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The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS.
Maklashina E, Rajagukguk S, Iverson TM, Cecchini G
(2018) J Biol Chem 293: 7754-7765
MeSH Terms: Bacterial Proteins, Catalysis, Crystallography, X-Ray, Electron Transport Complex II, Escherichia coli, Flavoproteins, Humans, Models, Molecular, Oxidation-Reduction, Protein Conformation, Protein Subunits, Reactive Oxygen Species
Show Abstract · Added April 1, 2019
Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.
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Crystal structure of an assembly intermediate of respiratory Complex II.
Sharma P, Maklashina E, Cecchini G, Iverson TM
(2018) Nat Commun 9: 274
MeSH Terms: Crystallography, X-Ray, Electron Transport Complex II, Escherichia coli, Escherichia coli Proteins, Flavin-Adenine Dinucleotide, Oxidoreductases
Show Abstract · Added April 1, 2019
Flavin is covalently attached to the protein scaffold in ~10% of flavoenzymes. However, the mechanism of covalent modification is unclear, due in part to challenges in stabilizing assembly intermediates. Here, we capture the structure of an assembly intermediate of the Escherichia coli Complex II (quinol:fumarate reductase (FrdABCD)). The structure contains the E. coli FrdA subunit bound to covalent FAD and crosslinked with its assembly factor, SdhE. The structure contains two global conformational changes as compared to prior structures of the mature protein: the rotation of a domain within the FrdA subunit, and the destabilization of two large loops of the FrdA subunit, which may create a tunnel to the active site. We infer a mechanism for covalent flavinylation. As supported by spectroscopic and kinetic analyses, we suggest that SdhE shifts the conformational equilibrium of the FrdA active site to disfavor succinate/fumarate interconversion and enhance covalent flavinylation.
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Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.
Gilliam LA, Lark DS, Reese LR, Torres MJ, Ryan TE, Lin CT, Cathey BL, Neufer PD
(2016) Am J Physiol Endocrinol Metab 311: E293-301
MeSH Terms: Animals, Antineoplastic Agents, Breast Neoplasms, Catalase, Disease Models, Animal, Doxorubicin, Electron Transport Complex I, Electron Transport Complex II, Female, Hydrogen Peroxide, Mice, Mice, Transgenic, Mitochondria, Muscle, Muscle Contraction, Muscle, Skeletal, Oxidation-Reduction, Proteins
Show Abstract · Added October 17, 2016
The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction.
Copyright © 2016 the American Physiological Society.
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17 MeSH Terms
Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II.
Maklashina E, Rajagukguk S, Starbird CA, McDonald WH, Koganitsky A, Eisenbach M, Iverson TM, Cecchini G
(2016) J Biol Chem 291: 2904-16
MeSH Terms: Electron Transport Complex II, Escherichia coli, Escherichia coli Proteins, Flavin-Adenine Dinucleotide
Show Abstract · Added January 26, 2016
Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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4 MeSH Terms
Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress.
Nazarewicz RR, Dikalova AE, Bikineyeva A, Dikalov SI
(2013) Am J Physiol Heart Circ Physiol 305: H1131-40
MeSH Terms: Animals, Aorta, Blood Pressure, Cell Respiration, Cells, Cultured, Diazoxide, Electron Transport Complex I, Electron Transport Complex II, Endothelial Cells, Humans, Membrane Glycoproteins, Membrane Potential, Mitochondrial, Mice, Mitochondria, NADPH Oxidase 2, NADPH Oxidases, Oxidative Stress, Potassium Channels, Superoxides, Vasodilator Agents
Show Abstract · Added March 30, 2014
Superoxide (O2(·-)) production by the NADPH oxidases is implicated in the pathogenesis of many cardiovascular diseases, including hypertension. We have previously shown that activation of NADPH oxidases increases mitochondrial O2(·-) which is inhibited by the ATP-sensitive K(+) channel (mitoKATP) inhibitor 5-hydroxydecanoic acid and that scavenging of mitochondrial or cytoplasmic O2(·-) inhibits hypertension. We hypothesized that mitoKATP-mediated mitochondrial O2(·-) potentiates cytoplasmic O2(·-) by stimulation of NADPH oxidases. In this work we studied Nox isoforms as a potential target of mitochondrial O2(·-). We tested contribution of reverse electron transfer (RET) from complex II to complex I in mitochondrial O2(·-) production and NADPH oxidase activation in human aortic endothelial cells. Activation of mitoKATP with low dose of diazoxide (100 nM) decreased mitochondrial membrane potential (tetramethylrhodamine methyl ester probe) and increased production of mitochondrial and cytoplasmic O2(·-) measured by site-specific probes and mitoSOX. Inhibition of RET with complex II inhibitor (malonate) or complex I inhibitor (rotenone) attenuated the production of mitochondrial and cytoplasmic O2(·-). Supplementation with a mitochondria-targeted SOD mimetic (mitoTEMPO) or a mitochondria-targeted glutathione peroxidase mimetic (mitoEbselen) inhibited production of mitochondrial and cytoplasmic O2(·-). Inhibition of Nox2 (gp91ds) or Nox2 depletion with small interfering RNA but not Nox1, Nox4, or Nox5 abolished diazoxide-induced O2(·-) production in the cytoplasm. Treatment of angiotensin II-infused mice with RET inhibitor dihydroethidium (malate) significantly reduced blood pressure. Our study suggests that mitoKATP-mediated mitochondrial O2(·-) stimulates cytoplasmic Nox2, contributing to the development of endothelial oxidative stress and hypertension.
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20 MeSH Terms
Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.
Tomasiak TM, Archuleta TL, Andréll J, Luna-Chávez C, Davis TA, Sarwar M, Ham AJ, McDonald WH, Yankovskaya V, Stern HA, Johnston JN, Maklashina E, Cecchini G, Iverson TM
(2011) J Biol Chem 286: 3047-56
MeSH Terms: Catalysis, Electron Transport Complex II, Enzyme Inhibitors, Escherichia coli, Escherichia coli Proteins, Fumarates, Models, Chemical, Models, Molecular, Oxidoreductases, Substrate Specificity
Show Abstract · Added March 20, 2014
Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.
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10 MeSH Terms
Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system.
Friemann R, Lee K, Brown EN, Gibson DT, Eklund H, Ramaswamy S
(2009) Acta Crystallogr D Biol Crystallogr 65: 24-33
MeSH Terms: Catalytic Domain, Crystallization, Crystallography, X-Ray, Electron Transport Complex III, Flavin-Adenine Dinucleotide, Iron, Mixed Function Oxygenases, Models, Chemical, Multienzyme Complexes, NAD, Protein Binding, Pseudomonas putida, Substrate Specificity, Toluene
Show Abstract · Added August 31, 2017
Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
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14 MeSH Terms
Fetal programming alters reactive oxygen species production in sheep cardiac mitochondria.
von Bergen NH, Koppenhafer SL, Spitz DR, Volk KA, Patel SS, Roghair RD, Lamb FS, Segar JL, Scholz TD
(2009) Clin Sci (Lond) 116: 659-68
MeSH Terms: Animals, Antioxidants, Disease Models, Animal, Electron Transport Complex I, Electron Transport Complex III, Female, Fetal Development, Hydrogen Peroxide, Male, Mitochondria, Heart, Mitochondrial Membranes, Oxidative Phosphorylation, Oxygen Consumption, Reactive Oxygen Species, Sheep
Show Abstract · Added February 22, 2016
Exposure to an adverse intrauterine environment is recognized as an important risk factor for the development of cardiovascular disease later in life. Although oxidative stress has been proposed as a mechanism for the fetal programming phenotype, the role of mitochondrial O(2)(*-) (superoxide radical) production has not been explored. To determine whether mitochondrial ROS (reactive oxygen species) production is altered by in utero programming, pregnant ewes were given a 48-h dexamethasone (dexamethasone-exposed, 0.28 mg.kg(-1) of body weight.day(-1)) or saline (control) infusion at 27-28 days gestation (term=145 days). Intact left ventricular mitochondria and freeze-thaw mitochondrial membranes were studied from offspring at 4-months of age. AmplexRed was used to measure H(2)O(2) production. Activities of the antioxidant enzymes Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and catalase were measured. Compared with controls, a significant increase in Complex I H(2)O(2) production was found in intact mitochondria from dexamethasone-exposed animals. The treatment differences in Complex I-driven H(2)O(2) production were not seen in mitochondrial membranes. Consistent changes in H(2)O(2) production from Complex III in programmed animals were not found. Despite the increase in H(2)O(2) production in intact mitochondria from programmed animals, dexamethasone exposure significantly increased mitochondrial catalase activity, whereas Mn-SOD and GPx activities were unchanged. The results of the present study point to an increase in the rate of release of H(2)O(2) from programmed mitochondria despite an increase in catalase activity. Greater mitochondrial H(2)O(2) release into the cell may play a role in the development of adult disease following exposure to an adverse intrauterine environment.
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15 MeSH Terms
Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.
Patel SP, Gamboa JL, McMullen CA, Rabchevsky A, Andrade FH
(2009) Invest Ophthalmol Vis Sci 50: 180-6
MeSH Terms: Abdominal Muscles, Animals, Blotting, Western, Electron Transport, Electron Transport Complex I, Electron Transport Complex II, Electron Transport Complex III, Electron Transport Complex IV, Energy Metabolism, Male, Membrane Potential, Mitochondrial, Mitochondria, Muscle, Mitochondrial Membranes, Oculomotor Muscles, Oxygen Consumption, Rats, Rats, Sprague-Dawley
Show Abstract · Added April 25, 2016
PURPOSE - The constant activity of the extraocular muscles is supported by abundant mitochondria. These organelles may enhance energy production by increasing the content of respiratory complexes. The authors tested the hypothesis that extraocular muscle mitochondria respire faster than do mitochondria from limb muscles because of the higher content of respiratory complexes.
METHODS - Inner mitochondrial membrane density was determined by stereological analysis of triceps surae (a limb muscle) and extraocular muscles of adult male Sprague-Dawley rats. The authors measured respiration rates of isolated mitochondria using a Clark-type electrode. The activity of respiratory complexes I, II, and IV was determined by spectrophotometry. The content of respiratory complexes was estimated by Western blot.
RESULTS - States 3, 4, and 5 respiration rates in extraocular muscle mitochondria were 40% to 60% lower than in limb muscle mitochondria. Extraocular muscle inner mitochondrial membrane density was similar to that of other skeletal muscles. Activity of complexes I and IV was lower in extraocular muscle mitochondria (approximately 50% the activity in triceps), but their content was approximately 15% to 30% higher. There was no difference in complex II content or activity or complex III content. Finally, complex V was less abundant in extraocular muscle mitochondria.
CONCLUSIONS - The results demonstrate that extraocular muscle mitochondria respire at slower rates than mitochondria from limb muscles, despite similar mitochondrial ultrastructure. Instead, differences were found in the activity (I, IV) and content (I, IV, V) of electron transport chain complexes. The discrepancy between activity and content of some complexes is suggestive of alternative subunit isoform expression in the extraocular muscles compared with limb muscles.
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17 MeSH Terms
Determining Rieske cluster reduction potentials.
Brown EN, Friemann R, Karlsson A, Parales JV, Couture MM, Eltis LD, Ramaswamy S
(2008) J Biol Inorg Chem 13: 1301-13
MeSH Terms: Binding Sites, Computer Simulation, Crystallography, X-Ray, Electrochemistry, Electrodes, Electron Transport Complex III, Ferredoxins, Hydrogen-Ion Concentration, Oxidation-Reduction, Pseudomonas, Solvents
Show Abstract · Added August 31, 2017
The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.
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11 MeSH Terms