Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells.

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics.

Effects of trichlorotelluro-dypnones on mitochondrial bioenergetics and their relationship to the reactivity with protein thiols.

The effect of four trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4, on the bioenergetics of isolated rat liver mitochondria (RLM) and cells was investigated. In a dose-dependent manner, the studied organotelluranes promoted Ca(2+)-dependent mitochondrial swelling inhibited by cyclosporine A and were associated with a decrease of the total mitochondrial protein thiol content. These effects characterize the opening of the classical mitochondrial permeability transition pore. Despite the reactivity with mitochondrial protein thiol groups, these compounds did not promote significant glutathione depletion. In the absence of Ca(2+), the organotelluranes promoted mitochondrial loss of ΔΨ in RLM concomitant with respiratory control decrease due to an increase of the state 4 respiration rate. In these conditions, mitochondrial swelling was absent, and thiol content was higher than that in the presence of Ca(2+). The differentiated effects observed in the presence and absence of Ca(2+) are probably related to the effects of that ion on membrane structure, with repercussions for the exposure of specific reactive protein thiol groups. In smooth muscle cells, these compounds promoted the loss of mitochondrial ΔΨ and apoptosis. The loss of ΔΨ was not preceded by a decrease of cell viability that is consistent with mitochondria as the primary targets for the action of these organotelluranes.

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 (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK(a) ~ 6.9). Conversely, ΔpH generated by enhanced pH(in) of PCPECL at a pH(out) 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 pH(in) at a pH(out) of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), 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 ΔΨ(M) blocks inner mitochondrial membrane fusion during apoptosis.

Specific effects of reactive thiol drugs on mitochondrial bioenergetics.

In this minireview, the more recent findings about the effects of peculiar reactive thiol drugs on mitochondria are presented. These include the following compounds: metallo meso-tetrakis porphyrins, palladacycles, telluranes and phenothiazines. Metallo meso-tetrakis porphyrins can exhibit both beneficial and deleterious effects on mitochodria that are modulated by the central metal, cell location, and availability of axial ligands. Therefore, these compounds have the versatility to be used for cell and mitochondria protection and death. The antioxidant activity of manganese porphyrins is related to a glutathione peroxidase-like activity. By attacking exclusively the membrane protein thiol groups without glutathione depletion, palladacycles are able to induce mitochondrial permeability transition (MPT) and cytochrome c release in the absence of oxidative stress. In hepatoma cells, the mitochondrial action of palladacycles was able to induce apoptotic death. As opposed to palladacycles, telluranes and phenothiazines are able to conjugate the capacity to promote the MPT in a dose-dependent manner in association with efficient antioxidant activity toward lipids. These studies demonstrated that the action of drugs on mitochondrial bioenergetics can be modulated by peculiar reactivity with thiol groups. Therefore, they contribute to studies of toxicity as well as the design of new drugs.

Study of respiratory cytochromes in liposomes.

Important findings regarding the structure and function of respiratory cytochromes have been made from the study of these hemeproteins associated to liposomes. These studies contributed to the comprehension of the biological role of these proteins in the electron transfer process, the regulatory mechanisms, the energy transduction mechanisms, the protein sites that interact with mitochondrial membranes and the role played by the non-redox subunits present in the protein complexes of the respiratory chain of eukaryotes. In this chapter, the protocols developed to study cytochrome bc (1) activity in liposomes and the binding of cytochrome c to lipid bilayers is presented . The former protocol was developed to study the mechanism of energy transduction related to the topology of the components of bc (1) complex in the mitochondrial membrane. These studies were done with purified cytochrome bc (1) complexes reconstituted into potassium-loaded vesicles. The latter protocol was developed to study the influence of pH, DeltapH, and DeltaPsi on the interaction of cytochrome c with liposomes that mimic the inner mitochondrial membrane.

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.

Organotellurane-promoted mitochondrial permeability transition concomitant with membrane lipid protection against oxidation.

Organotelluranes exhibit potent antioxidant properties as well as the ability to react with protein thiol groups and, thereby, they are good models to study the mechanism of the mitochondrial permeability transition (MPT). We evaluated the effects of the concentration of organotelluranes, namely RT-03 and RT-04, on rat liver mitochondria. At the concentration range of 0.25-1.0 microM, organotelluranes did not cause any mitochondrial dysfunction. At the concentration range of 5-10 microM, RT-03 and RT-04 caused the Ca2+-dependent opening of the (MPT) pore, regulated by Cyclosporin A. At the concentration range of 15-30 microM the swelling was not inhibited by Cyclosporin A and in the absence of Ca2+, a significant decrease of respiratory control ratio was observed due to concomitant phosphorylation impairment and uncoupling, transmembrane potential disruption, depletion of mitochondrial reduced thiol groups, and alterations in the bilayer fluidity. Above 100 microM, the organotelluranes caused complete inhibition of respiratory chain. Over the whole studied concentration range, RT-03 and RT-04 did not induce mitochondrial oxidative stress assessed by using the reactive oxygen and nitrogen species indicator 2',7'-dichlorodihydrofluorescein diacetate. Further, the organotelluranes also exhibited protective effect against t-butyl hydroperoxide-induced oxidative stress as well as against Fe2+/citrate-induced peroxidation of mitochondrial membranes and PCPECL liposomes. These results point out that MPT pore opening can involve damage exclusively to mitochondrial membrane proteins. The exclusive antioxidant activity observed at nanomolar range is also an interesting new finding described in this work.