Heterolytic Reduction of Fatty Acid Hydroperoxides by Cytochrome c/Cardiolipin Complexes: Antioxidant Function in Mitochondria

Abstract

Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H(2)O(2) was considered as a source of oxidative equivalents for this reaction, which is essential for programmed cell death. Here we report that peroxidase cyt c/CL complexes can utilize free fatty acid hydroperoxides (FFA-OOH) at exceptionally high rates that are approximately 3 orders of magnitude higher than for H(2)O(2). Similarly, peroxidase activity of murine liver mitochondria was high with FFA-OOH. Using EPR spin trapping and LC-MS techniques, we have demonstrated that cyt c/CL complexes split FFA-OOH predominantly via a heterolytic mechanism, yielding hydroxy-fatty acids, whereas H(2)O(2) (and tert-butyl hydroperoxide, t-BuOOH) undergo homolytic cleavage. Computer simulations have revealed that Arg(38) and His(33) are important for the heterolytic mechanism at potential FFA-OOH binding sites of cyt c (but not for H(2)O(2) or t-BuOOH). Regulation of FFA-OOH metabolism may be an important function of cyt c that is associated with elimination of toxic FFA-OOH and synthesis of physiologically active hydroxy-fatty acids in mitochondria.

Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E, Tocopherol Succinate and Tocopherol Phosphate

“…The resultant peroxidase activity displays significant specificity toward CLs, yielding its highly diversified oxygenated species and their hydrolysis products, predominantly mono-lyso-CLs and oxygenated fatty acids, with signaling modalities (49 -51). CL hydroperoxides can be utilized by the peroxidase complex as an alternative (to H 2 O 2 ) source of oxidizing equivalents, thus "perpetuating" the functioning of the peroxidase cycle (43,47). Although the significance of the peroxidase activation of cytochrome c in apoptosis has been documented (18,20), the uniqueness of CLs as endogenous activators is not unequivocal.…”

“…As an alternative to the H 2 O 2 source of oxidizing equivalents, we tested the fatty acid hydroperoxide, 13 S-hydroperoxy-9Z,11E-octadecadienoic acid (FFA-OOH). FFA-OOH and H 2 O 2 have different binding sites on the cytochrome c molecule: H 2 O 2 was found to occupy a site in the proximity to His 18 , whereas FFA-OOH binds to a different site, with the hydroperoxy group located in proximity to Arg 38 and His 33 (43). In line with previously reported data, FFA-OOHs were much better peroxidase substrates for cytochrome c/TOCL than H 2 O 2 , whereas the peroxidase activity of cytochrome c was only slightly increased by ␣-TOS and remained essentially unaffected by ␣-TOP (Fig.…”

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

“…H 2 O 2 is thought to be the ROS mediating CL-peroxidation, which is catalyzed by the peroxidase activity of the complex CL-Cyt C. 42,43 A recent work by Belikova et al, 63 however, revealed that fatty acid hydroperoxides can also provide the oxidizing equivalents to the peroxidase complex that results in CL-peroxidation. Therefore, it is possible that fatty acid hydroperoxides hydrolyzed by phospholipase-mediated hydrolysis once translocated from the ER to the mitochondria, can directly mediate CL-peroxidation.…”

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

“…CL-bound cytc shows a non-native tertiary structure, a disrupted heme-Fe-Met80 distal bond [12][13][14][15][16][17], and a drastically reduced midpoint potential, which falls out of the range required for its physiological role [18]. Further, the cleavage of the distal Fe-Met80 bond endows cytc with a high affinity for CO and NO [19,20] and peroxidase activity [21][22][23][24][25][26][27].…”

“…The high reactivity of the penta-coordinated CL-cytc complex [11,19,20,26] prompted us to investigate the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)). In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization, even though it has been shown that peroxynitrite may induce the very slow nitration (30 min) of the solvent-exposed Tyr74 residue; this leads to the cleavage of the Fe-Met80 bond, which is substituted by a weak Fe-Lys72 ligation [29].…”