Ubiquinone Reductase Activity Research Articles

Succinate Ubiquinone Reductase Activity Predicts Multiple Myeloma Sensitivity to Venetoclax

Multiple myeloma (MM) is a plasma cell malignancy estimated to account for 12,590 deaths and 30,280 new diagnoses in the US in 2017. Despite the use of next generation immunomodulatory drugs (IMiDs), proteasome inhibitors and newer targeted therapies, the development of chemo-resistant MM is common, with approximately 20% of patients succumbing to aggressive treatment-refractory disease within a short period of diagnosis. Resistance is primarily associated with evasion of apoptosis connected to the inability to release sufficient pro-apoptotic BCL-2 proteins above a threshold required to elicit apoptosis. Thus, finding alternative strategies to shift a resistant cell closer to the apoptotic threshold could potentially circumvent resistance.Metabolites contribute to the development of resistance to apoptosis by regulating pro and anti-apoptotic BCL-2 family members. We previously reported that depriving cells of glutamine enhanced binding of BIM to BCL-2 thereby sensitizing myeloma cell lines and patient samples to the BH3 mimetic venetoclax (ABT-199), a highly selective, potent BCL-2 antagonist. ABT-199 is however effective only in 40% of MM patients exhibiting the (11;14) translocation, warranting investigation of strategies to increase the applicability of ABT-199. We were interested to explore if there were metabolic differences in ABT-199 sensitive t(11;14) and resistant cells that could inform us of a) predictors of sensitivity; and b) metabolic targets that could be inhibited to sensitize resistant cells to ABT-199.Given that, tumor cells differentially rely on glycolysis and mitochondrial oxidative phosphorylation we first evaluated oxygen consumption rates (OCR) in ABT-199 sensitive and resistant MM lines. All ABT-199 sensitive lines exhibited lower OCR in contrast to the resistant lines. Metabolite profiling and isotope tracing flux analyses of glutamine deprived myeloma cell lines revealed specific reduction of TCA cycle metabolites including succinate. TCA cycle intermediates are linked to mitochondrial respiration via electron transport chain (ETC) complexes (I-V). Among the five ETC complexes, only Complex II/succinate dehydrogenase (SDH) is directly connected to the TCA cycle and ETC. SDH facilitates the oxidation of succinate to fumarate in the TCA cycle through its subunit SDHA, and transfers the released electrons to ubiquinone via its SDHB, C and D subunits supporting the succinate ubiquinone reductase (SQR) activity of Complex II. Therefore, we sought to further interrogate succinate dehydrogenase in facilitating ABT-199 sensitization. We evaluated SDH ubiquinone reductase activity in ABT-199 sensitive and resistant myeloma lines and demonstrated that all ABT-199 sensitive cell lines had significantly lower SQR activity. Furthermore, inhibition of the SDH ubiquinone reductase with the Qp site inhibitor thenoyltrifluoroacetone (TTFA) sensitized resistant MM cells to ABT-199. Inhibition of Complex I with piericidin or the SDHA subunit with 3-NPA were not as effective in sensitizing MM to ABT-199. Protein expression and co-immunoprecipitation analyses demonstrated an induction of BIM and enhanced BIM-BCL-2 binding upon SDH inhibition with TTFA. TTFA treated cells exhibited a reduction of FOXO3a phosphorylation and concomitant nuclear localization upon SDH inhibition suggestive of FOXO3a playing a role in BIM induction. TTFA + ABT-199 co-treatment induced apoptosis in ABT-199 resistant myeloma patient cells without impacting viability of the associated normal populations.Our study thus identifies a tumor selective approach of metabolically-driven synthetic lethality that relies on inhibition of the mitochondrial enzyme SDH and use of the BCL-2 antagonist, ABT-199 for myeloma therapy. In addition, our results importantly suggest SDH activity predicts ABT-199 sensitivity and can be interrogated in MM patients as a predictive marker of ABT-199 sensitivity particularly within t(11;14) patients currently being enrolled for ABT-199 treatment. DisclosuresNooka:Amgen, Novartis, Spectrum, Adaptive: Consultancy. Boise:Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

Electron transport chain activity is a predictor and target for venetoclax sensitivity in multiple myeloma

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the ‘primed’ state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.

The Features of Energy Metabolism in Hepatocytes of Rats that Receive Diets with Different Nutrient Contents

Abstract—The activities of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and NADH:ubiquinone reductase in the mitochondrial fraction from the liver tissue of rats fed diets with different sucrose and protein contents have been measured. In animals that received a high-sucrose diet, isocitrate dehydrogenase and malate dehydrogenase activities tend to decline and α-ketoglutarate dehydrogenase activity decreased by a factor of 2.5. Interestingly, the NADH:ubiquinone reductase activity remains at the control level in animals that are fed the high-sucrose diet. In the mitochondrial fraction from the liver tissue of rats that receive a high-sucrose–low-protein diet, the activities of NAD+-dependent dehydrogenases of the Krebs cycle decrease considerably, and the activity of NADH:ubiquinone reductase decreased as well, being more than 2.5 times lower than in the control. This may be one of the steps in the mechanism by which energy generated in the cell is regulated when the sucrose : protein ratio in the diet is imbalanced. These results can be used to develop a strategy for the correction of metabolic disturbances associated with nutrient contents in the diet.

Abstract 2715: Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma

Abstract Multiple myeloma (MM) is the second most common hematologic malignancy. In 2017, MM accounted for approximately 30,770 new diagnoses and 12770 deaths in the US. Although, advancements in treatment options have increased survival rates and life expectancy, MM remains incurable due to development of resistance. Venetoclax is a highly selective, potent BCL-2 antagonist, currently in phase I/II trials for MM and FDA approved for the treatment of CLL patients exhibiting 17p deletion. Venetoclax is effective in eliciting cell death as a single agent in a subset of MM with the (11;14) translocation (which we henceforth designate “sensitive”) in contrast to the majority of MM that is resistant (which we henceforth designate “resistant”). We previously reported that glutamine deprivation increases BIM binding to BCL-2 thereby sensitizing MM to venetoclax, while alpha-ketoglutarate supplementation reversed this sensitivity. We were therefore interested to explore if there was a metabolic basis for t(11;14)-myeloma sensitivity to single agent venetoclax to aid in 1) identifying venetoclax sensitive MM and; 2) inform us of metabolic targets that could be inhibited to sensitize resistant MM to venetoclax. We first queried the CoMMpass MM patient RNAseq trial data and cell lines for electron transport chain (ETC) and TCA cycle gene expression differences in t(11;14) vs non-t(11;14) patients and cell lines. All sensitive cells exhibited varied suppression of TCA/ETC genes, and reduced TCA cycle metabolites and oxygen consumption rates (OCR) compared to resistant cells. Examination of TCA cycle activities connected to the ETC and OCR showed significant decrease in Complex II Succinate ubiquinone reductase (SQR) activity in sensitive cell lines and purified CD38+ve MM patient cells and elevated SQR activity in resistant cells. Furthermore, inhibition of SQR with thenoyltrifluoroacetone (TTFA) sensitized resistant cells to venetoclax. Consistent with SQR inhibition leading to ABT-199 sensitivity, overexpression of an SQR mutant (SDHCR72C) in SDHC-knockout resistant MM, increased venetoclax sensitivity, identifying a unique role for SQR in regulating BCL-2 dependence. In interrogating the mechanistic underpinnings of SQR inhibition-induced sensitivity to venetoclax, we identified increased expression of ATF4 and BIM upon SQR inhibition. ATF4KD or BIMKO restored viability in TTFA and venetoclax co-treated MM further confirming the role of SQR inhibition-induced ATF4 and BIM in venetoclax sensitivity. In testing translationally relevant compounds, we determined that the histone deacetylase inhibitor, panobinostat reduced SQR activity in a dose dependent manner and sensitized MM cells to venetoclax. Our study thus identifies SQR as a novel target and predictive marker to aid in identifying ABT-199-responsive MM patients in a functional biomarker informed manner. Citation Format: Richa Bajpai, Abhinav Achreja, Changyong Wei, Arusha Siddiqa, Shannon M. Matulis, Vikas Gupta, Samuel K. McBrayer, Anjali Mittal, Manali Rupji, Hsiao-Rong Chen, Jeanne Kowalski, Sagar Lonial, Ajay K. Nooka, Lawrence H. Boise, Deepak Nagrath, Mala Shanmugam. Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2715.

Acute and subchronic toxicity of inhaled toluene in male Long–Evans rats: Oxidative stress markers in brain

The effects of exposure to volatile organic compounds (VOCs), which are of concern to the EPA, are poorly understood, in part because of insufficient characterization of how human exposure duration impacts VOC effects. Two inhalation studies with multiple endpoints, one acute and one subchronic, were conducted to seek effects of the VOC, toluene, in rats and to compare the effects between acute and subchronic exposures. Adult male Long–Evans rats were exposed to toluene vapor (n=6 per group) at a concentration of 0 or 1019±14ppm for 6h in the acute study and at 0±0, 10±1.4, 97±7, or 995±43ppm for 6h/d, 5d/week for 13 weeks in the subchronic study. For the acute study, brains were dissected on ice within 30min of the end of exposure, while for the subchronic study, brains were dissected 18h after the last exposure. Frontal cortex, hippocampus, cerebellum, and striatum were assayed for a variety of oxidative stress (OS) parameters including total aconitase (TA), protein carbonyls, glutathione peroxidase (GPX), glutathione reductase (GRD), glutathione transferase (GST), γ-glutamylcysteine synthetase (GCS), superoxide dismutase (SOD), total antioxidants (TAS), NADPH quinone oxidoreductase-1 (NQO1), and NADH ubiquinone reductase (UBIQ-RD) activities using commercially available kits. Following acute exposure, UBIQ-RD, GCS and GRD were increased significantly only in the cerebellum, while TAS was increased in frontal cortex. On the other hand, subchronic exposure affected several OS markers including increases in NQO1 and UBIQ-RD. The effect of subchronic toluene exposure on SOD and TAS was greater in the striatum than in the other brain regions. TA activity (involved in maintaining iron homeostasis and an indicator of DNA damage) was inhibited in striatum and cerebellum, increased in hippocampus, and unchanged in frontal cortex. Protein carbonyls increased significantly in both the frontal cortex and cerebellum. In general, the results showed that acute exposure to toluene affected OS parameters to a lesser extent than did subchronic exposure. These results suggest that toluene exposure induces OS in the brain and this may be a component of an adverse outcome pathway for some of the neurotoxic effects reported following toluene exposure.

Активність NADH-убіхінонредуктази та сукцинатдегідрогенази печінки щурів за умов токсичного гепатиту,

Активність NADH-убіхінонредуктази та сукцинатдегідрогенази печінки щурів за умов токсичного гепатиту,

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.

Novel ETF dehydrogenase mutations in a patient with mild glutaric aciduria type II and complex II‐III deficiency in liver and muscle

We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoA dehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G > T (exon 7) and p.P534L: c.1601 C > T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation.

Mitochondrial Dehydrogenases in the Aerobic Respiratory Chain of the Rodent Malaria Parasite Plasmodium yoelii yoelii

In the intraerythrocytic stages of malaria parasites, mitochondria lack obvious cristae and are assumed to derive energy through glycolysis. For understanding of parasite energy metabolism in mammalian hosts, we isolated rodent malaria mitochondria from Plasmodium yoelii yoelii grown in mice. As potential targets for antiplasmodial agents, we characterized two respiratory dehydrogenases, succinate:ubiquinone reductase (complex II) and alternative NADH dehydrogenase (NDH-II), which is absent in mammalian mitochondria. We found that P. y. yoelii complex II was a four-subunit enzyme and that kinetic properties were similar to those of mammalian enzymes, indicating that the Plasmodium complex II is favourable in catalysing the forward reaction of tricarboxylic acid cycle. Notably, Plasmodium complex II showed IC(50) value for atpenin A5 three-order of magnitudes higher than those of mammalian enzymes. Divergence of protist membrane anchor subunits from eukaryotic orthologs likely affects the inhibitor resistance. Kinetic properties and sensitivity to 2-heptyl-4-hydroxyquinoline-N-oxide and aurachin C of NADH: ubiquinone reductase activity of Plasmodium NDH-II were similar to those of plant and fungus enzymes but it can oxidize NADPH and deamino-NADH. Our findings are consistent with the notion that rodent malaria mitochondria are fully capable of oxidative phosphorylation and that these mitochondrial enzymes are potential targets for new antiplasmodials.

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.

Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.

Ubiquinone-binding Site Mutations in the Saccharomyces cerevisiae Succinate Dehydrogenase Generate Superoxide and Lead to the Accumulation of Succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo. We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.

Role of the Conserved Arginine 274 and Histidine 224 and 228 Residues in the NuoCD Subunit of Complex I from Escherichia coli

The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fast-relaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.

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.

Effects of site-directed mutations inEscherichia colisuccinate dehydrogenase on the enzyme activity and production of superoxide radicalsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Escherichia coli succinate dehydrogenase (SdhCDAB) catalyzes the oxidation of succinate to fumarate in the Krebs cycle, and during turnover, it produces superoxide radicals. SdhCDAB is a good model system for the succinate dehydrogenase (Sdh) found in the mitochondrial respiratory chain (complex II), as the subunits are structural homologues. Although mutations in sdh genes are reportedly associated with a variety of mitochondria-related diseases, the molecular mechanism of these diseases is poorly understood. We have investigated the effects of site-directed mutations around the heme (SdhD-H71L and SdhC-H91L), and at the ubiquinone-binding site (Q site; SdhC-I28E), on enzyme activity and production of superoxide radicals. The mutations SdhD-H71L and SdhC-I28E, but not SdhC-H91L, significantly reduce the succinate-ubiquinone reductase activity of the enzyme. All 3 mutant enzymes produce more superoxide than the wild-type enzyme, indicating that disturbance of the heme or the Q site can enhance superoxide production. The presence of a Q-site inhibitor reduces superoxide production significantly. Furthermore, the yield of superoxide is substrate dependent and increases with succinate concentration from 0.1 to 10 mmol/L. Our results indicate that, in SdhCDAB, the Q site with bound ubiquinone is an important source of superoxide radicals.

Activation of Isolated NADH:Ubiquinone Reductase I (Complex I) from Escherichia coli by Detergent and Phospholipids. Recovery of Ubiquinone Reductase Activity and Changes in EPR Signals of Iron−Sulfur Clusters

NADH:ubiquinone oxidoreductase (NDH-1 or complex I) from Escherichia coli was purified using a combination of anion exchange chromatography and centrifugation in sucrose density gradient. The dependence of enzyme activity on detergent and phospholipids was studied. Artificial hexaammineruthenium reductase activity was not affected by dodecyl maltoside (DDM) and asolectin. Ubiquinone reductase activity had a bell-shape dependence on DDM concentration; 7-10-fold activation could be achieved. Treatment with asolectin subsequently yields additional 2-fold activation with a corresponding increase in the apparent V(max) and without significant changes in apparent K(m). Comparative EPR studies of complex I reduced with NADH, "as prepared" and "activated by asolectin" showed an increase in the signals derived mainly from two [4Fe-4S] clusters in the activated enzyme. One of these signals could be simulated with an axial spectrum with g values of g(xyz)= 1.895, 1.904, 2.05, which corresponds to the parameters reported for the N2 cluster. This data indicates conformational rearrangements of catalytic importance in complex I upon binding of phospholipids.

Histidine 129 in the 75-kDa Subunit of Mitochondrial Complex I from Yarrowia lipolytica Is Not a Ligand for [Fe4S4] Cluster N5 but Is Required for Catalytic Activity

Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore.

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.

Functional Significance of Conserved Histidines and Arginines in the 49-kDa Subunit of Mitochondrial Complex I

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.

Opening of the Mitochondrial Permeability Transition Pore Induces Reactive Oxygen Species Production at the Level of the Respiratory Chain Complex I

We have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H(2)O(2) production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H(2)O(2) production was measured again in the presence of various amounts of NADH. PTP opening-induced H(2)O(2) production began when NADH concentration was higher than 50 microm and reached a maximum at over 300 microm. At such concentrations of NADH, the maximal H(2)O(2) production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H(2)O(2) production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H(2)O(2) production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death.