Iseli L. Nantes

Effect of Heme Iron Valence State on the Conformation of Cytochrome c and Its Association with Membrane Interfaces A CD AND EPR INVESTIGATION

Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochromec in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300–600 and 200–320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe3+. The studies also include the effect of Pi, NaCl, and CaCl2. Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome cwith charged interfaces promotes conformational changes in the α-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochromec. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl2, NaCl, and Pi, in response to the increase of the ionic strength.

Baccharis dracunculifolia, the main source of green propolis, exhibits potent antioxidant activity and prevents oxidative mitochondrial damage

Baccharis dracunculifolia DC (Asteraceae) is the main botanical source used by honeybees to produce Brazilian green propolis whose hepatoprotective properties have been already described. In this work we investigated the protective effects of the glycolic extract of B. dracunculifolia (GEBd) against oxidative stress in isolated rat liver mitochondria (RLM). The GEBd was prepared by fractionated percolation using propylene glycol as solvent. The total phenols and flavonoids, which are substances with recognized antioxidant action, were quantified in GEBd and the phytochemical analysis was carried out by HPLC. GEBd exhibited significant scavenger activity towards DPPH radicals and superoxide anions in a concentration-dependent manner, and also a Fe2+ chelating activity. GEBd decreased the basal H2O2 generation and the Fe2+- or t-BuOOH-induced ROS production in isolated mitochondria. Lipid oxidation of mitochondrial membranes, protein thiol groups and GSH oxidation were also prevented by GEBd. This shows that B. dracunculifolia exhibit potent antioxidant activity protecting liver mitochondria against oxidative damage and such action probably contribute to the antioxidant and hepatoprotective effects of green propolis.

Modulation of cytochrome c spin states by lipid acyl chains: a continuous- wave electron paramagnetic resonance (CW-EPR) study of haem iron

This work is a systematic study, showing a clear correlation between the nature of the lipid acyl chain and the spin states of cytochrome c interacting with different types of lipid membranes. According to the lipid acyl chain type, and the head group charge present in the bilayer, three spin states of cytochrome c were observed in different proportions: the native cytochrome c low spin state with rhombic symmetry (spin 1/2, g axially=3.07 and g radially=2.23), a low spin state with less rhombic symmetry (spin 1/2, g(1)=2.902, g(2)=2.225, and g(3)=1.510) and the high spin state (spin 5/2, g axially=6.0 and g radially=2.0). The proportion of the spin states of cytochrome c bound to bilayers was also dependent on the lipid/protein ratio, suggesting the existence of two or more protein sites interacting with the lipids. The lipid-induced alterations in the symmetry and spin states of cytochrome c exhibited partial reversibility when the ionic strength was increased, which reinforces the crucial role played by the electrostatic interaction with the lipid bilayer. Different cytochrome c spin states exhibited corresponding modifications in the haemprotein UV/visible spectra, particularly in the Q-band associated with loss of the 695 nm band and appearance of a band in the region of 600-650 nm. The observed reactivity of cytochrome c with oxidized forms of unsaturated lipids reinforces the possibility of the acyl chain insertion in the haemprotein structure.

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C.

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.

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.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca2+ dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 microM focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the PTP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since PTZ give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that PTZ bury in the inner mitochondrial membrane and the chemically generated PTZ cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MPT induction and may have implications for the cell death induced by PTZ.

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.

Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.

Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III

Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (K(d)), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K(d) values were 17 nM and 100 nM respectively. The CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K(d) values. The synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-d-glucosamine; G, beta-d-glucuronic acid; A*, N,3,6-O-sulphated alpha-d-glucosamine; I, 2-O-sulphated alpha-l-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500 Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.