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[Interaction of paramagnetic probes with submitochondrial particle membranes].

Redox-transformations of some paramagnetic probes differing in chemical structure have been studied during their interaction with the membranes of submitochondrial particles. Dependence of the probe restoration kinetics on the conditions of functioning of the electron-transport chain, presence of oxygen, respiration substrate, electron transport inhibitors (antimycin, alpha tenoiltriftoracetate, potassium cyanide) in the reaction mixture is shown. Water-soluble as well as strongly hydrophobic spin probes are restored when the respiration substrate (succinate) is present; they interact with the lipid-soluble carrier ubiquinone.

Antimycin A↗
PubMed1977

[Influence of pH on the steady state kinetics of electron transfer through the cytochrome chain of submitochondrial particles. Kinetic model for regulating the activity of carriers by the local concentration of hydrogen ions in the membrane].

The kinetic parameters of the submitochondrial particles cytochrome chain obtained from steady-state kinetics were studied for pH dependence. The life-times of the activated states (tau) for cytochrome pairs b leads to c1 and a leads to a3 are shown to bear dissimilar dependence on pH of the medium, while for cytochrome pairs c1 leads to c and c leads to a they display practically no pH dependence at all. The rate constants of the non-activated state (alphai-kiCo) decreased for the pair b leads to c1 and increased for a leads to a3 with the increase of pH from 6.5 to 8.5. The apparent pK values obtained therefrom were 7.2 and 8.9, respectively. A kinetic model is proposed suggesting that local pH in the mitochondrial membrane, dependent on the rate of electron transfer, may be a controlling factor for the ratio of activated and non-activated carrier states. The model is in good consistence with the experimental dependences of k'i on V and the pH dependences of alpha2 for b leads to c1 and a leads to a3. It also gives a qualitative prediction for the pH dependences of the ordinate intercepts of the straight lines in l/(k'i--alphai) vs. l/V plots. The rate constants for the diffusion of hydrogen and hydroxyl ions in the membrane are estimated on the basis of our kinetic data to be 10(4)--10(5) s-1 and 10(2)--10(3) s-i, respectively.

Cytochromes↗
PubMed1976

A method for determining the adenosine triphosphatase content of energy-transducing membranes. reaction of 4-chloro-7-nitrobenzofurazan with the adenosine triphosphatase of bovine heart submitochondrial particles.

1. Modification of a single amino acid residue by introduction of the nitrobenzofurazan group inactivates mitochondrial ATPase (adenosine triphosphatase) when membrane-bound in submitochondrial particles. The similarity between the reactions of both membrane-bound and isolated ATPase with 4-chloro-7-nitrobenzofurazan indicates that the single essential tryosine residue identified in the isolated enzyme [Ferguson, Loyd, Lyons & Radda (1975) Eur. J. Biochem. 54, 117-126] Is also a feature of the membrane-bound ATPase. 2. A procedure is presented for estimating the ATPase content of the inner mitochondrial membrane. It is based on the specificity of the incorporation of the nitrobenzofurazan group, and the ready removal of this group by compounds that contain a thiol group. This method indicates that 8.5% of the membrane protein is ATPase. The procedure should be applicable to the titration of the energy-transducing ATPases of bacterial plasma membranes and of the thylakoid membranes of chloroplasts. 3. Combination of the data obtained on the ATPase content of the bovine heart inner mitochondrial membrane with a titration of the cytochrome bc1 complex with antimycin indicates that these two components of the membrane are present in approximately equal amounts.

Adenosine Triphosphatases↗
PubMed2001

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III.

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing </=0.06 mol of Q/mol of complex III, and Q-replenished SMP. The results showed that Q was not required for electron transfer from ISP to b, a reaction that was inhibited by antimycin (also by myxothiazol or MOA-stilbene as reported elsewhere). It was also shown that antimycin did not inhibit electron transfer from b (b(H)) to Q, in clear contrast to the assumption of the Q-cycle hypothesis regarding the site of antimycin inhibition.

Animals↗
PubMed1970

Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria.

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD(+) NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.Oligomycin and bis(hexafluoroacetonitryl) acetone, a potent uncoupler of oxidative phosphorylation, were without effect on oxygen uptake but did influence the ATP-stimulated onset of respiration when succinate was substrate. Fresh particles were markedly inhibited by oxtylguanidine, indicating energy conservation, but this inhibition decreased on storage of the particles.Spectra show the presence of cytochrome components the same as those of the intact mung bean mitochondrion, but present at higher concentrations. The molar concentrations of the particle cytochromes were two to three times those of the intact mitochondrion and the molar ratios were calculated as 0.9:1.0:1.0:2.8 for cytochromes a:b:c:flavoprotein, respectively.

Journal Article↗
PubMed1999

Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.

Adenosine Triphosphatases↗
PubMed1976

On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in "sonic" submitochondrial particles.

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.

Animals↗
PubMed2001

Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles.

The aim of this study was to investigate the effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of complex III and on the cardiolipin content in bovine-heart submitochondrial particles (SMP). ROS were produced by treatment of nicotinamide adenine dinucleotide (NADH) respiring SMP with rotenone. This treatment resulted in a production of superoxide anion, detected by the epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial-mediated ROS generation resulted in a marked loss of complex III activity and in a parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD + catalase. Exogenous added cardiolipin was able to almost completely prevent the ROS-mediated loss of complex III activity. No effect was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, or with peroxidized cardiolipin. The results demonstrate that mitochondrial-mediated ROS generation affects the activity of complex III via peroxidation of cardiolipin, which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those physiopathological conditions characterized by an increase in the basal production of ROS such as aging, ischemia/reperfusion and chronic degenerative diseases.

Journal Article↗
PubMed1980

Observations on the oxidoreduction of the two cytochromes b in cytochrome c-deficient mitochondria and submitochondrial particles.

1. In cytochrome c depleted mitochondria cytochrome bT is reduced rapidly upon addition of ATP or slowly during state 4 respiration, but cytochrome bK is effectively reduced in such mitochondira respiring upon glutamate plus malate in all energy states. In mitochondria or in submitochondrial particles oxidized NADH or succinate, cytochromes bK and bT were always reduced and oxidized independently. 2. Difference spectra for the two b cytochromes were obtained in the presence of respiratory chain inhibitors. Reduced cytochrome bK in the presence of cyanide can be reoxidized by CoQ2. Cytochrome bT reduced in the presence of antimycin can be reoxidized by O2 if rotenone is added to an NADH-reduced sysem or malonate to a succinate-reduced system. There is no evidence for electron transfer between the two b cytochromes. 3. It is suggested that there is no electron transfer from cytochrome bT to cytochrome bK, but that a cytochrome bKbT dimer accepts electrons from the CoQ pool jointly with cytochrome c1 and another acceptor, perhaps the FeS centre. The major steady state species is b2K+b3T+, and a Q-loop occurs with reduction of CoQ by the fully reduced species b2K+b2T+. All proposed interactions between CoQ and Complex III are 2-electron processes and the change from 2-electrons to 1-electron transfer occurs within Complex III itself.

Antimycin A↗
PubMed1971

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.

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.

Cycloheximide↗
PubMed1968

A comparison of the effects of NN'-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enrgy-linked reactions in mitochondria and submitochondrial particles.

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.

Adenine Nucleotides↗
PubMed2001

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.

Animals↗
PubMed1979

[Interaction of 3-(3H)-2-n-nonyl-4-hydroxyquinoline-N-oxide with submitochondrial particles of beef heart. III. Correlation between the binding of 3H-NQNO and inhibition of the respiratory activity].

The present work is an extension of our precedent papers (1-3). In the present report the relationship was studied between the inhibition by 3H-NQNO, 3-3H-2-n-nonyl-4-hydroxy-quinoline-N-oxide, of the respiratory activity induced in submitochondrial particles from beef heart by NADH and the binding of 3H-NQNO to the specific binding site. The experiments showed that the concentrations of inhibition sites and of specific binding sites are identical. Titrating the inhibition of NADH-oxidase activity with increasing amounts of 3H-NQNO a close connection was observed between the decrease of the respiratory activity and the increase of the specific binding of 3H-NQNO. When nearly full inhibition (85%) was reached, also the specific binding was saturated. We may conclude that 3H-NQNO behaves like an ideally simple inhibitor: the inhibition of electron transfer by 3H-NQNO is linear with the saturation of the specific binding site.

Animals↗
PubMed1981

[Effect of tyrosine-74-modified cytochrome C on electron transfer in Keilin-Hartree submitochondrial particles].

The tyrosine-74 modified cytochrome c is obtained by the interaction of cytochrome c with N-(2, 2, 5, 5-tetramethyl-3-carboxy-piroline-oxyl) Imidazol. The absorbtion spectrum of the modified derivative at the visible region does not differ substantially from the native protein. The band of absorbtion at 695 nm, which shows that the performed modification is soft and the conformation of the modified cytochrome c is close to that of the native protein. Redox potential of the modified cytochrome c, determined by spectrophotometric titration, does not differ from that of the native protein. Submitochondrial particles, deficient of cytochrome c, are reconstructed with modified protein. In both substrates of biologic oxidation succinate and ascorbate the electron transport is restored by the modified protein in considerably higher concentrations than those of the native cytochrome c. Inspite of small conformation differences and unchanged redox potential of the modified cytochrome c substantial differences are observed in its electron-carrying properties as well as its interaction with the membrane cytochrome c reductase and oxydase.

Animals↗
PubMed2006

Reduction of manganese porphyrins by flavoenzymes and submitochondrial particles: a catalytic cycle for the reduction of peroxynitrite.

The reduction of manganese(III) meso-tetrakis((N-ethyl)pyridinium-2-yl)porphyrin (MnTE-2-PyP) to manganese(II) was catalyzed by flavoenzymes such as xanthine oxidase and glucose oxidase, and by Complex I and Complex II of the mitochondrial electron transport chain. The reduced manganese porphyrin has been previously shown to react rapidly with superoxide and carbonate radical anion. Herein, we describe the reaction of a reduced manganese porphyrin with peroxynitrite that proceeds as a two-electron process, has a rate constant greater than 7 x 10(6) M(-1) s(-1) (at pH 7.25 and 37 degrees C), and produces nitrite and the Mn(IV)Porphyrin. The Mn(II)/Mn(IV) redox cycle was used to divert peroxynitrite from the inactivation of succinate dehydrogenase. In a typical experiment, 5 microM MnTE-2-PyP in the presence of excess succinate was able to protect the succinate dehydrogenase and succinate oxidase activities of submitochondrial particles challenged with a cumulative dose of 140 microM peroxynitrite infused in the course of 2 h. Other MnPorphyrins that are reduced more slowly do not provide as much protection underscoring the rate limiting character of the reduction step. The data presented here serve to rationalize the pharmacological action of MnPorphyrins as peroxynitrite reduction catalysts in vivo and opens avenues for the development of MnPorphyrins to protect mitochondria from oxidative damage.

Animals↗