Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Abstract

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c's conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria--its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition--are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.

Schema 1

Possible cardiolipin (CL) binding sites on cytochrome c. Structure of native cyt c (1HRC). Several domains that are likely involved in interactions with CL and acting as heme-iron ligands include the following amino acid residues: Lys72, Lys73, Lys86 and Lys87 (A-site), Asn52 (C-site) and Lys22, Lys25, His26, Lys27 and His33 (L-site). Met80 and His18 form coordination bonds with heme. Intrinsic fluorescence of Trp59 is quenched by the proximity to the heme moiety.

Schema 2

Proposed structural model of alternative (un)folding of cyt c bound to anionic membrane surface. The model explains reported physico-chemical properties of cyt c including its peroxidase activity. A – side view, membrane interface is at the bottom of the protein; B – view from the membrane side. Protein backbone is encoded by color in accord with unfolding energy (in the order from low to high energy: white, red, yellow, green, and blue [37]). Amino acid residues important for binding with the membrane and peroxidase activation are highlighted.

Schema 3

Catalytic mechanism of horseradish peroxidase. A – ferric enzyme with H₂O₂ bound as a ligand in the 6th coordination position of iron; B – oxoferryl iron and porphyrin centered radical (Compound I).

Schema 4

Oxidative lipidomics “Hit-map” of small intestine from mice subjected to total body irradiation. A non-random, cyt c-driven mechanism is involved in the catalysis of γ-irradiation induced peroxidation of intestinal phospholipids. Selective oxidation of CL followed by oxidation of PS takes place after γ-irradiation and is a part of the intestinal apoptosis in vivo.

Schema 5

Cross-roads of mitophagy and apoptosis. Autophagy and apoptosis are two processes that may be mutually inhibitory; autophagy usually precedes apoptosis while triggering of apoptosis is associated with blocked autophagy. It is likely that these two pathways are intrinsically interconnected via molecular switches that turn on the autophagy process (with still inhibited apoptosis) followed by activation of apoptosis (with turned off autophagy). Both processes function as parts of an essential combined mechanism of elimination of irreparably damaged cells. Phospholipid signaling, particularly deregulation of characteristic for normal cells asymmetry of phospholipids, has been discovered as one of important factors in both autophagy and apoptosis. Collapse of cardiolipin asymmetry in mitochondria and covalent association of phosphatidylethanolamine (or phosphatidylserine) with LC3₁ are the two major events in signaling, culminating in apoptotic cell death and mitophagy, respectively. It is possible that changes of cardiolipin asymmetry in mitochondria are at the center of the chain of events leading to cell death. This chain includes several consecutive levels of regulation: 1. Synthesis of cardiolipin (CL) and its molecular speciation with a balance of poly- and mono-unsaturated molecular forms as well as saturated CLs). 2. Scramblase-3 (SCR-3) is inactive; maintenance of asymmetry of CL between the inner and outer mitochondrial membranes; CL and cyt c are spatially separated. 3. Regulation of cyt c/CL interactions via cyt c phosphorylation hindering binding of negatively charged CL to cyt c. 4. Low levels of H₂O₂ production leading to insufficiency of oxidizing equivalents. 5. Cyt c can undergo phosphorylation of its Tyr 97 (in the heart) [168] Tyr 48 (in the liver) [169] likely via a cAMP dependent pathway. Phosphorylation of Y97 is associated with changes of the absorbance at 695 nm which suggests subtle structural changes in the heme environment [168]. Peroxidase activity of cyt c/CL complex involves formation of Tyr radicals [20]. It is possible that phosphorylation of Y97 and Y48 affects binding of cyt c with CL as well as its peroxidase activity. 6. During initiation of autophagy, SCR phosphorylation (resulting in its activation) moves CL to the outer mitochondrial membrane and stimulates (turns on) mitophagy. There is no CL oxidation at this time, because cyt c may be phosphorylated and H₂O₂ is still unavailable. As damage develops, cyt c can be dephosphorylated and more avidly binds with CL. This disrupts electron transport and stimulates H₂O₂ production. As a result, CL gets oxidized, thus initiating apoptosis and the end of autophagy (mitophagy).

Schema 6

Inhibition of CL-activated peroxidase activity of cyt c and prevention of CL oxidation in mitochondria leading to suppression of apoptosis. Peroxidase activity of cyt c/CL complexes leads to CL oxidation and accumulation of products required for the release of pro-apoptotic factors from mitochondria. Consequently, agents and factors that inhibit the peroxidase activity and prevent CL oxidation may act as anti-apoptotic agents. A new approach to regulate the cyt c peroxidase activity is based on the use of modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). NBD-CL forms high-affinity complexes with cyt c and blocks cyt c-catalyzed oxidation of several peroxidase substrates, cyt c self-oxidation, and, most importantly, inhibits cyt c-dependent oxidation of polyunsaturated CL and accumulation of CL hydroperoxides. Mitochondrial targeting of such agents may lead to discovery of new potent drugs. Several options shown on the schema include mitochondria-targeted conjugates of nitroxide radicals (TEMPO) with hemi-gramicidin S (GS) or triphenyl-phosphonium. Specifically, GS-TEMPO is selectively accumulated in mitochondria where it acts as an electron scavenger capable of preventing superoxide formation and its dismutation into H₂O₂ that is necessary for CL oxidation. GS-TEMPO is also an effective anti-apoptotic agent. Mitochondria-targeted donors of nitric oxide (NO˙) – such as 2-(hydroxyamino-vinyl)-triphenyl-phosphonium (HVTP) - activatable by peroxidase activity of cyt c owe their anti-apoptotic potency to the NO˙-dependent reduction of reactive intermediates of the peroxidase cycle.