Diode-like behaviour of a mitochondrial electron-transport enzyme

Sucheta, Artur; Ackrell, Brian A.C.; Cochran, Bruce; Armstrong, Fräser A.

doi:10.1038/356361a0

Cited by 181 publications

(144 citation statements)

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“…1B), as the E m of the cytochrome b of the P. denitrificans enzyme 5 is much lower than that of the fumarate:succinate couple at pH 7.4 (5 mV) (36). Therefore, it is not reduced appreciably (ϳ8%; data not shown) by succinate (see also Fig.…”

Section: Resultsmentioning

confidence: 91%

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Waldeck¹,

Stowell²,

Lee³

et al. 1997

Journal of Biological Chemistry

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Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic "signatures" accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochrome b, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4 -10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate-and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity ( 2 nm) of the high potential 3Fe-4S cluster, situated in the succinate dehydrogenase part of the enzyme, and the low potential, low spin b-heme in the membrane anchor of the enzyme.Succinate:ubiquinone oxidoreductase, SQR, 1 is the only membrane-bound enzyme in the tricarboxylic acid cycle. As "complex II," it performs the two-electron oxidation of succinate to produce fumarate, while transferring the electrons to quinone (Q) to yield quinol (QH 2 ). The reverse process is mediated by quinol:fumarate oxidoreductases (QFR), which occur in anaerobic and some facultative organisms. The two enzymes are related and are capable of catalyzing their respective reverse reactions under suitable conditions (1, 2).SQR contains three or four polypeptides depending on the organism. The largest subunit, a flavoprotein, contains the dicarboxylate binding site and one flavin moiety (FAD); the latter is covalently bound in most cases. The iron-sulfur protein is intermediate in size and contains three iron-sulfur clusters of type 2Fe-2S, 4Fe-4S, and 3Fe-4S, often referred to as S-1, S-2, and S-3, respectively, in the case of SQR. These two hydrophilic subunits protrude into the cytosol (prokaryotic enzyme) or the mitochondrial matrix (eukaryotic enzyme), and together catalyze the succinate dehydrogenase activity of the enzyme. They are anchored to the membrane by one or two hydrophobic quinone polypeptides (QP), which may contain zero, one, or two b-heme(s). These anchoring subunits confer reactivity with the bound Qs; for SQR from bovine heart (3, 4) and a variety of higher plants (5), the existence of two Q sites has been established. S...

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Section: Resultsmentioning

confidence: 91%

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Waldeck¹,

Stowell²,

Lee³

et al. 1997

Journal of Biological Chemistry

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“…In the context of our previous suggestion, i.e. that substrate binding or release at SDH is much more favourable for the oxidised enzyme, the phenomenon may also be viewed from a kinetic standpoint [2]. Thus, the higher the rate that electrons are supplied to the enzyme, the shorter becomes the lifetime of the oxidised active form.…”

Section: Methodsmentioning

confidence: 99%

“…At this pH, oxidation of succinate does not proceed readily (c.f. [2]); however, succinate was included in order to stabilise the enzyme. In both cases the voltammogram is very unusual, being dominated by a pair of reduction peaks which effectively overlay in the directions of increasing negative or increasing positive potential.…”

Section: Methodsmentioning

confidence: 99%

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Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay

et al. 1993

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Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of -80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases.For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and 'succinate dehydrogenase' from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The 'tunnel-diode' effect may thus represent an evolutionary adaptation to aerobic metabolism.

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“…The reason for the irreversible response may be as follows: firstly, some enzymes in the living cells assist in the unidirectional electron transfer process; 12 secondly, formation and generation of coenzymes are also responsible for electron transfer between living cells and graphite electrode.…”

Section: Psa and CV Response Of Mcf-7 Cellsmentioning

confidence: 99%

Electrochemical Study of Breast Cancer Cells MCF-7 and Its Application in Evaluating the Effect of Diosgenin

Liu

Guo

et al. 2005

ANAL. SCI.

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Cell growth and the effect of some drugs on cells can be evaluated by traditional morphological observation methods or by biological technology.However, to perform these experiments, complex instruments and professionals are always required.Recent studies indicated that electrochemical methods, such as CV, scanning electrochemical microscopy, electric impedance and oxygen electrode, could also provide useful information on the living cell because the cell viability has positive relation with its electrode behavior. 1-4As a plant steroid produced by secondary metabolism, diosgenin shows many important biological functions in plants and animals, acting as a regulator of developmental and physiological processes in animal system. 5It is also widely used to synthesize new steroidal drugs. 6 Recently, some studies have concerned its utilization in cancer therapy. 7 However, to our knowledge, few reports about the effect of diosgenin on the breast cancer cells can be found in the literature.Potential stripping analysis, first introduced by Jagner and Granile in 1976, is a highly sensitive electroanalysis technology including two steps: (1) preconcentration of the analyte by electrodeposition; and (2) stripping of the analyte by an appropriate oxidant. 8 It has been applied to metal analysis and bioanalysis. 9,10 In this work, electrochemical methods, i.e., potentiometric stripping analysis (PSA) and cyclic voltammetry (CV), were used to investigate the electrochemical behavior of breast cancer cells and to evaluate the influence of diosgenin on the viability and proliferation of breast cancer cells. Trypan Blue dyeing tests were also employed to verify the results obtained by PSA. Experimental ChemicalsDiosgenin was from the Institute of Life Science and Technology of Hunan University. A stock solution of 1.0 × 10 -2 M diosgenin was prepared with ethanol and diluted to desired concentrations with water. Phosphate buffer solution (PBS) composed of 136.7 mM NaCl, 2.7 mM KCl, 9.7 mM Na2HPO4 and 1.5 mM KH2PO4 was used as the background electrolyte in all experiments. Other chemicals are of analytical grade. InstrumentationAll electrochemical measurements were performed at a CHI 660A electrochemical analyzer (Chenhua Company, China) by applying a three-electrode system, with a graphite electrode (0.45 cm 2 ) as working electrode, platinum as counter electrode and Ag/AgCl electrode as reference electrode. Cell culture and cell treatmentBreast cancer cell line MCF-7 was kindly provided by Dr. K. Q. Deng (Institute of Life Science and Technology of Hunan University) and was routinely cultured in RPMI-1640 medium (Invitrogen Corporation) + 10% fetal bovine serum (Sigma) + 0.05 mg mL -1 gentamicin (Sigma) + 100 U mL -1 penicillin (Sigma) at 37˚C in a CO2 incubator containing 5% CO2.After the breast cancer cells were cultured for five days, they were collected by trypsinization with 0.05% (v/v) trypsin (Sigma) and centrifugation at 1200 rpm for 10 min, and then suspended in PBS. It should be mentioned that PBS-containing cells s...

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Diode-like behaviour of a mitochondrial electron-transport enzyme

Cited by 181 publications

References 5 publications

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay

Electrochemical Study of Breast Cancer Cells MCF-7 and Its Application in Evaluating the Effect of Diosgenin

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