Farnesylacetone, a sesquiterpenic hormone of Crustacea, inhibits electron transport in isolated rat liver mitochondria

Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.     

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.     

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 10⁹ M^–1 sec^–1 corresponding to a diffusion coefficient in the range of 10^–6 cm²/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10^–9 cm²/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK _m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.     

An Evaluation of the Measurement of the Activities of Complexes I-IV in the Respiratory Chain of Human Skeletal Muscle Mitochondria

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-β-D-maltoside. Complex I activity is measured in the presence of 2.5 mg · ml⁻¹ bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b₅ reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.     

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.     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

Enzymatic and structural modifications of mitochondrial NADH-ubiquinone reductase with autolysis as experimental model

Complex I (nicotinamide adenine dinucleotide-ubiquinone reductase) is a complex enzyme system located in the inner mitochondrial membrane. It has the ability to catalyze several different enzymatic reactions in electron transport, and is known to be one of the respiratory chain components most sensitive to ischaemia. Mitochondria and two complexes I (complex IA and complex IB) were isolated from normal and ischaemic myocardial tissue. Enzymatic activities, polypeptide composition, as well as other components such as non-haem iron, acid-labile sulphur and ubiquinone, were determined. The results indicated that complex IB reflected the enzymatic changes in the mitochondria during myocardial ischaemia, but complex IA did not. The lesion that resulted from ischaemia was localised as altered enzymatic activities due to a different polypeptide composition, as well as loss of ubiquinone and non-haem iron from complex IB.     

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.     

Significance of respirasomes for the assembly/stability of human respiratory chain complex I

We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.     

Nitric Oxide-Mediated Inhibition of the Mitochondrial Respiratory Chain in Cultured Astrocytes

Abstract: The Ca²⁺-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon-γ (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II–III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor N ^G-monomethyl- l -arginine. The lipopolysaccharide/interferon-γ treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.     

Coenzyme Q₁₀ reverses mitochondrial dysfunction in atorvastatin-treated mice and increases exercise endurance

Statins are cholesterol-lowering drugs widely used in the prevention of cardiovascular diseases; however, they are associated with various types of myopathies. Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and thus decrease biosynthesis of low-density lipoprotein cholesterol and may also reduce ubiquinones, essential coenzymes of a mitochondrial electron transport chain, which contain isoprenoid residues, synthesized through an HMG-CoA reductase-dependent pathway. Therefore, we hypothesized that statin treatment might influence physical performance through muscular mitochondrial dysfunction due to ubiquinone deficiency. The effect of two statins, atorvastatin and pravastatin, on ubiquinone content, mitochondrial function, and physical performance was examined by using statin-treated mice. Changes in energy metabolism in association with statin treatment were studied by using cultured myocytes. We found that atorvastatin-treated mice developed muscular mitochondrial dysfunction due to ubiquinone deficiency and a decrease in exercise endurance without affecting muscle mass and strength. Meanwhile, pravastatin at ten times higher dose of atorvastatin had no such effects. In cultured myocytes, atorvastatin-related decrease in mitochondrial activity led to a decrease in oxygen utilization and an increase in lactate production. Conversely, coenzyme Q(10) treatment in atorvastatin-treated mice reversed atorvastatin-related mitochondrial dysfunction and a decrease in oxygen utilization, and thus improved exercise endurance. Atorvastatin decreased exercise endurance in mice through mitochondrial dysfunction due to ubiquinone deficiency. Ubiquinone supplementation with coenzyme Q(10) could reverse atorvastatin-related mitochondrial dysfunction and decrease in exercise tolerance.     

Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.     

Complex I binds several mitochondrial NAD-coupled dehydrogenases

NADH:ubiquinone reductase (complex I) of the mitochondrial inner membrane respiratory chain binds a number of mitochondrial matrix NAD-linked dehydrogenases. These include pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, mitochondrial malate dehydrogenase, and beta-hydroxyacyl-CoA dehydrogenase. No binding was detected between complex I and cytosolic malate dehydrogenase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, lipoamide dehydrogenase, citrate synthase, or fumarase. The dehydrogenases that bound to complex I did not bind to a preparation of complex II and III, nor did they bind to liposomes. The binding of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and mitochondrial malate dehydrogenase to complex I is a saturable process. Based upon the amount of binding observed in these in vitro studies, there is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space. The possible metabolic significance of these interactions is discussed.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q_P) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q_P site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

Release of flavin from the mitochondrial NADH-dehydrogenase complex

The conditions leading to the release of flavin mononucleotide from the NADH-dehydrogenase Complex I of the mitochondrial respiratory chain were studied. Upon incubation of a mitochondrial suspension for 1-4 h, a spontaneous release of flavin mononucleotide to the aqueous phase was observed. The release abruptly increased even at insignificant damage of mitochondria (by heating, very small quantities of detergents or washings, NaHCO3, and acidic or alkaline pH). This was detected by the enhancement in the intensity of flavin fluorescence and the degree of its depolarization. The loss of flavin mononucleotide leads to the loss of the NADH:ubiquinone reductase activity (but not the NADH: ferricyanide reductase activity). It is suggested that the loss of flavin mononucleotide from Complex I by the action of temperature, detergents, alkali and other agents can be a cause of many mitochondrial diseases.     

Syntheses of biologically active ubiquinone derivatives

Various 6-alkylubiquinone or 6-(omega-haloalkyl)ubiquinone derivatives were synthesized through a radical coupling reaction between alkanoyl or omega-haloalkanoyl peroxides and ubiquinone 0. The latter was synthesized from 2-methoxy-4-methylphenol via nitration, methylation, reduction, and oxidation by modifications of the reported methods. 6-(omega-Haloalkyl)ubiquinones were converted to 6-(omega-hydroxyalkyl)ubiquinones by a mercuric-assisted solvolysis technique. The 6-(omega-hydroxyalkyl)ubiquinones were then esterified with carboxylic acid anhydrides or carboxylic acid bearing reporting groups, such as a photoaffinity label, N-(4-azido-2-nitrophenyl)-beta-alanine, or a spin-label, 3-carboxy-2,2,5,5-tetramethyl-3-pyrrolinyl-1-oxy. The esterification was catalyzed by dicyclohexylcarbodiimide and pyridine, and the esters were purified by preparative silica gel thin-layer chromatography, developed by 3% ethanol in benzene. The spectral properties and biological functions of the synthesized ubiquinone derivatives were studied. The biological function of the synthesized compounds was followed by the ability to serve as an electron acceptor, donor, or mediator in the isolated mitochondrial electron transfer complexes of succinate-Q reductase, ubiquinol-cytochrome c reductase, and succinate-cytochrome c reductase, respectively. The concentration effect of these ubiquinone derivatives on the electron transfer reaction was compared with that of ubiquinone 10. The study of the inhibitory effect of synthesized arylazidoubiquinone on succinate-cytochrome c reductase after photolysis confirmed the existence of specific Q-binding proteins in this segment of the respiratory chain. The specific interaction between ubiquinone and protein has also gained support from the immobilization of the spin-label of a synthesized spin-labeled ubiquinone derivative.     

Crystal structure of mitochondrial respiratory membrane protein complex II

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.     

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.