Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors

The effects of both nitric oxide (NO) and peroxynitrite on complexes I (NADH dehydrogenase) and III (cytochrome c reductase) isolated from bovine heart have been examined. EPR signals ("g=2.01") previously detected in association with loss of complex I and III activities in cultured cells and isolated mitochondria subjected to nitrosative stress are shown not to arise from these particular enzymes. Neither NO nor peroxynitrite (ONO(2)(-)) reacts to any appreciable extent with the oxidized forms of flavin mononucleotide, iron-sulfur clusters, or heme moieties found in complexes I and III. However, ONO(2)(-) is readily able to abstract electrons from the reduced forms of both complexes I and III, without any apparent modification of the enzyme cofactors. While no attempt was made in the present study to catalog all the possible modifications, it is clear that ONO(2)(-) can react with the protein moieties of the enzymes. For example, when added in excess, ONO(2)(-) derivatizes a select few tyrosine residues in both complexes I and III forming 3-nitrotyrosine as detected by immunoblots. In the case of complex I, we find a minimum of 3 out of the 46 subunits present were modified (49, approximately 18, and approximately 15kDa); whereas in complex III, 4 out of the 13 subunits stained for 3-nitrotyrosine (46, 27, 7, and 6kDa). Significant irreversible inhibition of activity required the addition of >10(2)-fold excesses of ONO(2)(-) to the enzymes. At 10(3)-fold excess of added ONO(2)(-), the activity of complex I was only diminished by approximately 18%, while a 60% loss of activity was observed for complex III.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Respiratory chain supercomplexes in plant mitochondria

Supercomplexes are defined associations of protein complexes, which are important for several cellular functions. This "quintenary" organization level of protein structure recently was also described for the respiratory chain of plant mitochondria. Except succinate dehydrogenase (complex II), all complexes of the oxidative phosphorylation (OXPOS) system (complexes I, III, IV and V) were found to form part of supercomplexes. Compositions of these supramolecular structures were systematically investigated using digitonin solubilizations of mitochondrial fractions and two-dimensional Blue-native (BN) polyacrylamide gel electrophoresis. The most abundant supercomplex of plant mitochondria includes complexes I and III at a 1:2 ratio (I1 + III2 supercomplex). Furthermore, some supercomplexes of lower abundance could be described, which have I2 + III4, V2, III2 + IV(1-2), and I1 + III2 + IV(1-4) compositions. Supercomplexes consisting of complexes I plus III plus IV were proposed to be called "respirasome", because they autonomously can carry out respiration in the presence of ubiquinone and cytochrome c. Plant specific alternative oxidoreductases of the respiratory chain were not associated with supercomplexes under all experimental conditions tested. However, formation of supercomplexes possibly indirectly regulates alternative respiratory pathways in plant mitochondria on the basis of electron channeling. In this review, procedures to characterize the supermolecular organization of the plant respiratory chain and results concerning supercomplex structure and function are summarized and discussed.     

Supercomplexes in the respiratory chains of yeast and mammalian mitochondria

Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.     

Hybridization in sunfish influences the muscle metabolic phenotype

Hybridization has the potential to exert pleiotropic effects on metabolism. Effects on mitochondrial enzymes may arise through incompatibilities in nuclear- and mitochondrial-encoded subunits of the enzyme complexes of oxidative phosphorylation. We explored the metabolic phenotype of bluegill (Lepomis macrochirus), pumpkinseed (Lepomis gibbosus), and their unidirectional F(1) hybrids (male bluegill × female pumpkinseed). In hybrids, glycolytic enzyme activities were indistinguishable from (aldolase, pyruvate kinase) or intermediate to (lactate dehydrogenase, phosphoglucoisomerase) parentals, but complex IV activities aligned with pumpkinseed, both 30% lower than bluegill. In isolated mitochondria, the specific activities of complexes I, II, and V were indistinguishable between groups. However, both complex III and IV showed indications of depressed activities in hybrid mitochondria, though no effects on mitochondrial state 3 or state 4 respiration were apparent. The patterns in complex IV activities were due to differences in enzyme content rather than enzyme V(max); immunoblots comparing complex IV content with catalytic activity were indistinguishable between groups. The sequence differences in complex IV catalytic subunits (CO1, CO2, CO3) were minor in nature; however, the mtDNA-encoded subunit of complex III (cytochrome b) showed eight differences between bluegill and pumpkinseed, several of which could have structural consequences to the multimeric enzyme, contributing to the depressed complex III catalytic activity in hybrids.     

Identification and characterization of respirasomes in potato mitochondria

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.     

Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Acrolein is an air pollutant from cigarette smoking and other pollutions and also a by-product of lipid peroxidation. Studies have demonstrated that acrolein causes cytotoxicity and genotoxicity, including liver damage and death of hepatocytes. However, the toxic effects and the underlying mechanisms of acrolein on mitochondria, especially, on liver mitochondria, have not been well studied. In the present study, we investigated the toxic effects and mechanisms of acrolein on mitochondria isolated from rat liver by examining mitochondrial respiration, dehydrogenases, complex I, II, III, IV and V, permeability transition, and protein oxidation. Acrolein incubation (10-1000 microM, or 0.02-2 micromol/mg protein) with mitochondria caused dose-dependent inhibition of NADH- and succinate-linked mitochondrial respiration chain, change of mitochondrial permeability transition, increase in protein carbonyls, and selective enzyme inhibition of mitochondrial complex I, II, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, but no effects on mitochondrial complex III, IV, V and malate dehydrogenase. These results suggest that acrolein is a mitochondrial toxin and that mitochondrial dysfunction caused by acrolein may play an important role in acrolein toxicity such as hepatotoxicity and also smoking-related diseases.     

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Malonaldehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines to modify proteins to inactivity enzymes and also modify nucleosides to cause mutagenicity. Mitochondrial dysfunction is a major contributor to aging and age-associated diseases. We hypothesize that accumulated MDA due to mitochondrial dysfunction during aging targets mitochondrial enzymes to cause further mitochondrial dysfunction and contribute to aging and age-associated diseases. We investigated the effects of MDA on mitochondrial respiration and enzymes (membrane complexes I, II, III and IV, and dehydrogenases, including alpha-ketoglutaric dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH)) in isolated rat liver mitochondria. MDA showed a dose-dependent inhibition on mitochondrial NADH-linked respiratory control ratio (RCR) and ADP/O ratio declined from the concentrations of 0.2 and 0.8 micromol/mg protein, respectively, and succinate-linked mitochondrial RCR and ADP/O ratio declined from 1.6 and 0.8 micromol/mg protein. MDA also showed dose-dependent inhibition on the activity of PDH, KGDH and MDH significantly from 0.1, 0.2 and 2 micromol/mg protein, respectively. Activity of the complexes I and II was depressed by MDA at 0.8 and 1.6 micromol/mg protein. However, MDA did not affect activity of complexes III and IV in the concentration range studied (0-6.4 micromol/mg protein). These results suggest that MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes examined to MDA is in the order of PDH>KGDH>complexes I and II>MDH>complexes III and IV.     

Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.     

Mitochondria are an essential mediator of nitric oxide/cyclic guanosine 3',5'-monophosphate blocking of glucose depletion induced cytotoxicity in human HepG2 cells

It is well known that glucose is a major energy source in tumors and that mitochondria are specialized organelles required for energy metabolism. Previous studies have revealed that nitric oxide (NO) protects against glucose depletion-induced cytotoxicity in mouse liver cells and in rat hepatocytes, but the detailed mechanism is not well understood. Therefore, we investigated the involvement of mitochondria in the NO protective effect in human hepatoma HepG2 cells. In this study, we showed that glucose depletion resulted in a time-dependent decrease in intracellular NO and in the protein expression of NO synthases. This glucose depletion-induced decrease in NO was blocked by NO donors. Next, we showed that the cytoprotective effect of NO is via a cyclic guanosine 3',5'-monophosphate-dependent pathway. Additionally, SNP blocked a glucose depletion-induced decrease in mitochondrial mass, mitochondrial DNA copies, and ATP level in HepG2 cells. Moreover, glucose depletion decreased the expression of various mitochondrial proteins, including cytochrome c, complex I (NADH dehydrogenase), complex III (cytochrome c reductase), and heat shock protein 60; these glucose depletion-induced effects were blocked by SNP. Furthermore, we found that rotenone and antimycin A (mitochondria complex I and III inhibitors, respectively) blocked SNP cytoprotection against glucose depletion-induced cytotoxicity. Taken together, our results indicated that the mitochondria serve as an important cellular mediator of NO during protection against glucose deprivation-induced damage.     

Functional and molecular analysis of mitochondria in thyroid oncocytoma

We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes I (NADH dehydrogenase), II (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.     

Effect of endogenous nitric oxide on energy metabolism of rat heart mitochondria during ischemia and reperfusion

The effects of ischemia and postischemic reperfusion on the functions of the heart and its mitochondria were studied with special attention to the effect of nitric oxide (NO) by treatment of rat hearts with the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) or its noninhibitory isomer N^G-nitro-D-arginine methyl ester (D-NAME). NO generated during reperfusion caused increase in coronary flow (CF), but had no effect on the left ventricular pressure (LVP) or heart rate (HR). The ATP level of the heart decreased during ischemia and was not completely restored by introduction of oxygen during reperfusion due to damage of complexes I and II of the respiratory chain of mitochondria by NO. Inhibition of the respiratory chain resulted in generation of hydrogen peroxide, and NO and NO-derived species generated after production of NO caused further damage of various proteins in mitochondria, such as complexes I and II of the respiratory chain and pyruvate dehydrogenase (PDH). These results suggested that NO generated on reperfusion was the primary cause of mitochondrial dysfunction by damage of complexes I and II of the respiratory chain, with consequent increase of CF in the heart.     

Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.     

Functional and molecular analysis of mitochondria in thyroid oncocytoma.

We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes 1 (NADH dehydrogenase), 11 (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.     

Free radical chemistry in biological systems

Mitochondria are an active source of the free radical superoxide (O2-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O2 uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O2. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O2 uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O2- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

Nitric oxide-mediated mitochondrial damage: A potential neuroprotective role for glutathione

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II–III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, l-buthionine-[S,r]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II–III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-γ for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II–III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.     

Structure, function, and assembly of heme centers in mitochondrial respiratory complexes

The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O(2), is mediated by protein-bound redox cofactors. In mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc(1) complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a(3), with Cu(B), form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc(1), whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Selective inhibition of NADH-CoQ oxidoreductase (complex I) of rat brain mitochondria by arachidonic acid

The effects of arachidonic acid on the enzyme complexes in the electron transport system were investigated using submitochondrial particles from rat brain. Arachidonic acid irreversibly inhibited NADH-CoQ oxidoreductase (complex I) activity, but had no effect on the activities of succinate-CoQ oxidoreductase (complex II), CoQH2-cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), ATPase (complex V), glutamate dehydrogenase, and malate dehydrogenase up to 50 microM. The inhibition was dose-dependent with an IC50 value of 110 nmol/mg protein. The Lineweaver-Burk plot revealed that the inhibition by arachidonic acid was noncompetitive against CoQ with a Ki value of 33 microM and uncompetitive against NADH with a Ki value of 22 microM.     

Astrocyte-derived nitric oxide causes both reversible and irreversible damage to the neuronal mitochondrial respiratory chain

Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.