Reactions of heme peroxidases with peroxynitrite

The interaction of peroxynitrite, produced by ozonation of azide, with two heme peroxidases (horseradish peroxidase and lactoperoxidase) was studied. Enzymes retained full activity after incubation with peroxynitrite at neutral pH. Lactoperoxidase alone was found to catalyze peroxynitrite decomposition, whereas horseradish peroxidase accelerated peroxynitrite decomposition only in the presence of certain substrates. For example, in the presence of guaiacol the catalyzing effect was clear, but in the presence of trolox was only noticeable.     

The formation of peroxynitrite in the applied physiology of mitochondrial nitric oxide

Mitochondria require nitric oxide (NO) to exert a delicate control of metabolic rate as well as to regulate life functions, cell cycle activation and arrest, and apoptosis. All activities depend on the matrical NO steady state concentration as provided by mitochondrial (mtNOS) and cytosolic sources (eNOS) and reduced by forming superoxide anion and H₂O₂ and a low peroxynirite (ONOO⁻) yield. We review herein the biochemical pathways involved in the control of NO mitochondrial level and its biological and physiological significance in hormone effects and aging. At high NO, the cost of this physiological regulation is that ONOO⁻ excess will lead to nitrosation/nitration and oxidization of mitochondrial and cell proteins and lipids. The disruption of NO modulation of mitochondrial respiration supports then, a platform for prevalent neurodegenerative and metabolic diseases.     

Reactions of manganese porphyrins with peroxynitrite and carbonate radical anion

We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO-) and carbonate radical anion (CO3.) using stopped-flow and pulse radiolysis, respectively. Rate constants for the reactions of Mn(III) porphyrins with ONOO- ranged from 1 x 10(5) to 3.4 x 10(7) m(-1) s(-1) and correlated well with previously reported kinetic and thermodynamic data that reflect the resonance and inductive effects of the substituents on the porphyrin ring. Rate constants for the reactions of Mn(III) porphyrins with CO3. ranged from 2 x 10(8) to 1.2 x 10(9) m(-1)s(-1) at pH 相似文献   

Degradation of extracellular matrix by peroxynitrite/peroxynitrous acid

The extracellular matrix (ECM) provides strength and elasticity to tissues and plays a key role in regulating cell behavior; damage to this material is believed to be a major factor in many inflammatory diseases. Peroxynitrite/peroxynitrous acid, which is generated at elevated levels at sites of inflammation, is believed to play a role in ECM damage; however, the mechanisms involved are poorly understood. Here we examined the reactions of bolus peroxynitrite, and that generated in a time-dependent manner by SIN-1 decomposition, with ECM isolated from a vascular smooth muscle cell line and porcine thoracic aorta. Bolus peroxynitrite caused the release of ECM glycosaminoglycans and proteins, the formation of 3-nitroTyr, and the detection of ECM-derived radicals (by immuno-spin trapping) in a concentration-dependent manner. Release and nitration of ECM components were modulated by the local pH and bicarbonate. SIN-1 caused the release of glycosaminoglycan, but not protein, from vascular smooth muscle cell-derived ECM in a concentration-, time-, and pH-dependent manner. The data presented here suggest that peroxynitrite-mediated damage to ECM occurs via a radical-mediated pathway. These reactions may contribute to ECM damage at sites of inflammation and play a role in disease progression, including rupture of atherosclerotic lesions.     

Reactions of peroxynitrite with globin proteins and their possible physiological role

It is now widely accepted that, besides their well-established function in O(2) transport, hemoglobin and myoglobin also undergo several redox reactions aimed to scavenge toxic free radicals and reactive oxygen and nitrogen species. At least some of these reactions are believed to play an important physiological role in the defense against oxidative stress. This aspect is exemplified by the recently discovered neuroglobin, a globin expressed in the brain. Rather than being considerably involved in reversible O(2) binding, neuroglobin is likely to undergo redox reactions to protect neurons against oxidative and potentially pathogenic pathways, as those operating after episodes of tissue hypoxia or ischemia. A major part of the cellular damage occurring under such conditions has been ascribed to formation of peroxynitrite, that originates from the reaction between two biologically important free radicals, nitric oxide (NO ) and superoxide. Here we review the current knowledge of the reactions of different forms of hemoglobin, myoglobin, and neuroglobin with peroxynitrite and discuss their physiological role on the basis of measured rate constants and on the probability of occurrence of these reactions in vivo.     

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia.

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.     

Protein export from the mitochondrial matrix

Assembly of a functional mitochondrion requires import of proteins from the cytosol and export of proteins from the matrix. Most previous studies have focused on the import pathway followed by nucleus-encoded proteins. However, it is now clear that proteins encoded in the nucleus as well as those encoded in the mitochondrion also move from the matrix into and across the inner membrane, a process defined here as export. These exported proteins are found in at least three cellular locations: the inner mitochondrial membrane, the intermembrane space and the cell surface. Here, we consider the pathways for export and the relationships between import and export.     

Superoxide released into the mitochondrial matrix

Reactive oxygen species (ROS) that are produced by mitochondria are released toward the mitochondrial matrix or the intermembrane space. Each ROS pool is likely involved in different cellular mechanisms and damage. Unfortunately, it is difficult to distinguish the provenance and effects of ROS. Here we introduce a method to semiquantitate the steady-state levels of superoxide produced in the matrix of mitochondria. Superoxide produced during cellular respiration is capable of oxidizing hydroethidine, a probe that is membrane permeant. The poor membrane permeability of the hydroethidine oxidation products causes accumulation of these fluorescent products within the mitochondria. After isolation of mitochondria, a method based on the capillary electrophoretic separation of individual organelles and their detection by laser-induced fluorescence detection is used to determine their fluorescent contents. Use of this method for the analysis of organelle fractions obtained from cells treated with antimycin A or rotenone confirms that the detected fluorescence is associated with superoxide produced by mitochondria. Furthermore, using this method the superoxide levels in the mitochondrial matrix of a cytoplasmic hybrid (cybrid) cell line (DeltaH2-1) and one of its parent cell lines (143B) were compared.     

Methamphetamine-induced inhibition of mitochondrial complex II: roles of glutamate and peroxynitrite

High-dose methamphetamine (METH) is associated with long-term deficits in dopaminergic systems. Although the mechanism(s) which contributes to these deficits is not known, glutamate and peroxynitrite are likely to play a role. These factors are hypothesized to inhibit mitochondrial function, increasing the free radical burden and decreasing neuronal energy supplies. Previous studies suggest a role for the mitochondrial electron transport chain (ETC) in mediating toxicity of METH. The purpose of the present studies was to determine whether METH administration selectively inhibits complex II of the ETC in rats. High-dose METH administration (10 mg/kg every 2 h x 4) rapidly (within 1 h) decreased complex II (succinate dehydrogenase) activity by approximately 20-30%. In addition, decreased activity of complex II-III, but not complex I-III, of the mitochondrial ETC was also observed 24 h after METH. This inhibition was not due to direct inhibition by METH or METH-induced hyperthermia and was specific to striatal brain regions. METH-induced decreases in complex II-III were prevented by MK-801 and the peroxynitrite scavenger 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-sulphonatophenyl) porphinato iron III. These findings provide the first evidence that METH administration, via glutamate receptor activation and peroxynitrite formation, selectively alters a specific site of the ETC.     

Reactions of *NO, *NO2 and peroxynitrite in membranes: physiological implications

Nitric oxide (^·NO) and nitrogen dioxide (^·NO₂) are hydrophobic gases. Therefore, lipid membranes and hydrophobic regions of proteins are potential sinks for these species. In these hydrophobic environments, reactive nitrogen species will exhibit different chemistry than in aqueous environments due to higher local concentrations and the lack of hydrolysis reactions. The peroxynitrite anion (ONOO^-) and peroxynitrous acid (ONOOH) can freely pass through lipid membranes, making peroxynitrite-mediated reactions in a hydrophobic environment also of extreme relevance. The reactions observed by these reactive nitrogen species in a hydrophobic milieu include oxidation, nitration and even potent chain-breaking antioxidant reactions. The physiological and toxicological relevance of these reactions is discussed.     

Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.     

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000muM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.     

Charge properties of mitochondrial matrix proteins.

Proteins of the rat liver mitochondrial matrix have been separated into anionic (acidic), cationic(basic), and neutral groups by electrophoresis. These groups represent 69, 8, and 23% of the total matrix protein, respectively, compared to 69, 21, and 10% for the cytosol protein. The acidic nature of the mitochondrial matrix proteins has been confirmed by cellulose ion-exhange chromatography, isoelectric focusing in sucrose gradients, and amino acid analysis. The anionic, cationic, and neutral matrix proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 18, 6, and 5 bands, respectively, compared to 22 bands for the total fraction. The significance of the charge properties of these proteins in terms of mitochondrial biogenesis is discussed.     

Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Delta cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.     

Purification and immunohistochemical study of actin in mitochondrial matrix

Actin was purified to apparent homogeneity from the matrix of ultra-pure mitochondria of rat livers by DNase-I affinity chromatography and HPLC gel filtration. The mitochondrial actin was immunologically identified by an anti-actin antibody, and its apparent molecular weight was 43 KDa, as determined by SDS-polyacrylamide gel electrophoresis. The immunohistochemical study revealed the localization of the mitochondrial actin in the matrix space and on the internal surface of inner membrane. The actin fraction eluted from a DNase-I column by KCl-EGTA solution underwent polymerization and bundling in vitro.     

Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5'-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5' bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that 2'-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2'-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2'-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.     

Apoptosis induces efflux of the mitochondrial matrix enzyme deoxyguanosine kinase

Deoxyguanosine kinase (dGK) initiates the salvage of purine deoxynucleosides in mitochondria and is a key enzyme in mitochondrial DNA precursor synthesis. The active form of the enzyme is a 60-kDa protein normally located in the mitochondrial matrix. Here we describe the subcellular distribution of dGK during apoptosis in human epithelial kidney 293 cells and human lymphoblast Molt-4 cells. Immunological methods were used to monitor dGK as well as other mitochondrial proteins. Surprisingly, dGK was found to relocate to the cytosolic compartment at a similar rate as cytochrome c, a mitochondrial intermembraneous enzyme known to enter the cytosol early in apoptosis. The redistribution of dGK from the mitochondria to the cytosol may be of importance for the activation of apoptotic purine nucleoside cofactors such as dATP and demonstrates that mitochondrial matrix proteins may selectively leak out during apoptosis.     

Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite

Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O(2)(.-)) as a central mediator. However, mitochondrial O(2)(.-) and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O(2)(.-) through different techniques, along with the oxidants derived from it. Mitochondrial aconitase, a sensitive target of O(2)(.-), was inactivated 60% in BAECs incubated in 30 mM glucose (hyperglycemic condition) with respect to cells incubated in 5 mM glucose (normoglycemic condition). Under hyperglycemic conditions, increased oxidation of the mitochondrially targeted hydroethidine derivative (MitoSOX) to hydroxyethidium, the product of the reaction with O(2)(.-), could be specifically detected. An 8.8-fold increase in mitochondrial O(2)(.-) steady-state concentration (to 250 pM) and formation rate (to 6 microM/s) was estimated. Superoxide formation increased the intracellular concentration of both hydrogen peroxide, measured as 3-amino-2,4,5-triazole-mediated inactivation of catalase, and nitric oxide-derived oxidants (i.e., peroxynitrite), evidenced by immunochemical detection of 3-nitrotyrosine. Oxidant formation was further evaluated by chloromethyl dichlorodihydrofluorescein (CM-H(2)DCF) oxidation. Exposure to hyperglycemic conditions triggered the oxidation of CM-H(2)DCF and was significantly reduced by pharmacological agents that lower the mitochondrial membrane potential, inhibit electron transport (i.e., myxothiazol), and scavenge mitochondrial oxidants (i.e., MitoQ). In BAECs devoid of mitochondria (rho(0) cells), hyperglycemic conditions did not increase CM-H(2)DCF oxidation. Mitochondrial O(2)(.-) formation in hyperglycemic conditions was associated with increased glucose metabolization in the Krebs cycle and hyperpolarization of the mitochondrial membrane.