Cintia Kawai

Protective role of mitochondrial unsaturated lipids on the preservation of the apoptotic ability of cytochrome C exposed to singlet oxygen.

Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c (cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PCPECL) liposomes (50/30/20%): totally saturated lipids (tsPCPECL), totally unsaturated lipids (tuPCPECL), partially unsaturated (80%) lipids, with unsaturation in the PC and PE content (puPCPECL80), and partially unsaturated (20%) lipids, with unsaturation in the CL content (puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. In the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage (lipid-protected cytochrome c). In CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. The presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function.

pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pKa values and site L containing ionizable groups with pKaobs values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, we demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pH-independent binding (microscopic dissociation constant Ksapp2, approximately 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pKa of approximately 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on Ksapp1 was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed Ksapp1 values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site L ionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Not Only Oxidation of Cardiolipin Affects the Affinity of Cytochrome c for Lipid Bilayers

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

Towards the mechanisms involved in the antioxidant action of MnIII [meso-tetrakis(4-N-methyl pyridinium) porphyrin] in mitochondria

Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, MnIIITMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of MnIIITMPyP. SOD and cytochrome c antioxidant activities were also investigated. MnIITMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, MnIIITMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for MnIIITMPyP and do not exclude cytochrome c-like activity. However, considering that para MnIIITMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the MnIITMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of MnIITMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of MnIIITMPyP, whose efficiency is dependent on the NADPH/GSH content in cells.

Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes

The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pHout) of PCPECL liposomes, with an internal pH (pHin) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pKa ~ 6.9). Conversely, ΔpH generated by enhanced pHin of PCPECL at a pHout of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pHin at a pHout of 8.0. At bulk acidic pH, ΔΨ generated by Na+ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pHout, the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨM blocks inner mitochondrial membrane fusion during apoptosis.

Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso-Tetrakis Porphyrin Free Bases

The photodynamic effects of the cationic TMPyP (meso‐tetrakis [N‐methyl‐4‐pyridyl]porphyrin) and the anionic TPPS4 (meso‐tetrakis[4‐sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D2O, and was impaired by sodium azide and sorbic acid. The effect of D2O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long‐lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D2O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.

Interaction and Reaction of the Antioxidant Mn III [Meso-Tetrakis(4-NMethyI Pyridinium) Porphyrin] with the Apoptosis Reporter Lipid Phosphatidylserine

In the present study, the binding of the cationic MnTMPyP [meso-tetrakis (4-N-methyl pyridinium) porphyrin] to negatively charged membrane models containing phosphatidylcholine (PC)/ phosphatidylserine (PS) and the porphyrin reactivity with PS-derived peroxides (LOOH) were evaluated. This investigation is relevant due to the participation of PS in cell signaling and apoptosis. Addition of PC/PS liposomes to MnTMPyP solutions led to spectral changes of the porphyrin electronic absorption spectrum suggestive of electrostatic interactions with the negatively charged interface provided by PS. The binding of MnTMPyP to PC/PS liposomes was corroborated by the quenching of the fluorophore merocyanine 540. In addition total energy calculations of saturated and unsaturated PS based on the spinpolarized variant of KS-DFT were used to support some experimental data. Cyclic voltammetry analysis revealed that the association to PC/PS liposomes shifted the redox potential of the Mn/Mn (+87 mV vs NHE, in aqueous buffered solution) couple to a more positive value to (+110 mV vs NHE). This event favors the oxidation of O2 -• to O2 by the porphyrin. MnTMPyP was able to react with the lipid-derived peroxides as evidenced by spectral changes of the porphyrin consistent with the generation of a high valence state (Mn=O) of the catalyst. The spectral changes were not observed when biological lipids containing unsaturated PC and PS were replaced by the corresponding dipalmitoyl (DP)-