The effect of some sulfhydryl reagents on the respiratory chain activity

The observation that in cation transport experiments N-ethylmaleimide (NEM) behaves as uncoupler and as a respiratory inhibitor at the same time, the effect of sulfhydryl reagents on the redox state of respiratory chain, has been studied. Spectra of mitochondrial suspension in the span 300-630 nm have revealed that NEM promotes the oxidation of all the respiratory intermediates, cytochrome a included. Azide completely reverses the oxidation effect of NEM, suggesting that it cannot be ascribed to an irreversible damage of mitochondrial intactness. Mersalyl, which shares the highly sensitive SH reagent and specific inhibitor of Pi transport properties of NEM, gives completely different results. It is proposed that, besides the generally accepted inhibitory effect on primary dehydrogenases reacting with their SH groups, NEM may also behave as an oxidizing agent which can promote the release of reducing equivalents from the respiratory chain.     

Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria

The free radical EPR signals of ubisemiquinone in mitochondria and submitochondrial particles (SMP) were investigated. One of the signals observed under the conditions of the respiratory chain highly oxidized and characterized by an unusually short time of the spin-lattice relaxation has previously been termed as SQ-2. The intensity of SQ-2 in SMP strongly depends on pH, the maximal concentration of QH. is reached at about 8.5. The signal is absent in the succinate dehydrogenase-depleted SMP and is highly sensitive to specific inhibitors of succinate: CoQ-oxidoreductase, such as alpha-thenoyltrifluoroacetone and carboxin. In SMP SQ-2 disappears in the presence of low concentrations of ferricyanide, while in mitochondria this non-penetrating oxidant provokes the appearance of SQ-2. The data obtained suggest that SQ-2 belongs to a stable ubisemiquinone which forms a complex with a FeS center of succinate dehydrogenase, is localized at the M-side of the membrane, and is kinetically isolated from the cytochrome chain. Oxidation of the terminal segment of the respiratory chain of mitochondria and SMP reduced by succinate in the presence of antimycin, is in some cases accompanied by an appearance of a strong free radical EPR signal which is stable at 77K but disappears rapidly in the frozen samples at -30- -40 degrees C. It is suggested that the signal is generated by an antimycin-insensitive oxidation of QH2 to QH. via the branch of the respiratory chain comprised of the Rieske FeS-protein and cytochrome c1. The mechanisms of how the two-electron oxidation-reduction of CoQ is coupled with the one-electron transfer through the cytochromes and FeS centers in the respiratory chain are discussed.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Superoxide dismutase enhances chain-breaking antioxidant capability of hydroquinones.

2-tert-butyl-(1), 2,6-dimethyl-(2), 2,5-dimethyl-(3), trimethyl-(4), and 2,3-dimethoxy-5-methyl-(5) substituted p-hydroquinones (QH2) were tested as a chain-breaking antioxidant during the oxidation of methyl linoleate (ML) in dodecyl sulfate micellar solution, pH 7.40, at 37 degrees C. In the absence of superoxide dismutase (SOD), all the studied QH2 displayed very moderate if any antioxidant capability. When 5-25 U/ml SOD was added, QH2 showed a pronounced ability to inhibit ML oxidation. The stoichiometric factor of inhibition was found to be about one for all the tested QH2 in the presence of SOD. The reactivities of QH2 to the ML peroxy radical increase in the order QH2 5 < QH2 3 < QH2 1 approximately QH2 2 < QH2 4; reactivity of QH2 4 exceeds that reported for the majority of phenolic antioxidants. The features of QH2 as an antioxidant in aqueous environment is likely associated with the reactivity of semiquinone (O.-) formed due to attack of the peroxy radical to QH2. O.- reacts readily with molecular oxygen with formation of superoxide (O2.-); in turn, O2.- attacks both to QH2 and ML (likely, as HO2.) that results in fast depleting QH2 and chain propagation, respectively. The addition of SOD results in purging a reaction mixture from O2.- and, as a corollary, in depressing undesirable reactions with the participation of O2.-. Under these conditions, QH2 displays the theoretically highest inhibitory activity which is determined solely by the reactivity of QH2 to the peroxy radical.     

Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores.

Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the 'pulsed' form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.     

Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate

The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined. It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair. Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor. Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors. At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b. Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + O2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic. The plot of log (Q/QH2) against log (b3+/b2+) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4. This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5. On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4. BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value. Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b. It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH2 of two species of cytochromes b-562.     

Identification of two different Q-binding sites in QH2-cytochrome c oxidoreductase, using the Q analogue n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone

The pK and mid-point redox potential of the Q-analogue 7-(n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ) in aqueous medium are so low that under the experimental conditions used for studying the inhibition of electron transfer in submitochondrial particles only the oxidized, anionic form is present. The KD of the analogue, determined by comparing its inhibitory effect with that of n-heptyl-4-hydroxyquinoline N-oxide, is (0.003 + 0.24 x mg protein/ml) microM. The inhibition of succinate oxidation is pH dependent, due to a pH-dependent change in the overcapacity of the QH2-oxidizing system above the Q-reducing system. If the terminal part of the respiratory chain is reduced with ascorbate, the analogue inhibits the reduction of cytochrome b by substrate in the presence of antimycin with a similar KD value. In the absence of ascorbate the KD value is 100-times higher. The reduction of cytochrome b by substrate in particles treated with 2,3-dimercaptopropanol (BAL) + O2 is also sensitive to HMHQQ, with a KD value in between the two values given above. It is concluded that the QH2 oxidase system contains two different sites for interaction with ubiquinone. The site responsible for the inhibition of steady-state electron transfer is near the Fe-S cluster, as is shown by the sensitivity to the redox state of this cluster and by the effect of HMHQQ on the EPR signal of the reduced cluster. The second site, which is similar to the antimycin-binding site, is occupied only at higher concentrations of inhibitor. The affinity of HMHQQ for this site is not affected by the redox state of the Fe-S cluster.     

The enzyme activity of energy metabolism in antibiotic-resistant staphylococci

The rates of lactate accumulation and glucose oxidation by the clinic strains of Staphylococcus aureus were determined. The inhibition grade of the glucose oxidation by cyanide indicates that respiratory chain of staphylococci participates in the oxidizing processes. During glucose oxidation the respiratory chain generates the difference of electric potentials -156 divided by -179 mV. The comparative assessment have shown that the antibiotic-resistant staphylococci are characterized by a higher rate of glucose oxidation as well as by value of the greater transmembrane difference of electric potentials.     

Carbon monoxide oxygenase activity of cytochrome cd1

Cytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa catalyzes the oxygenation of carbon monoxide by dioxygen. A minimum estimate of the turnover number for this activity is 7 mol of carbon dioxide produced per hour per mole of cytochrome subunit at 30 degrees C and pH 7. The reaction is 98% inhibited by 2.5 mM cyanide, but catalase has no effect. The reaction accounts for the unusual reduction of ferric cytochrome in the presence of carbon monoxide, but no additional reducing agent. The reaction is independent of the steady-state oxidation level of the cytochrome during turnover. Under anaerobic conditions, ferricyanide plus water may substitute for dioxygen as the source of oxidizing equivalents and atomic oxygen.     

Substituted p-hydroquinones as inhibitors of lipid peroxidation

The technique based on monitoring oxygen consumption was applied to study 12 alkyl- and methoxy-substituted p-hydroquinones (QH(2)) as a chain-breaking antioxidant during the oxidation of styrene and methyl linoleate (ML) in bulk as well as ML oxidation in micellar solution of sodium dodecyl sulfate (SDS) at 37 degrees C. The antioxidant activities of QH(2) were characterized by two parameters: the rate constant k(1) for reaction of QH(2) with the peroxy radical LO(2)*: QH(2)+LO(2)*-->QH*+LOOH and the stoichiometric factor of inhibition, f, which shows how many kinetic chains may be terminated by one molecule of QH(2). In the case of styrene and ML oxidation in bulk, f values never exceed two; for the majority of QH(2), f was found to be significantly less than two due to the interaction of QH* with molecular oxygen. In the absence of superoxide dismutase (SOD), all the studied QH(2) displayed a very moderate if any antioxidant capability during ML oxidation in SDS micelles. When 20U/ml SOD was added, the majority of QH(2) showed a pronounced ability to inhibit ML oxidation, f parameter being ca. one. The features of QH(2) as an antioxidant in aqueous environment are suggested to associate with the reactivity of semiquinone (Q*(-)). Q*(-) reacts readily with molecular oxygen with formation of superoxide (O(2)*(-)); further reactions of O(2)*(-) result in fast depleting QH(2) and chain propagation. The addition of SOD results in purging a reaction mixture from O(2)*(-) and, as a corollary, in depressing undesirable reactions with the participation of O(2)*(-). With all the oxidation models, QH(2) were found to be very reactive to LO(2)*. The rate constants k(1) decreased progressively when going from the oxidation of styrene to ML oxidation in bulk and further to ML oxidation in SDS micelles.     

An indirect and highly sensitive method for the determination of the flow of reducing equivalents in the respiratory chain

Reactions leading to oxido-reduction of TMPD have shown that, in its oxidized form, this compound has among others an extinction maximum at 610 nm; with the exception of cytochrome a, at this wavelength none of the respiratory chain intermediates has the ability to absorb the incident light. This property together with the one of reacting with cytochrome c, has given us the possibility to use TMPD as a "probe" of the reducing equivalents flow in the respiratory chain. Added to mitochondrial suspension, TMPD undergoes redox cycles in relation to the activity of the respiratory chain, modulated by increasing concentrations of succinate or respiratory inhibitors. NEM-induced reversible oxidation on the respiratory intermediates can also be determined by following the TMPD oxidation. The preliminary data obtained are thus consistent with the hypothesis that in appropriate conditions, the TMPD redox state can be used as a probe of the respiratory chain activity.     

The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain

In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti-Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q2, Q4, Q6 and Q10 and an analogue, 2,3-dimethoxyl-5-n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1-6) or both, of QH2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o.     

Oxidative Phosphorylation in Fractionated Bacterial Systems: Effect of Chloramphenicol

Chloramphenicol was found to have a direct effect on the respiratory chain of Mycobacterium phlei cells grown in the presence of this drug. Analysis of the respiratory chain components revealed that the presence of chloramphenicol during growth resulted in a partial inhibition in the synthesis of the cytochromes. However, a stimulation in oxidative phosphorylation was observed with the cell-free extract of cells grown in the presence of chloramphenicol. The oxidation of succinate was found to be stimulated 20 to 130%, depending on the particular extract, whereas the oxidation of reduced nicotinamide adenine dinucleotide (NADH) was found to be similar to that of extracts obtained from cells grown in the absence of the drug. Of particular interest was the finding that the cell-free extract of cells grown in the presence of the drug exhibited an increased level of phosphorylation (17 to 100%) when NADH was used as the electron donor. Chloramphenicol appears to affect a component of the respiratory chain between the flavoprotein and cytochrome c. Fractionation of the electron transport particles revealed an increased level of cytochrome b in the fractions which exhibited a stimulation in oxidative phosphorylation.     

Assembly of the mitochondrial membrane system. Cytoplasmic mutants of Saccharomyces cerevisiae with lesions in enzymes of the respiratory chain and in the mitochondrial ATPase.

Mutants of Saccharomyces cervisiae with defects in enzymes of the electron transfer chain and in the rutamycin-sensitive ATPase have been isolated. Some of the mutants are specifically affected in either cytochrome oxidase, coenzyme QH2-cytochrome c reductase or ATPase. Other strains are deficient in both cytochrome oxidase and coenzyme QH2-cytochrome c reductase but still have rutamycin-sensitive ATPase. All the mutants reported in this study fail to be complemented by a rho0 tester derived from a respiratory competent strain. The meiotic spore progeny obtained by mating the mutants to a respiratory competent haploid yeast, when scored for growth on glycerol, show a non-Mendelian segregation of the phenotype. These two genetic tests indicate the mutations to be cytoplasmically inherited.     

Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.     

The effect of captopril on nitric oxide formation and on generation of radical forms of mitochondrial respiratory chain compounds in ischemic rat heart.

The increase of radical forms of mitochondrial respiratory chain compounds (MRCC) is an indicator of an increased risk of the formation of oxygen radicals. Using electron paramagnetic resonance (EPR), we found an increase of signals corresponding to ubisemichinone radical (.QH) and ironsulfur proteins radical forms (-FeS) of these respiratory chain compounds during ischemia in the isolated perfused rat heart (.QH increased from 1.51 to 3.08, .FeS1 from 1.14 to 2.65 arbitrary units). During the 5-min reperfusion, the signals returned to normoxic levels. In isolated mitochondria exposed to anoxia and reoxygenation the radical forms of .QH and FeS2 changed in a similar manner as in the intact heart. A combination of in vivo captopril treatment and in vitro L-arginine administration significantly decreased the levels of MRCC radicals in the isolated myocardium (.QH from 2.61 to 1.72 and .FeS, from 1.82 to 0.46 under normoxia; .QH from 4.35 to 2.66 and .FeS1 from 1.93 to 1.35 during ischemia). This decrease in MRCC radical forms was associated with increased NO levels in the perfusate, determined as NO2- / NO3-, as well as tissue NO levels determined using EPR as the dinitrosyl iron complex (DNIC). These results provide new information about the cardioprotective effects of ACE inhibitors and L-arginine.     

The Respiratory Chain of Plant Mitochondria. I. Electron Transport Between Succinate and Oxygen in Skunk Cabbage Mitochondria

The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and the cytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobic with succinate have been measured spectrophotometrically and fluorimetrically in the absence of respiratory inhibitor and in the presence of cyanide or antimycin A. No component identifiable by these means was oxidized rapidly enough in the presence of one or the other inhibitor to qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoprotein and ubiquinone obtained by injecting 12 mum oxygen into the anaerobic mitochondrial suspension were kinetically indistinguishable in the presence of cyanide or antimycin A, implying that these 2 components are part of a respiratory pathway between succinate and oxygen which does not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. The cytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and 0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence of cyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex shows an induction period of 1 sec and a half-time of 3.5 sec. A split respiratory chain with 2 terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinone is proposed for these mitochondria.     

The oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.     

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.     

Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.