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Interaction of a mitochondria-specific anionic phospholipid, cardiolipin (CL), with an intermembrane protein, cytochrome c (cyt c), yields a peroxidase complex. During apoptosis, the complex induces accumulation of CL oxidation products that are essential for detachment of cyt c from the mitochondrial membrane, induction of permeability transition, and release of proapoptotic factors into the cytosol. Therefore, suppression of the peroxidase activity and prevention of CL oxidation may lead to discovery of new antiapoptotic drugs. Here, we report a new approach to regulate the cyt c peroxidase activity by using modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). We demonstrate that 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 tetralinoleoyl CL (TLCL) and accumulation of TLCL hydroperoxides. Electrospray ionization mass spectrometry and fluorescence analysis revealed that oxidation and cleavage of the NBD moiety of NBD-CL underlie the inhibition mechanism. We conclude that modified CL combining a nonoxidizable monounsaturated trioleoyl CL with a C(12)-NBD fragment undergoes a regiospecific oxidation thereby representing a novel inhibitor of cyt c peroxidase activity.

Activation of peroxidase catalytic function of cytochrome c (cyt c) by anionic lipids is associated with destabilization of its tertiary structure. We studied effects of several anionic phospholipids on the protein structure by monitoring (1) Trp59 fluorescence, (2) Fe-S(Met80) absorbance at 695 nm, and (3) EPR of heme nitrosylation. Peroxidase activity was probed using several substrates and protein-derived radicals. Peroxidase activation of cyt c did not require complete protein unfolding or breakage of the Fe-S(Met80) bond. The activation energy of cyt c peroxidase changed in parallel with stability energies of structural regions of the protein probed spectroscopically. Cardiolipin (CL) and phosphatidic acid (PA) were most effective in inducing cyt c peroxidase activity. Phosphatidylserine (PS) and phosphatidylinositol bisphosphate (PIP2) displayed a significant but much weaker capacity to destabilize the protein and induce peroxidase activity. Phosphatidylinositol trisphosphate (PIP3) appeared to be a stronger inducer of cyt c structural changes than PIP2, indicating a role for the negatively charged extra phosphate group. Comparison of cyt c-deficient HeLa cells and mouse embryonic cells with those expressing a full complement of cyt c demonstrated the involvement of cyt c peroxidase activity in selective catalysis of peroxidation of CL, PS, and PI, which corresponded to the potency of these lipids in inducing cyt c's structural destabilization.

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.