Reaction of electron-transfer flavoprotein ubiquinone oxidoreductase with the mitochondrial respiratory chain

Submitochondrial particles catalyze the reduction of electron-transfer flavoprotein (ETF) by NADH and succinate under anaerobic conditions in reactions that are totally inhibited by rotenone and thenoyl trifluoroacetone, respectively. The particles also catalyze the ATP-dependent reduction of NAD+ by enzymatically reduced ETF. The latter reaction is inhibited by rotenone and carbonyl cyanide chlorophenylhydrazone and all three reactions are inhibited by antibody to electrontransfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). These observations indicated that ETF-QO reacts with the pool of ubiquinone that is reduced by NADH and succinic dehydrogenases. Consistent with this hypothesis, NADH- and succinic-ETF reductase activities are inhibited 99% in ubiquinone-depleted particles, and reincorporation of exogenous ubiquinone restores at least 90% of these activities. Reduction of the bc1 complex by ETF and acyl CoA oxidase activity are also inhibited by antibody to ETF-QO. Myxothiazole and antimycin which inhibit the quinonol oxidation and quinone reduction sites, respectively, in the bc1 complex also inhibit electron transport from ETF-QO through the complex according to current models of the Q-cycle (Rich, P.R. (1986) J. Bioenerg. Biomembranes 18, 145-156). The results show that ETF-QO is an obligatory component of the electron transport pathway between ETF and the ubiquinone pool and suggest a mechanism for the steady-state turnover of ETF-QO.     

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.     

Role of coenzyme Q in the mitochondrial respiratory chain. Reconstitution of activity in coenzyme Q deficient mutants of yeast.

The reduction of cytochrome c by the reduced form of the 6-decyl analogue of coenzyme Q follows first-order kinetics with respect to cytochrome c and increases in a linear manner with added mitochondrial protein. The activity is completely sensitive to antimycin A in whole cell extracts of yeast as well as in isolated mitochondria and fractionates with markers for the mitochondrial electron-transport chain. The presence of both cytochrome b and c1 in an approximately 2:1 ratio appears essential for enzymatic activity. Reduced coenzyme Q-cytochrome c reductase obeys Michaelis-Menten kinetics when assayed in mitochondria obtained from a yeast strain lacking coenzyme Q. Both reduced nitotinamide adenine dinucleotide and succinate:cytochrome c reductase activities were not detectable in six coenzyme Q deficient strains tested, but were restored after addition of the oxidized form of the coenzyme Q analogue. No marked difference in the concentration of the analogue required to restore the two activities was observed.     

Cooperativity and flexibility of the protonmotive activity of mitochondrial respiratory chain

Functional and structural data are reviewed which provide evidence that proton pumping in cytochrome c oxidase is associated with extended allosteric cooperativity involving the four redox centers in the enzyme . Data are also summarized showing that the H+/e- stoichiometry for proton pumping in the cytochrome span of the mitochondrial respiratory chain is flexible. The DeltapH component of the bulk-phase membrane electrochemical proton gradient exerts a decoupling effect on the proton pump of both the bc1 complex and cytochrome c oxidase. A slip in the pumping efficiency of the latter is also caused by high electron pressure. The mechanistic and physiological implications of proton-pump slips are examined. The easiness with which bulk phase DeltapH causes, at least above a threshold level, decoupling of proton pumping indicates that for active oxidative phosphorylation efficient protonic coupling between redox complexes and ATP synthase takes place at the membrane surface, likely in cristae, without significant formation of delocalized DeltamuH+. A role of slips in modulating oxygen free radical production by the respiratory chain and the mitochondrial pathway of apoptosis is discussed.     

Decylubiquinol impedes mitochondrial respiratory chain complex I activity

In this study, indirect immunofluorescence labeling was used to examine the cellular dynamic distribution of Thr11 phosphorylated H3 at mitosis in MCF-7 cells. The Thr11 phosphorylation was observed beginning at prophase at centromeres. Upon progression of mitosis, fluorescence signal was enhanced in the central region of the metaphase plate and maintained till anaphase at centromeres. During telophase, the fluorescent signal of Thr11 phosphorylated H3 disappears from centromeres, but the signal appears again at the midbody during cytokinesis, which suggests that the modified histones may take part in the formation of the midbody and play a crucial role in cytokinesis. Chromatin immunoprecipitation (ChIP) was used to confirm that Thr11 phosphorylated H3 is specifically associated with centromere DNA at prophase to metaphase, which is coincident with the results observed by immunofluorescence. In conclusion, there was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and stages of chromatin condensation. The timing of Thr11 phosphorylation and dephosphorylation in mitosis were similar to that reported for Ser10 phosphorylation of H3. The Thr11 phosphorylated H3 localized at centromeres during mitosis, which was different from the Ser10 phosphorylated H3 localized at telomere regions and Thr3 phosphorylated H3 localized along the chromosome arms. The results suggest that the Thr11 phosphorylation of histone H3 may play a specific role which was different from Ser10 and Thr3 phosphorylation in mitosis.     

Effects of respiratory chain inhibitors on mitochondrial ATPase activity

The effects of six respiratory chain inhibitors (amytal, rotenone, piericidin A, antimycin A, 2-N-heptyl-4-hydroxyquinoline N-oxide, and cyanide) on mitochondrial ATPase activity have been investigated. Each of these compounds inhibited the ATPase activity of intact mitochondria induced by uncoupling agents, ionophores, or alterations in ionic composition; the effects were variable depending upon the type and concentration of uncoupling agents or inhibitors utilized. The ATPase activity of sonicated submitochondrial particles was also diminished by respiratory inhibitors, but the isolated ATPase enzyme was not affected. We conclude from these results that these respiratory inhibitors interfere with the energy coupling mechanism of oxidative phosphorylation. The experimental observations tend to support the “chemical” theory, and appear to be less consistent with the “chemiosmotic” hypothesis of oxidative phosphorylation.     

Effects of alkyl side chain modification of coenzyme Q10 on mitochondrial respiratory chain function and cytoprotection

The effect of the alkyl side chain length of coenzyme Q₁₀ on mitochondrial respiratory chain function has been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were identified that exhibited significantly improved effects on mitochondrial oxygen consumption and mitochondrial membrane potential, and also conferred significant cytoprotection on cultured mammalian cells in which glutathione had been depleted by treatment with diethyl maleate. The analogues also exhibited lesser inhibition of the electron transport chain than idebenone. The results obtained provide guidance for the design of CoQ₁₀ analogues with improved activity compared to that of idebenone (1), the latter of which is undergoing evaluation in the clinic as a therapeutic agent.     

Determination of the cDNA sequence for the human mitochondrial 75-kDa Fe-S protein of NADH-coenzyme Q reductase

A human-hepatoma cDNA lambda gt11 expression library was probed with an antibody to holoenzyme complex I (NADH-CoQ reductase) of the respiratory chain. One of the 30 antibody positive clones was purified to homogeneity, amplified by the polymerase chain reaction (PCR), subcloned and sequenced. It proved to be highly similar to the cDNA sequence for the bovine 75-kDa Fe--S protein. Using the sequence obtained from this library, both sense and antisense oligonucleotides were constructed and used to probe a human kidney cDNA library using PCR amplification with oligonucleotides that flank the polylinker region of the lambda phage. Two further cDNA clones were obtained which overlapped and covered the entire cDNA sequence of 2526 bp. The encoded protein of 727 amino acids has 21 amino acids that differ from the bovine-protein sequence. Northern blot analysis of mRNA from fibroblasts of complex-I deficient patients revealed no abnormalities. We show that this Fe--S protein has significant similarity with (a) the gamma chain of the hydrogen hydrogenase of Alcaligenes eutrophus and (b) the A chain of the formate dehydrogenase of Methanobacterium formicum.     

Supersizing the mitochondrial respiratory chain

The complexes of the mitochondrial respiratory chain assemble into higher-order structures called supercomplexes or respirasomes that are thought to be important in channeling electron flow and controlling ROS production. A number of recent papers identify the first protein factors necessary for supercomplex assembly and stability.     

Dependence of yeast mitochondrial unselective channel activity on the respiratory chain

The dependence of yeast mitochondrial unselective channel activity on the respiratory chain was investigated. Modulation of the respiratory chain with different substrates and inhibitors showed that channel activity was dependent on the electron flow rate through the chain and that external NADH only could provide a sufficient rate to activate the channel. These results support the hypothesis that the yeast mitochondrial unselective channel may be involved in the oxidation of cytosolic NADH without coupling to ATP synthesis.     

Supercomplex organization of the mitochondrial respiratory chain and the role of the Coenzyme Q pool: pathophysiological implications

In this review we examine early and recent evidence for an aggregated organization of the mitochondrial respiratory chain. Blue Native Electrophoresis suggests that in several types of mitochondria Complexes I, III and IV are aggregated as fixed supramolecular units having stoichiometric proportions of each individual complex. Kinetic evidence by flux control analysis agrees with this view, however the presence of Complex IV in bovine mitochondria cannot be demonstrated, presumably due to high levels of free Complex. Since most Coenzyme Q appears to be largely free in the lipid bilayer of the inner membrane, binding of Coenzyme Q molecules to the Complex I-III aggregate is forced by its dissociation equilibrium; furthermore free Coenzyme Q is required for succinate-supported respiration and reverse electron transfer. The advantage of the supercomplex organization is in a more efficient electron transfer by channelling of the redox intermediates and in the requirement of a supramolecular structure for the correct assembly of the individual complexes. Preliminary evidence suggests that dilution of the membrane proteins with extra phospholipids and lipid peroxidation may disrupt the supercomplex organization. This finding has pathophysiological implications, in view of the role of oxidative stress in the pathogenesis of many diseases.     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.     

Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity

Eugenol (1) is an active principle of Rhizoma acori graminei, a medicinal herb used in Asia for the treatment of symptoms reminiscent of Alzheimer's disease (AD). It has been shown to protect neuronal cells from the cytotoxic effect of amyloid beta peptides (Abetas) in cell cultures and exhibit antidepressant-like activity in mice. Results from this study show that eugenol inhibits monoamine oxidase A (MAOA) preferentially with a K(i)=26 microM. It also inhibits MAOB but at much higher concentrations (K(i)=211 microM). In both cases, inhibition is competitive with respect to the monoamine substrate. Survey of compounds structurally related to eugenol has identified a few that inhibit MAOs more potently. Structure activity relationship reveals structural features important for MAOA and MAOB inhibition. Molecular docking experiments were performed to help explain the SAR outcomes. Four of these compounds, two (1, 24) inhibiting MAOA selectively and the other two (19, 21) inhibiting neither MAOA nor MAOB, were tested for antidepressant-like activity using the forced swim test in mice. Results suggest a potential link between the antidepressant activity of eugenol and its MAOA inhibitory activity.