Effects of tamoxifen on the electron transport chain of isolated rat liver mitochondria

Tamoxifen (and 4-hydroxytamoxifen), a nonsteroidal triphenylethylene antiestrogenic drug widely used in the treatment of breast cancer, interacts strongly with the respiratory chain of isolated rat liver mitochondria. The drug acts as both an uncoupling agent and a powerful inhibitor of electron transport. Tamoxifen brings about a collapse of the membrane potential. Enzymatic assays and spectroscopic studies indicate that tamoxifen inhibits electron transfer in the respiratory chain at the levels of complex III (ubiquinol–cytochrome-c reductase) and, to a lesser extent, of complex IV (cytochrome-c oxidase). The activities can be restored by the addition of diphosphatidylglycerol, a phospholipid implicated in the functioning of the respiratory chain complexes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain

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Adenylate control of respiration in plants: the contribution of rotenone-insensitive electron transport to ADP-limited oxygen consumption by soybean mitochondria

The aim was to test the hypothesis that rotenone-insensive electron transport (bypass of complex I) may underlie rapid state 4 (ADP-limited) mitochondrial respiration. A comparison of mitochondria from soybean ( Glycine max L. cv. Bragg) cotyledons and nodules showed that ADP-sufficient (state 3) malate plus pyruvate oxidation by mitochondria from 7-day-old cotyledons was inhibited 50% by rotenone and state 4 rates were rapid, whereas nodule mitochondria were 80% inhibited by rotenone and had slower state 4 rates of malate plus pyruvate oxidation. Respiration of malate alone (pH 7.6) by cotyledon mitochondria was slow, especially in the absence of ADP; subsequent addition of pyruvate dramatically increased state 4 oxygen uptake concomitant with a rapid rise in mitochondrial NADH (determined by fluorimetry). Rotenone had no effect on this increased rate of state 4 respiration. The rate of malate oxidation by nodule mitochondria was relatively rapid compared with cotyledon mitochondria. The addition of pyruvate in state 4 caused a slow increase in matrix NADH and only a slight stimulation of oxygen uptake. Rotenone inhibited state 4 malate plus pyruvate oxidation by 50% in these mitochondria. From a large number of cotyledon and nodule mitochondrial preparations, a close correlation was found between the rate of state 4 oxygen uptake and rotenone-resistance. During cotyledon development increased rotenone-resistance was associated with an increase in the alternative oxidase. Addition of pyruvate to cotyledon mitochondria, during state 4 oxidation of malate in the presence of antimycin A, significantly stimulated O₂ uptake and also almost eliminated respiratory control. Such combined operation of the rotenone-insensitive bypass and the alternative oxidase in vivo will significantly affect the extent to which adenylates control the rate of electron transport.     

Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism

Mitochondria play important roles in generation of free radicals, ATP formation, and in apoptosis. We studied the levels of mitochondrial electron transport chain (ETC) complexes, that is, complexes I, II, III, IV, and V, in brain tissue samples from the cerebellum and the frontal, parietal, occipital, and temporal cortices of subjects with autism and age-matched control subjects. The subjects were divided into two groups according to their ages: Group A (children, ages 4-10 years) and Group B (adults, ages 14-39 years). In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p<0.05), of complex I in the frontal cortex (p<0.05), and of complexes II (p<0.01), III (p<0.01), and V (p<0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism. In the cerebellum and temporal cortex, no overlap was observed in the levels of these ETC complexes between subjects with autism and control subjects. In the frontal cortex of Group A, a lower level of ETC complexes was observed in a subset of autism cases, that is, 60% (3/5) for complexes I, II, and V, and 40% (2/5) for complexes III and IV. A striking observation was that the levels of ETC complexes were similar in adult subjects with autism and control subjects (Group B). A significant increase in the levels of lipid hydroperoxides, an oxidative stress marker, was also observed in the cerebellum and temporal cortex in the children with autism. These results suggest that the expression of ETC complexes is decreased in the cerebellum and the frontal and temporal regions of the brain in children with autism, which may lead to abnormal energy metabolism and oxidative stress. The deficits observed in the levels of ETC complexes in children with autism may readjust to normal levels by adulthood.     

A generic model of electron transport in mitochondria

In this paper, a simplified, generic model of mitochondrial metabolism is explored. In particular the following question is addressed: To what extent are phenomena observed in experiments and simulations of mitochondrial metabolism generic, in the sense that they must occur in all models with this basic structure? Of particular interest are the electron transport chain and oxidative phosphorylation, and how flux through the system and the redox states of intermediates respond to physiologically important stimuli. These stimuli include changes in substrate supply (NADH/FADH(2)), in oxygenation, and in membrane proton gradient/ATP demand. Analytical techniques are used to show that certain experimentally observed effects must occur in the generic model. These include the responses of both flux and redox states to changed substrate and oxygen concentrations. At the same time other effects, such as the responses of redox states to changes in proton gradient, are dependent on the details of the model, and are not common to every model with the same basic structure. The phenomenon of saturation in response to large inputs is also discussed.     

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

The inhibition of photosynthetic electron transport by methyl parathion

The effect of methyl parathion (metacid-50), an organophosphorous insecticide, on the Hill reactions of isolated mesophyll chloroplasts ofSorghum vulgare was studied. The pesticide was found to inhibit the Hill reaction with all the Hill oxidants tested, namely potassium ferricyanide,2,6-dichlorophenol indophenol and para-benzoquinone. The concentration of the pesticide required to inhibit 50% of the control Hill activity (I₅₀value) was found to vary with the different Hill oxidants.     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.     

Transplasma membrane electron transport in plants

The presence of transplasma membrane electron transport in a variety of plant cells and tissues is reported. It is now agreed that this property of eukaryotic cells is of ubiquitous nature. Studies with highly purified plasma membranes have established the presence of electron transport enzymes. Two types of activities have been identified. One, termed Standard reductase, is of general occurrence. The other, inducible under iron deficiency and relatively more active, is Turbo reductase. However, the true nature of components participating in electron transport and their organization in the plasma membrane is not known. The electron transport is associated with proton release and uses intracellular NAD(P)H as substrate. The electron flow leads to changes in intracellular redox status, pH, and metabolic energy. The responsiveness of this system to growth hormones is also observed. These findings suggest a role for electron flow across the plasma membrane in cell growth and regulation of ion transport. Involvement of this system in many other cellular functions is also argued.     

Factors limiting respiration by isolated cauliflower mitochondria

Oxygen consumption by isolated cauliflower mitochondria oxidising malate, succinate or NADH was less than when a combination of any two substrates was supplied. The rates obtained with any two were less than the aggregate of the individual rates. Only with the combination of malate plus succinate did the presence of the NADH result in faster rates of oxygen uptake. Conversely, malate and succinate separately, or in combination, inhibited the oxidation of erogenous NADH. The rate-limiting step for the oxidation of these substrates lies within the respiratory chain but on that part used exclusively by each substrate. The addition of NADH with either malate or succinate, or both, saturates the chain and makes the rate limiting step a component common to all pathways. Malate and isocitrate oxidation were considerably greater in disrupted, than in intact mitochondria, eliminating substrate dehydrogenases as rate-limiting factors. Substrate entry was also eliminated in the case of malate oxidation. It is suggested that the tricarboxylate transporter may restrict the rate of isocitrate oxidation.     

Direct interaction between linear electron transfer chains and solute transport systems in bacteria

Abstract In studies on alanine and lactose transport in Rhodopseudomonas sphaeroides we have demonstrated that the rate of solute uptake in this phototrophic bacterium is regulated by the rate of lightinduced cyclic electron transfer.
In the present paper the interaction between linear electron transfer chains and solute transport systems was studied in Rhodopseudomonas sphaeroides and Escherichia coli .
The results demonstrate that the activities of alanine transport in Rps. sphaeroides and lactose and proline transport in E. coli are directly controlled by the electron transfer activity in the respiratory chain, under conditions that the proton-motive force remains constant.     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport.

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μ ) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μ -succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μ ; 4 μ -antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μ ) and antimycin A (4 μ ) by 110%. Azide (1 m ) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 m -KCN ( ). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

线粒体电子传递系统的组装及其生物学意义

电子传递链亦称呼吸链,由位于线粒体内膜的I、II、III、IV 4种复合物组成,负责电子传递和产生质子梯度。电子主要从复合物I进入电子传递链,经复合物III传递至复合物IV。电子传递系统的组装是一个十分复杂的过程,目前已知主要有约69个结构亚基以及至少16个组装因子参与了人类复合物I、III、IV的组装,这些蛋白质由核基因组与线粒体基因组共同编码。对线粒体电子传递系统的蛋白质组成及其结构已研究得较为清楚,但对它们的组装了解得还比较初步。许多人类线粒体疾病是由于电子传递系统的功能障碍引起的,其中又有许多是由于该系统中一个或多个部件的错误组装引起的。研究这些缺陷不仅能够加深对线粒体疾病发病机理的了解,也有助于揭示线粒体功能的调控机制。将着重对电子传递系统复合物的组装及其与人类疾病关系的研究进展进行综述。     

Phosphate transport in rat liver mitochondria: Location of sulfhydryl groups essential for transport activities

The membrane orientation and symmetry of protein thiol group(s) necessary for transport of P_i in rat liver mitochondria have been assessed by comparing inhibition of transport in intact mitochondria to that in inverted vesicles of purified inner membrane. The permeability characteristics of a variety of inhibitors have been determined under specified conditions. The sensitivities of the uptake pathways in mitochondria and in inverted vesicles appear thus far to be identical. By comparing results with permeant and nonpermeant inhibitors, or sequential treatment with different inhibitors, arguments can be made in favor of a single reorienting site of thiol sensitivity.DABS p-(diazonium)-benzenesulfonic acid - IMV inner membrane vesicles - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - GSH reduced glutathione - TMPD N,N,NN-tetramethyl-p-phenylenediamine - EGTA ethylene glycol-bis (-aminoethyl ether) - p-CMB p-chloromercuribenzoate - NEM N-ethylmaleimide - FCCP p-trifluoromethoxycarbonyl cyanide phenylhydrazone