Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.     

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.     

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.     

Effects of Q metabolites and related compounds on mitochondrial succinate and NADH oxidase systems

The effects of Q metabolites (Q acid-I, Q acid-II) and related compounds (dihydro Q acid-I, dehydro Q acid-II, QS-n, and their esters) on mitochondrial succinate and NADH oxidase systems were investigated. The activity restoring succinate oxidation in acetone-treated beef heart mitochondria was found to decrease with descending order of carbon number (n) of the side chain of the Q metabolites; activity was restored with Q acid-I (n = 7) to one-third as much as that with Q-7 and Q-10, but Q acid-II (n = 5) did not restore any activity. Of the related compounds with a carboxyalkyl group (QS-n), QS-16-QS-18 (n = 16–18) were found to be most active, and their activities were also correlated with n. The relationship between the restoration of activity and the partition coefficient was considered. NADH oxidation in pentane-treated beef heart submitochondrial particles could be restored with esters of low molecular weight quinones to the same extent as with Q-10, but not with the metabolites.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase and succinate dehydrogenase complexes contain plant specific subunits

Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9–10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis. Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Mitochondrial proteome: cancer-altered metabolism associated with cytochrome c oxidase subunit level variation

Shifts in metabolism associated with tumorigenesis were first noted by Otto Warburg in the 1920s. In the ensuing decades many examples of the phenomenon have been elucidated while the underlying molecular mechanism has remained elusive. As the enzyme complex at the crux of oxidative phosphorylation, cytochrome c oxidase is uniquely positioned to have a very high impact on cellular metabolism. In this study, we test the hypothesis that there is a specific association between altered cytochrome c oxidase subunit levels and altered metabolism by combining the technique of reverse-phase protein microarray with radiolabeled glucose metabolic studies. Such a relationship is observed with five different cell lines, two of which (1542N and 1542T) are a matched set of normal and tumor-based lineages derived from the same prostate gland. By measuring the [(14)C]carbon dioxide production of a cell line metabolizing [1-(14)C]glucose and comparing those measurements to values obtained for the same cell line metabolizing [6-(14)C]glucose, we determined the relative utilization of the hexose monophosphate shunt and glycolysis progressing through the Krebs cycle metabolic pathway in each cell line. In all cases there is an increased utilization of hexose monophosphate shunt relative to glycolysis progressing through the Krebs cycle in tumor derived relative to normal derived cell lines. Additionally, there is an associated increase in the ratio of nuclear encoded cytochrome c oxidase subunits to mitochondrially encoded cytochrome c oxidase subunits in the tumor-derived cell lines. These results demonstrate an alteration in subunit levels of a single enzyme complex (cytochrome c oxidase) commensurate with tumor-altered metabolism.     

Superoxide dismutase does not inhibit the oxidation of cytochrome c and cytochrome oxidase.

An electrometric system was used to measure Ca⁺⁺ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca⁺⁺ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca⁺⁺ concentration below 10^?6 M.     

Effects of a transition from normoxia to anoxia on yeast cytochrome c oxidase and the mitochondrial respiratory chain: Implications for hypoxic gene induction

Previous studies have demonstrated that the mitochondrial respiratory chain and cytochrome c oxidase participate in oxygen sensing and the induction of some hypoxic nuclear genes in eukaryotes. In addition, it has been proposed that mitochondrially-generated reactive oxygen and nitrogen species function as signals in a signaling pathway for the induction of hypoxic genes. To gain insight concerning this pathway, we have looked at changes in the functionality of the yeast respiratory chain as cells experience a shift from normoxia to anoxia. These studies have revealed that yeast cells retain the ability to respire at normoxic levels for up to 4 h after a shift and that the mitochondrial cytochrome levels drop rapidly to 30-50% of their normoxic levels and the turnover rate of cytochrome c oxidase (COX) increases during this shift. The increase in COX turnover rate cannot be explained by replacing the aerobic isoform, Va, of cytochrome c oxidase subunit V with the more active hypoxic isoform, Vb. We have also found that mitochondria retain the ability to respire, albeit at reduced levels, in anoxic cells, indicating that yeast cells maintain a functional mitochondrial respiratory chain in the absence of oxygen. This raises the intriguing possibility that the mitochondrial respiratory chain has a previously unexplored role in anoxic cells and may function with an alternative electron acceptor when oxygen is unavailable.     

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50?μM KCN for 24?h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD⁺) deficit combined with a replicative state, rather than oxidative stress and energy depletion.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Changes induced by hydrazine in optical spectra of cytochrome oxidase

Casein kinase-TS (Ck-TS), a type-2 casein kinase purified from rat liver cytosol which phosphorylates seryl and threonyl residues N-terminal to acidic clusters, is specifically inhibited by polyglutamyl peptides which are ineffective both on type-1 casein kinase and on cAMP-dependent protein kinase. The inhibition is competitive toward the protein substrate and non-competitive toward ATP. Among the polyglutamates tested (Glu)70 is the most effective (Ki 0.11 microM). (Glu)10 and (Glu)5 are also inhibitors, though less powerful than (Glu)70, while (Glu)3, (Glu)2 and free glutamic acid up to 5 mM are ineffective. These results disclose the possibility that naturally occurring polypeptides containing long stretches of acidic residues may act as physiological inhibitors of type-2 casein kinases.     

Inner mitochondrial membranes bound to concanavalin A-sepharose display succinate dehydrogenase, ATPase, and cytochrome oxidase activity

A fraction (15-20% of the total protein) of a preparation of bovine submitochondrial particles (SMPs) binds to concanavalin A-sepharose. The bound membranes displayed succinate dehydrogenase, cytochrome oxidase, and ATPase activity, which, as in SMPs, were inhibited by malonate, cyanide, and oligomycin, respectively. These results indicate that the bound membranes are inner mitochondrial membranes and that they contain a glycoprotein which was recognized by concanavalin A. It was possible to repeatedly perform the three enzyme assays, one after the other, in the same gel with the bound membranes. Long-term stability tests (22 days) showed that cytochrome oxidase was much more stable in the membranes bound to the gel than in SMPs, while the ATPase activity decayed at a similar rate in the two conditions. Thus, inner mitochondrial membranes bound to ConA-Sepharose appear to be a potentially interesting model for the study of immobilized multienzymatic complexes.     

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.     

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.     

Study of the interaction of cadmium with membrane-bound succinate dehydrogenase

Cadmium ions inhibit membrane-bound succinate dehydrogenase with a second-order rate constant of 10.42 mM^–1s^–1 at pH 7.35 and 25°C. Succinate and malonate protect the enzyme against cadmium ion inhibition. The protection pattern exerted by succinate and malonate suggests that the group modified by cadmium is located at the active site. The pH curve of inactivation by Cd²⁺ indicates the involvement of an amino acid residue with pKa of 7.23.