2). Recently, cyt c’s equally important life-terminating function as a trigger and regulator of apoptosis was identified. This destructive role is realized through the

Cytochrome c and apoptotic peroxidation

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Scheme 2. Normal functions of cyt c in normally functioning cells and apoptotic cells. Redox catalysis of oxidation of anionic phospholipids—CL in mitochondria and PS in plasma membrane—triggering the formation of MPTP and PS-signaling pathways, respectively.

relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in mediating caspase-9 activation. These two essential life–death functions of cyt c are mainly accomplished by pools of solubilized and electrostatically membrane-bound cyt c. Both pools of cyt c can be easily mobilized for translocation into the cytosol through the MPTP during apoptosis. Another pool of tightly membrane-bound cyt c avidly interacts with CL which results in changed properties and functions for cyt c. It is likely that this pool of cyt c is selected by its availability and its hydrophobic interactions with polyunsaturated molecular species of CL containing C20:4, C22:5, and C22:6 fatty acid residues. The CL-bound cyt c assumes the role of a membrane-bound peroxidase that can effectively catalyze oxidative stress and cause

oxidation of CL if a source of oxidizing equivalents such as H2O2, ONOO, and lipid hydroperoxides is activated. The accessibility of the source can launch and propagate an oxidative cascade of CL oxidation. In all probability, appearance of the signal is triggered by disruption of electron transport initiated by pro-apoptotic stimuli and production of ROS in amounts that overwhelm the capacities of ROS controlling mechanisms. Thus, CLbound cyt c acts as a mitochondrial death receptor transducing pro-apoptotic signals into executing oxidative cascades that produce an overload of oxidized polyunsaturated CL species, which, in turn, are required for both detachment of cyt c from the membrane and for the formation of MPTP through which cyt c is released into the cytosol. Upon arrival in the new cytosolic

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environment, cyt c interacts with numerous target molecules and participates in the formation of apoptosomes and with PS on the inner leaflet of the plasma membrane. The PS/cyt c complex, in a manner similar to that of the CL/cyt c complex, acts as a peroxidase ready to catalyze PS oxidation. It is likely that an excess of ROS generated by disrupted mitochondria contribute to sources of oxidizing equivalents that feed the PS/cyt c peroxidase reaction. Accumulation of oxidized PS facilitates its migration across the cell membrane, behaving as a bpseudo-scramblaseQ resulting in externalization of both PS and PSox. Finally, externalized molecular species of PS and PSox are appetizing beat-meQ signals for professional and nonprofessional phagocytes that engulf and digest apoptotic cells, thus concluding the catalytic role of cyt c as a mitochondrial redox-dependent life– death switch during apoptosis.

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APT — aminophospholipid translocase BHCL — bovine heart cardiolipin cyt c — cytochrome c CL — cardiolipin Clox — cardiolipin oxidized CRP — C-reactive protein DCFH2 — 2V7V-dichlorodihydrofluorescein DOPC — dioleoyl-phosphatidylcholine DOPS — dioleoyl-phosphatidylserine EPR — electron paramagnetic resonance MFG-E8 — milk fat globule protein MPTP — mitochondrial permeability transition pore NBD-PS — 1-palmitoyl-2-(6-((7-nitro-2,1,3-benxoxadiazol-4-yl)amino)hexanoyl)-sn-glycero-6-phosphoserine NO — nitric oxide PAPS — palmitoyl-arachidonoyl-phosphatidylserine PC — phosphatidylcholine PLSCR — phospholipid scramblase PS — phosphatidylserine PSox — phosphatidylserine oxidized PSR — phophatidylserine receptor PTPC — a permeability–transition–pore complex ROS — reactive oxygen species TOCL — tetraoleoyl-cardiolipin VDAC — voltage-dependent anion channels