scholarly journals Spectroscopic studies of flavoproteins and non-haem iron proteins of submitochondrial particles of Torulopsis utilis modified by iron- and sulphate-limited growth in continuous culture

1971 ◽  
Vol 124 (1) ◽  
pp. 171-187 ◽  
Author(s):  
C. I. Ragan ◽  
P. B. Garland
Keyword(s):  
Electron Transport ◽  
Respiratory Chain ◽  
Nadh Dehydrogenase ◽  
Difference Spectrum ◽  
Spectroscopic Studies ◽  
Submitochondrial Particles ◽  
Limited Growth ◽  
Nadph Dehydrogenase ◽  
Haem Iron

1. A spectroscopic resolution has been made of the components contributing to the ‘iron-flavoprotein’ trough extending from 450 to 520nm in the reduced-minus-oxidized difference spectrum of submitochondrial particles of Torulopsis utilis. 2. Seven components were identified other than cytochrome b, ubiquinone and succinate dehydrogenase. On the basis of the effects of iron- and sulphate-limited growth of cells on their subsequently derived electron-transport particles, and also by consideration of analytical measurements of the concentration of FMN, FAD, non-haem iron and acid-labile sulphide in the electron-transport particles in relation to the magnitude of the spectroscopic changes, it was possible to identify five of these components as follows: species 1a, the flavin of NADH dehydrogenase ferroflavoprotein; species 1b, the iron–sulphur component of NADH dehydrogenase ferroflavoprotein; species 1′, the flavin of an NADPH dehydrogenase; species 2, an iron–sulphur or ferroflavoprotein component; species 3, the flavin of l-3-glycerophosphate dehydrogenase. Two additional components were a fluorescent flavoprotein, probably lipoamide dehydrogenase, and a b-type cytochrome reducible by NADH or NADPH but not reoxidizable by the respiratory chain. 3. Species 1b and 2 were undetectable in electron-transport particles from iron- or sulphate-limited cells, but could be recovered in vivo under non-growing conditions. 4. The recovery in vivo of species 2 but not species 1b was inhibited by cycloheximide. 5. The recovery of species 1b correlates with the recovery of site 1 conservation. 6. The recovery of species 1b with species 2 correlates with the recovery of piericidin A sensitivity. 7. Evidence is presented for an NADPH dehydrogenase distinct from NADH dehydrogenase. The oxidation of NADH and NADPH by the respiratory chain is sensitive to piericidin A, and an iron–sulphur protein common to both pathways (species 2) is suggested as the piericidin A-sensitive component. 8. The approximate E′0 (pH7.0) values of species 1 (a and b, low potential) and species 2 (high potential) indicate that site 1 energy conservation occurs between the levels of species 1 (a and b) and species 2.

scholarly journals Effect of sulphate-limited growth on mitochondrial electron transfer and energy conservation between reduced nicotinamide–adenine dinucleotide and the cytochromes in Torulopsis utilis

1971 ◽  
Vol 124 (1) ◽  
pp. 155-170 ◽  
Author(s):  
B. A. Haddock ◽  
P. B. Garland
Keyword(s):  
Energy Conservation ◽  
Cytochrome B ◽  
Antimycin A ◽  
Alternative Route ◽  
Aerobic Incubation ◽  
Submitochondrial Particles ◽  
Limited Growth ◽  
Sulphide Concentration ◽  
Haem Iron ◽  
Acid Labile

1. Conditions have been established for the sulphate-limited growth of Torulopsis utilis in continuous culture. 2. Mitochondria prepared from sulphate-limited cells lack both piericidin A sensitivity and the first energy-conservation site (site 1). Sensitivity to antimycin A or cyanide and the second and third energy-conservation sites were apparently unaffected by sulphate-limited growth. 3. Aerobic incubation for 8h of sulphate-limited cells with a low concentration of sulphate (50μm or less) resulted in the recovery of mitochondrial piericidin A sensitivity and site 1. The use of higher concentrations of sulphate (250μm or more) still resulted in the recovery of mitochondrial piericidin A sensitivity and site 1, but also resulted in the appearance of a non-phosphorylating oxidase, which mediated oxidation of the respiratory chain at about the level of cytochrome b in an antimycin A- and cyanide-insensitive manner. Both this alternative route and the conventional normal route of respiration were shown to coexist and to intercommunicate at the level of cytochrome b. 4. Low-temperature spectroscopy failed to identify any new respiratory component to explain the alternative route. 5. The apparent affinity of the alternative route for oxygen was similar to that for the conventional route through cytochrome oxidase, namely half-maximal activity at 0.1μm-oxygen or less. 6. The non-haem iron concentration of submitochondrial particles was unaffected by sulphate limitation, whereas the acid-labile sulphide concentration was lowered tenfold. Marked increases (between four- and 30-fold) in the acid-labile sulphide concentration of submitochondrial particles were observed in sulphate-limited cells after aerobic incubation with various concentrations of sulphate. The lowest increase (fourfold) was observed without added sulphate, the highest (30-fold) with 1.0mm added sulphate. 7. The ratio of non-haem iron to acid-labile sulphide in submitochondrial particles varied with different growth conditions from a maximum of 15.0 to a minimum of 0.72. It is suggested that analytical measurements of non-haem iron are an inadequate guide to the concentration of iron–sulphur protein in complex systems. 8. The effects of sulphate-limited growth on site 1 and piericidin sensitivity are interpreted to indicate a role for iron–sulphur protein in these properties. 9. The aerobic incubation of sulphate-limited cells with cycloheximide resulted in the recovery by mitochondria of site 1 but not of piericidin sensitivity. 10. The appearance of the alternative route for cyanide- and antimycin-A (but not piericidin A-) insensitive respiration on incubating sulphate-limited cells with sulphate concentrations higher than 250μm indicates that the alternative route involves an iron–sulphur protein.

scholarly journals Electron-paramagnetic-resonance spectroscopy studies of iron-sulphur centres of submitochondrial particles from iron- and sulphur-deficient. Candida utilis

1975 ◽  
Vol 146 (1) ◽  
pp. 239-246 ◽  
Author(s):  
T A Gray ◽  
P B Garland ◽  
D J Lowe ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Nadh Dehydrogenase ◽  
Candida Utilis ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Submitochondrial Particles ◽  
Limited Growth ◽  
Paramagnetic Resonance ◽  
Dehydrogenase Reaction ◽  
Electron Paramagnetic

1. Measurements were made at 12 degrees K of the electron-paramagnetic-resonance (e.p.r.) spectra of submitochondrial particles from Candida utilis cells grown under conditions that alter the amount of the mitochondrial NADH dehydrogenase (EC 1.6.99.3). 2. Iron-limited growth decreases the extent of iron-sulphur e.p.r. signals to undetectable values that are less than 1 percent of those normally found with glycerol-limited growth. 3. Small but significant signals attributable to the NADH dehydrogenase were detected in submitochondrial particles from sulphate-limited cells. 4. Measurements made on submitochondrial particles prepared from these and other phenotypically modified cells lead us to conclude that the presence of low-temperature e.p.r.-detectable iron-sulphur centres attributable to the NADH dehydrogenase are necessary but not sufficient for the coupling of ATP synthesis to the NADH dehydrogenase reaction in the mitochondrial membrane of C. utilis. 6. The amplitude of the g=2.01 signal observed in non-reduced submitochondrial particles is approximately tenfold diminished by iron limitation but not significantly altered by sulphate limitation.

scholarly journals The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis

1971 ◽  
Vol 124 (5) ◽  
pp. 853-865 ◽  
Author(s):  
S. O. C. Tottmar ◽  
C. I. Ragan
Keyword(s):  
Respiratory Chain ◽  
Reductase Activity ◽  
Submitochondrial Particles ◽  
Ferricyanide Reduction ◽  
Paramagnetic Resonance ◽  
Ferricyanide Reductase ◽  
Starting Point ◽  
Nicotinamide Adenine Dinucleotide Dehydrogenase ◽  
Haem Iron ◽  
Acid Labile

1. An NADH–ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH–ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH–ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH–ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes.

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs

1992 ◽  
Vol 70 (11) ◽  
pp. 1529-1533 ◽  
Author(s):  
Laura Jill McCutcheon ◽  
Colin Robert Cory ◽  
Linda Nowack ◽  
Hua Shen ◽  
Medhi Mirsalami ◽  
...  
Keyword(s):  
Electron Transport ◽  
Dilated Cardiomyopathy ◽  
Respiratory Chain ◽  
Transport System ◽  
Nadh Dehydrogenase ◽  
Genetic Defect ◽  
Electron Transport System ◽  
Idiopathic Dilated Cardiomyopathy ◽  
Acid Oxidation ◽  
Doberman Pinscher

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca2+-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.Key words: dilated cardiomyopathy, congestive heart failure, mitochondria, electron transport system, myoglobin, NADH dehydrogenase.

scholarly journals Non-haem iron and the dissociation of piericidin A sensitivity from site 1 energy conservation in mitochondria from Torulopsis utilis

1971 ◽  
Vol 124 (1) ◽  
pp. 135-151 ◽  
Author(s):  
R. A. Clegg ◽  
P. B. Garland
Keyword(s):  
Energy Conservation ◽  
Electron Flow ◽  
Iron Concentration ◽  
Normal Function ◽  
Aerobic Incubation ◽  
Submitochondrial Particles ◽  
Limited Growth ◽  
Iron Deficient ◽  
Haem Iron ◽  
Acid Labile

1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100μg/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of 59Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ‘normal’ situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ‘normal’ non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5–0.8ng-atom/mg of protein. It is concluded that the concentration of iron–sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.

scholarly journals Evidence that the inhibition sites of the neurotoxic amine 1-methyl-4-phenylpyridinium (MPP+) and of the respiratory chain inhibitor piericidin A are the same

1991 ◽  
Vol 273 (2) ◽  
pp. 481-484 ◽  
Author(s):  
R R Ramsay ◽  
M J Krueger ◽  
S K Youngster ◽  
T P Singer
Keyword(s):  
Electron Transfer ◽  
Respiratory Chain ◽  
Nadh Dehydrogenase ◽  
Nadh Oxidation ◽  
Submitochondrial Particles ◽  
Alkyl Chains ◽  
Alkyl Derivatives ◽  
Derivatives Of ◽  
Respiratory Chain Inhibitors

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. Proof that these classical respiratory chain inhibitors and MPP+ react at the same site in the complex NADH dehydrogenase molecule has been difficult to obtain because none of these compounds bind covalently to the target. The 4′-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself, but still dissociate on washing the membrane preparations, with consequent re-activation of the enzyme. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin.

scholarly journals Anticancer gold(iii)-bisphosphine complex alters the mitochondrial electron transport chain to induce in vivo tumor inhibition

2021 ◽  
Author(s):  
Jong Hyun Kim ◽  
Samuel Ofori ◽  
Sean Parkin ◽  
Hemendra Vekaria ◽  
Patrick G. Sullivan ◽  
...  
Keyword(s):  
Electron Transport ◽  
Metal Complexes ◽  
Electron Transport Chain ◽  
Chemical Diversity ◽  
Mitochondrial Electron Transport Chain ◽  
Generate Functional ◽  
Tumor Inhibition ◽  
Transport Chain

Expanding the chemical diversity of metal complexes provides a robust platform to generate functional bioactive reagents.

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