Ubiquinone Oxidoreductase Activity Research Articles

Supercritical Fluid Extraction of Bioactive Components from Apple Peels and Their Modulation of Complex I Activity in Isolated Mitochondria.

Supercritical fluid extraction (SFE) was used to extract bioactive compounds from apple (Malus domestica) peel waste from three different Italian cultivars. The bioactive fractions were extracted applying a temperature of 60 °C and a pressure of 250 bar for 15 min with 20% ethanol as co-solvent, at a flow rate of 2 mL/min. The total polyphenol (TP), anthocyanin (TA), ascorbic acid (AA), and antioxidant activity contents (TACs) were measured, while chromatographic analyses were performed to highlight the differences between the extracts. The Stark cultivar had the highest levels of polyphenols, anthocyanins, and ascorbic acid, while the Royal Gala cultivar showed the highest total antioxidant activity. SFE extracts were then tested for their effect on the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) activity on mitochondria isolated from human embryonic kidney cells (HEK239). The Stark extract showed the most positive response in terms of NADH oxidation. The results obtained in this work highlight the potential of apple peel waste as a source of functional phytocompounds and suggest that Stark cultivar extracts may be exploited for pharmacological applications. This study supports the circular bioeconomy by promoting the use of waste products as a valuable resource.

Reverse Electron Transport at Mitochondrial Complex I in Ischemic Stroke, Aging, and Age-Related Diseases.

Stroke is one of the leading causes of morbidity and mortality worldwide. A main cause of brain damage by stroke is ischemia-reperfusion (IR) injury due to the increased production of reactive oxygen species (ROS) and energy failure caused by changes in mitochondrial metabolism. Ischemia causes a build-up of succinate in tissues and changes in the mitochondrial NADH: ubiquinone oxidoreductase (complex I) activity that promote reverse electron transfer (RET), in which a portion of the electrons derived from succinate are redirected from ubiquinol along complex I to reach the NADH dehydrogenase module of complex I, where matrix NAD+ is converted to NADH and excessive ROS is produced. RET has been shown to play a role in macrophage activation in response to bacterial infection, electron transport chain reorganization in response to changes in the energy supply, and carotid body adaptation to changes in the oxygen levels. In addition to stroke, deregulated RET and RET-generated ROS (RET-ROS) have been implicated in tissue damage during organ transplantation, whereas an RET-induced NAD+/NADH ratio decrease has been implicated in aging, age-related neurodegeneration, and cancer. In this review, we provide a historical account of the roles of ROS and oxidative damage in the pathogenesis of ischemic stroke, summarize the latest developments in our understanding of RET biology and RET-associated pathological conditions, and discuss new ways to target ischemic stroke, cancer, aging, and age-related neurodegenerative diseases by modulating RET.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

Study of the inhibitory effects of chrysin and its nanoparticles on mitochondrial complex II subunit activities in normal mouse liver and human fibroblasts

BackgroundMitochondrial complex ΙΙ has a unique biological role owing to its participation in both the citric acid cycle and the electron transport chain. Our goal was to evaluate the succinate dehydrogenase and ubiquinone oxidoreductase activity of mitochondrial complex II in the presence of chrysin and chrysin–chitosan nanoparticles. Chrysin chitosan nanoparticles were synthesized and characterized using ultraviolet spectroscopy, Fourier transform-infrared spectroscopy, X-ray diffraction, transmission electron microscopy, scanning electron microscopy, drug release, and zeta potential. The binding affinity of chrysin to complex II subunits was assessed by molecular docking. The IC50 values were measured in a suspension of mouse mitochondria, and the inhibitory effect of chrysin and chrysin chitosan nanoparticles on mitochondrial complex ΙΙ was determined. ResultsThe free energy of binding between chrysin and complex ΙΙ subunits A, B, C, and D was −4.9, −5, −8.2, and −8.4 kcal/mol, respectively. The characteristic peak of chrysin was confirmed at 348 nm. The chrysin chitosan nanoparticles contained characteristic bands of both chrysin and chitosan. The crystalline nature of chrysin chitosan nanoparticles was confirmed by X-ray powder diffraction measurements showing the characteristic Bragg peaks of (11.2°), (32.2°), (19.6°), (27.6°), and (31.96°). Transmission and scanning electron microscopy revealed their spherical shape and an average particle size of 49.7 ± 3.02 nm. Chrysin chitosan nanoparticles showed a burst release within the initial 2 h followed by a steady release at 8 h. Their zeta potential was positive, between +35.5 and +80 mV. The IC50 of chrysin, chitosan nanoparticles, chrysin chitosan nanoparticles, and 5-fluorouracil was 34.66, 184.1, 12.2, and 0.05 μg/mL, respectively, in adult mice liver and 129, 311, 156, and 8.07 μg/mL, respectively, in normal human fibroblasts. When comparing the inhibitory effects on complex ΙΙ activity, application of the IC50 of chrysin, chitosan nanoparticles, chrysin chitosan nanoparticles, and 5-fluorouracil resulted in 40.14%, 90.9%, 86.7%, and 89% decreases in SDH activity and 70.09%, 86.74%, 60.8%, and 80.23% decreases in ubiquinone oxidoreductase activity in normal adult mice, but 80.9%, 89.06%, and 90% significant decreases in SDH activity, and 90%, 85%, and 95% decreases in ubiquinone reductase after treatment with chrysin, chrysin chitosan nanoparticles, and 5-fluorouracil, in normal human fibroblasts, respectively. ConclusionsChrysin and CCNPs exhibit potent inhibitory effects on SDH activity ubiquinone oxidoreductase activity.

Dissection and Reconstitution Provide Insights into Electron Transport in the Membrane-Bound Aldehyde Dehydrogenase Complex of Gluconacetobacter diazotrophicus

Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH). Although the reducing equivalents from the substrate are transferred to ubiquinone in the membrane, intramolecular electron transport in ALDH is not understood. Here, we purified the AldFGH complex, the membrane-bound ALDH that is physiologically relevant to acetic acid fermentation in Gluconacetobacter diazotrophicus strain PAL5. The purified AldFGH complex showed acetaldehyde:ubiquinone (Q2) oxidoreductase activity. c-type cytochromes of the AldFGH complex (in the AldF subunit) were reduced by acetaldehyde. Next, we genetically dissected the AldFGH complex into AldGH and AldF units and reconstituted them. The AldGH subcomplex showed acetaldehyde:ferricyanide oxidoreductase activity but not Q2 reductase activity. The ALDH activity of AldGH was not found in membranes but was found in the soluble fraction of the recombinant strain, suggesting that the AldF subunit is responsible for membrane binding of the AldFGH complex. The absorption spectra of the purified AldGH subcomplex suggested the presence of an [Fe-S] cluster, which can be reduced by acetaldehyde. The AldFGH complex reconstituted from the AldGH subcomplex and AldF showed Q2 reductase activity. We propose a model in which electrons from the substrate are abstracted by a molybdopterin in the AldH subunit and transferred to the [Fe-S] cluster(s) in the AldG subunit, followed by electron transport to c-type cytochrome centers in the AldF subunit, which is the site of ubiquinone reduction in the membrane. IMPORTANCE Two membrane-bound enzymes of acetic acid bacteria, pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH), are responsible for vinegar production. Upon the oxidation of acetaldehyde, ALDH reduces ubiquinone in the cytoplasmic membrane. ALDH is an enzyme complex of three subunits. Here, we tried to understand how ALDH works by using a classical biochemical approach and genetic engineering to dissect the enzyme complex into soluble and membrane-bound parts. The soluble part had limited activity in vitro and did not reduce ubiquinone. However, the enzyme complex reconstituted from the soluble and membrane-bound parts showed ubiquinone reduction activity. The proposed working model of ALDH provides a better understanding of how the enzyme works in the vinegar fermentation process.

Barth Syndrome: Connecting Cardiolipin to Cardiomyopathy.

The Barth syndrome (BTHS) is caused by an inborn error of metabolism that manifests characteristic phenotypic features including altered mitochondrial membrane phospholipids, lactic acidosis, organic acid-uria, skeletal muscle weakness and cardiomyopathy. The underlying cause of BTHS has been definitively traced to mutations in the tafazzin (TAZ) gene locus on chromosome X. TAZ encodes a phospholipid transacylase that promotes cardiolipin acyl chain remodeling. Absence of tafazzin activity results in cardiolipin molecular species heterogeneity, increased levels of monolysocardiolipin and lower cardiolipin abundance. In skeletal muscle and cardiac tissue mitochondria these alterations in cardiolipin perturb the inner membrane, compromising electron transport chain function and aerobic respiration. Decreased electron flow from fuel metabolism via NADH ubiquinone oxidoreductase activity leads to a buildup of NADH in the matrix space and product inhibition of key TCA cycle enzymes. As TCA cycle activity slows pyruvate generated by glycolysis is diverted to lactic acid. In turn, Cori cycle activity increases to supply muscle with glucose for continued ATP production. Acetyl CoA that is unable to enter the TCA cycle is diverted to organic acid waste products that are excreted in urine. Overall, reduced ATP production efficiency in BTHS is exacerbated under conditions of increased energy demand. Prolonged deficiency in ATP production capacity underlies cell and tissue pathology that ultimately is manifest as dilated cardiomyopathy.

Mitochondrial Target of Nobiletin's Action

Nobiletin is an 0-methylated flavonoid found in citrus peels that have anticancer, antiviral, neuroprotective; anti-inflammatory activities and depending on the cell types exhibits both pro- or anti-apoptotic properties We have found that nobiletin decreases oxygen consumption by bovine brain isolated mitochondria in the presence of glutamate and malate and increases in the presence of succinate. In paralleli nobiletin increases NADH: oxidation, a-ketoghitarate dehydrogenase activities and through matrix substrate-level phosphorylation elevates the a-ketoglutarate-dependent-production-of ATP. In addition, nobiletin reduces the production of peroxides in the presence of complex I substrates and slightly enhances succinate-driven H(2)0(2) formation. Besides, nobiletin induces transient elevation of membrane potential followed by mild depolarization. Affinity purified, nobiletin binding proteins revealed one major anti-NDUFVl positive protein with 52kD and NADH: ubiquinone oxidoreductase activity. This fraction can produce peroxide that is inhibited by nobiletin. We propose that nobiletin may act as a mild "uncoupler", which through activation of a-ketoglutarate dehydrogenase (a-KGDH)-complex and acceleration of matrix substrate-level phosphorylation maintains membrane potential at an abnormal level. This switch in mitochondrial metabolism could elevate succinate-driven oxygen consumption that may underlay in both pro- and anti-apoptotic effects of nobiletin.

Rotenone Decreases Hatching Success in Brine Shrimp Embryos by Blocking Development: Implications for Zooplankton Egg Banks.

While many zooplankton species recover quickly after the treatment of water resources with the piscicide, rotenone, some fail to reach pretreatment population density or, in rare cases, do not reappear at all. The variable impact of rotenone on zooplankton populations could stem from differences in the capacity of species to switch entirely to anaerobic catabolic pathways in the presence of rotenone, which blocks mitochondrial electron transport. Alternatively, variable responses among species could originate from differences in permeability of dormant life-stages to lipophilic chemicals like rotenone. The purpose of the present study was to determine the effects of rotenone on development, emergence and hatching of zooplankton embryos that lack both the anaerobic capacity to develop in the presence of rotenone and a permeability barrier to prevent the entry of rotenone during dormancy. Post-diapause embryos of the brine shrimp, Artemia franciscana, were employed as a model system, because they are permeable to lipophilic compounds when dechorionated and require aerobic conditions to support development. Early development in this species is also well characterized in the literature. Brine shrimp embryos were exposed to rotenone while development was either slowed by chilling or suspended by anoxia. Development, emergence and hatching were then observed in rotenone-free artificial seawater. The data presented demonstrate that rotenone freely diffuses across the embryonic cuticle in a matter of hours, and prevents development and emergence after brief exposures to ecologically relevant concentrations (0.025–0.5 mg L-1) of the piscicide. Neither the removal of rotenone from the environment, nor the removal of embryonic water with a hypertonic solution, are sufficient to reverse this block on development and emergence. These data indicate that rotenone could impair recruitment from egg banks for species of zooplankton that lack both an embryonic barrier to the entry of lipophilic compounds and the anaerobic capacity to develop when NADH:ubiquinone oxidoreductase activity is inhibited by rotenone.

Expression of yeast NDI1 rescues a Drosophila complex I assembly defect.

Defects in mitochondrial electron transport chain (ETC) function have been implicated in a number of neurodegenerative disorders, cancer, and aging. Mitochondrial complex I (NADH dehydrogenase) is the largest and most complicated enzyme of the ETC with 45 subunits originating from two separate genomes. The biogenesis of complex I is an intricate process that requires multiple steps, subassemblies, and assembly factors. Here, we report the generation and characterization of a Drosophila model of complex I assembly factor deficiency. We show that CG7598 (dCIA30), the Drosophila homolog of human complex I assembly factor Ndufaf1, is necessary for proper complex I assembly. Reduced expression of dCIA30 results in the loss of the complex I holoenzyme band in blue-native polyacrylamide gel electrophoresis and loss of NADH:ubiquinone oxidoreductase activity in isolated mitochondria. The complex I assembly defect, caused by mutation or RNAi of dCIA30, has repercussions both during development and adulthood in Drosophila, including developmental arrest at the pupal stage and reduced stress resistance during adulthood. Expression of the single-subunit yeast alternative NADH dehydrogenase, Ndi1, can partially or wholly rescue phenotypes associated with the complex I assembly defect. Our work shows that CG7598/dCIA30 is a functional homolog of Ndufaf1 and adds to the accumulating evidence that transgenic NDI1 expression is a viable therapy for disorders arising from complex I deficiency.

Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclicTrypanosoma brucei

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

Mitochondrial release of the NADH dehydrogenase Ndi1 induces apoptosis in yeast

Saccharomyces cerevisiae NDI1 codes for the internal mitochondrial ubiquinone oxidoreductase, which transfers electrons from NADH to ubiquinone in the respiratory chain. Previously we found that Ndi1 is a yeast homologue of the protein apoptosis-inducing factor-homologous mitochondrion-associated inducer of death and displays potent proapoptotic activity. Here we show that S. cerevisiae NDI1 is involved in apoptosis induced by various stimuli tested, including H(2)O(2), Mn, and acetate acid, independent of Z-VAD-fmk (a caspase inhibitor) inhibition. Although Ndi1 also participates in respiration, its proapoptotic property is separable from the ubiquinone oxidoreductase activity. During apoptosis, the N-terminal of Ndi1 is cleaved off in the mitochondria, and this activated form then escapes out to execute its apoptotic function. The N-terminal cleavage appears to be essential for the manifestation of the full apoptotic activity, as the uncleaved form of Ndi1 exhibits much less growth-inhibitory activity. Our results thus indicate an important role of Ndi1 in the switch of life and death fates in yeast: during normal growth, Ndi1 assimilates electrons to the electron transport chain and initiates the respiration process to make ATP, whereas under stresses, it cleaves the toxicity-sequestering N-terminal cap, is released from the mitochondria, and becomes a cell killer.

PGC-1β mediates adaptive chemoresistance associated with mitochondrial DNA mutations

Primary mitochondrial dysfunction commonly leads to failure in cellular adaptation to stress. Paradoxically, however, nonsynonymous mutations of mitochondrial DNA (mtDNA) are frequently found in cancer cells and may have a causal role in the development of resistance to genotoxic stress induced by common chemotherapeutic agents, such as cis-diammine-dichloroplatinum(II) (cisplatin, CDDP). Little is known about how these mutations arise and the associated mechanisms leading to chemoresistance. Here, we show that the development of adaptive chemoresistance in the A549 non-small-cell lung cancer cell line to CDDP is associated with the hetero- to homoplasmic shift of a nonsynonymous mutation in MT-ND2, encoding the mitochondrial Complex-I subunit ND2. The mutation resulted in a 50% reduction of the NADH:ubiquinone oxidoreductase activity of the complex, which was compensated by increased biogenesis of respiratory chain complexes. The compensatory mitochondrial biogenesis was most likely mediated by the nuclear co-activators peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) and PGC-1β, both of which were significantly upregulated in the CDDP-resistant cells. Importantly, both transient and stable silencing of PGC-1β re-established the sensitivity of these cells to CDDP-induced apoptosis. Remarkably, the PGC-1β-mediated CDDP resistance was independent of the mitochondrial effects of the co-activator. Altogether, our results suggest that partial respiratory chain defects because of mtDNA mutations can lead to compensatory upregulation of nuclear transcriptional co-regulators, in turn mediating resistance to genotoxic stress.

Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms

The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into α and β subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740- and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.

Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ 1-pyrroline-5-carboxylate (P5C) dehydrogenase domains . In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a β 8α 8-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest k cat/ K m was obtained with CoQ 2. The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ 1, combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites.

Synergetic inhibition of the brain mitochondrial NADH: Ubiquinone oxidoreductase (Complex I) by fatty acids and Ca2+

The NADH:ubiquinone oxidoreductase (respiratory complex I) activity of inside-out pig brain submitochondrial particles is inhibited by endogenous or externally added free fatty acids in time-dependent fashion. The rate and degree of the inhibition is dramatically increased by Ca2+. The Ca2+-promoted, fatty acid-induced inhibition is pH dependent, this being particularly evident at pH > 8.0. The inhibition is completely reversed by either EGTA or by bovine serum albumin (BSA). BSA prevents previously described (Kotlyar, A. B., Sled, V. D., and Vinogradov, A. D. (1992) Biochim. Biophys. Acta, 1098, 144-150) inhibitory effect of Ca2+ and alkaline pH on the de-active-to-active form transition of complex I. A possible mechanism of synergetic inhibition on complex I by Ca2+ and fatty acids is discussed.

Complex I (NADH:ubiquinone oxidoreductase) is active in but non-essential for procyclic Trypanosoma brucei

The requirement of complex I (NADH:ubiquionone oxidoreductase) for respiration in Trypanosoma brucei is controversial. Recent identification of homologues of its subunits in mitochondrial proteome resolved a question of its presence or absence. However, with one exception, no data have been available concerning the function(s) of complex I or its subunits. Here we present a functional RNAi study of three (NUBM, NUKM, NUEM) putative subunits of this complex. Although no changes were detected in growth, mitochondrial membrane potential or reactive oxygen species production in cell lines depleted for target transcript, the NUBM and NUKM RNAi knock-downs showed decreased specific NADH:ubiquinone oxidoreductase activity. Moreover, glycerol gradients of all cell lines revealed the presence of two distinct peaks of NADH dehydrogenase activity, with shifted sensitivity to inhibitors of complex I upon RNAi induction. Thus complex I is not only present in the procyclic stage of T. brucei 29-13 strain, but it does participate in electron transport chain.

Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction

The formation of reactive oxygen species (ROS) and the change of the intracellular pH (pH(i)) are common phenomena during apoptosis. How they are interconnected, however, is poorly understood. Here we show that numerous anticancer drugs and cytokines such as Fas ligand and tumour necrosis factor α provoke intracellular acidification and cause the formation of mitochondrial ROS. In parallel, we found that the succinate:ubiquinone oxidoreductase (SQR) activity of the mitochondrial respiratory complex II is specifically impaired without affecting the second enzymatic activity of this complex as a succinate dehydrogenase (SDH). Only in this configuration is complex II an apoptosis mediator and generates superoxides for cell death. This is achieved by the pH(i) decline that leads to the specific dissociation of the SDHA/SDHB subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required for the SQR activity.

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ∼7Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q1 and Q2 that were coupled to proton pumping. Apparent Km values for Q1 and Q2 were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.

Kava, a Tonic for Relieving the Irrational Development of Natural Preventive Agents

The last two decades have witnessed explosive growth in the study of natural and other cancer chemopreventive agents ([1][1], [2][2]). Extensive preclinical data have been generated for natural agents, and many (such as green tea, curcumin, phenyl isothiocyanate, indole-3-carbinol, silibinin,

The iron–sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.