Inhibition of the mitochondrial bc1 complex by dibromothymoquinone

We have studied the effects of dibromothymoquinone (DBMIB) in various redox activities of the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations higher than 50 mol/mol of cytochrome c1 the inhibitor produces a bypass of electron transfer on the substrate side of the bc1 complex, because of its autooxidation capability. This induces an artifactual overestimation of the real inhibition titer of the redox activity of this enzyme, which has been found to be 3-6 mol/mol of cytochrome c1 by following the ubiquinol-cytochrome c reductase activity. This action is reversed by addition of excess of sulphydryl compounds like cysteine.     

The proton pump of the mitochondrial bc1 complex

The molecular mechanism of the proton pump activity by the respiratory chain bc1 complex is still unknown. This group has proposed since long time that protonation/deprotonation events in the apoproteins of the complex are cooperatively linked to the oxido-reduction reactions at the quinone catalytic centre. Protolytic residues in the apoproteins can thus provide proton transfer pathways between the bulk aqueons phases and the redox centre. A series of experiments has been carried out aimed at demonstrating a role of particular complex subunits in the pump process. In this paper recent results are reviewed which have evidenced a definite role of polypeptide carboxyl residues in the proton pump mechanism. In particular, experiments carried out with both the bovine and P. denitrificans purified enzymes have indicated a specific involvement of aspartic residue(s) in the Rieske Fe/S protein in the proton pump function.     

Independent lateral diffusion of cytochrome bc1 complex and cytochrome oxidase in the mitochondrial inner membrane

Distinct fluorophores have been conjugated to antibodies for cytochrome bc1 complex and cytochrome oxidase, two integral electron transferring proteins in the mitochondrial inner membrane. Addition of these fluorescent antibodies to preparations of mitochondrial inner membranes followed by appropriate secondary antibodies causes distinct and independent aggregation of the two cytochrome proteins. These results reveal that both cytochrome bc1 complex and cytochrome oxidase diffuse laterally in the membrane plane independent of one another consistent with the random collision model for electron transport in the mitochondrial inner membrane.     

Evidence for electron equilibrium between the two hemes bL in the dimeric cytochrome bc1 complex

Structural analysis of the dimeric mitochondrial cytochrome bc1 complex suggests that electron transfer between inter-monomer hemes bL-bL may occur during bc1 catalysis. Such electron transfer may be facilitated by the aromatic pairs present between the two bL hemes in the two symmetry-related monomers. To test this hypothesis, R. sphaeroides mutants expressing His6-tagged bc1 complexes with mutations at three aromatic residues (Phe-195, Tyr-199, and Phe-203), located between two bL hemes, were generated and characterized. All three mutants grew photosynthetically at a rate comparable to that of wild-type cells. The bc1 complexes prepared from mutants F195A, Y199A, and F203A have, respectively, 78%, 100%, and 100% of ubiquinol-cytochrome c reductase activity found in the wild-type complex. Replacing the Phe-195 of cytochrome b with Tyr, His, or Trp results in mutant complexes (F195Y, F195H, or F195W) having the same ubiquinol-cytochrome c reductase activity as the wild-type. These results indicate that the aromatic group at position195 of cytochrome b is involved in electron transfer reactions of the bc1 complex. The rate of superoxide anion (O2*) generation, measured by the chemiluminescence of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one hydrochloride-O2* adduct during oxidation of ubiquinol, is 3 times higher in the F195A complex than in the wild-type or mutant complexes Y199A or F203A. This supports the idea that the interruption of electron transfer between the two bL hemes enhances electron leakage to oxygen and thus decreases the ubiquinol-cytochrome c reductase activity.     

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (ΔpH) and membrane potential (Δψ) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of ΔpH in PBM of young developing nodules in the presence of the K⁺/H⁺ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased ΔpH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both ΔpH in the presence of nigericin in the medium and Δψ generated on PBM by H⁺-ATPase. A decrease in the effects of metabolites on ΔpH and the absent activity of the PBM H⁺ pump were observed in the aging nodules. The obtained data on the changes in ΔpH and Δψ caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.     

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (delta pH) and membrane potential (delta psi) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of delta psi in PBM of young developing nodules in the presence of the K+/H+ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased delta pH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both delta pH in the presence of nigericin in the medium and delta psi generated on PBM by H+-ATPase. A decrease in the effects of metabolites on delta pH and the absent activity of the PBM H+ pump were observed in the aging nodules. The obtained data on the changes in deltapH and dlta psi caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.     

The ubiquinol/bc1 redox couple regulates mitochondrial oxygen radical formation

The generation of oxygen radicals in biological systems and their sites of intracellular release were subject of numerous studies in the last decades. Based on these studies mitochondria were considered as the major source of intracellular oxygen radicals. Although this finding is more or less accepted the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps of the respiratory chain is the electron bifurcation at the bc1 complex. From recent studies with genetically mutated mitochondria it became clear that electron bifurcation from ubiquinol to the bc1 complex requires an underanged mobility of the head domain of the Rieske iron sulfur protein. On the other hand it is long known that inhibition of electron bifurcation by antimycin A causes the leakage of single electrons to dioxygen, which results in the release of O2*- radicals. These findings made us to prove whether the impediment of the interaction of ubiquinol with the bc1 complex is the regulator of single electron diversion to oxygen. Impediment of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the bc1 complex is inserted. Irrespectively, whether the fluidity of membrane lipids was elevated or decreased electron flow rates to the Rieske iron sulfur protein and to low potential cytochrome b were drastically reduced. Concomitantly O2*- radicals were released from these mitochondria, suggesting an effect on the mobility of the head domain of the Rieske iron sulfur protein. These results including the well known effect of antimycin A revealed the involvement of the ubiquinol bc1 redox couple in mitochondrial O2*- formation. The regulator which controls leakage of electrons to oxygen appears to be the electron branching activity of the bc1 complex.     

Simultaneous determination of membrane potential and pH gradient by photodiode array spectroscopy

Membrane potential (delta psi) and pH difference (delta pH) were simultaneously determined in liposomes using a photodiode array spectrophotometer. By the use of a cyanine dye (DiS-C3(5)) and 9-aminoacridine for delta psi and delta pH probes, respectively, both changes of delta psi and delta pH could be successfully determined by photodiode array spectrometry. Each dye did not disturb the fluorescence spectrum of the other probe when its concentration was lower than 5 microM. The K+-diffusion potential-driven, FCCP(protonophore)-mediated H+-influx process in the K+-loaded liposomes was analyzed by this method. Results indicate that the kinetic behavior of H+ influx changes at a FCCP concentration of approx. 30 nM. The rate of delta pH formation increased quantitatively with increasing concentrations of FCCP up to 30 nM, but was markedly enhanced at higher concentrations, although the maximal delta pH attained was about 3 pH units in any case when a K+-diffusion potential of -180 mV was applied.     

Structural basis of multifunctional bovine mitochondrial cytochrome bc1 complex

The mitochondrial cytochrome bc1 complex is a multifunctional membrane protein complex. It catalyzes electron transfer, proton translocation, peptide processing, and superoxide generation. Crystal structure data at 2.9 A resolution not only establishes the location of the redox centers and inhibitor binding sites, but also suggests a movement of the head domain of the iron-sulfur protein (ISP) during bc1 catalysis and inhibition of peptide-processing activity during complex maturation. The functional importance of the movement of extramembrane (head) domain of ISP in the bc1 complex is confirmed by analysis of the Rhodobacter sphaeroides bc1 complex mutants with increased rigidity in the ISP neck and by the determination of rate constants for acid/base-induced intramolecular electron transfer between [2Fe-2S] and heme c1 in native and inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovine heart mitochondrial bc1 complex by nonionic detergent at concentrations that inactivate electron transfer activity. This peptide-processing activity is shown to be associated with subunits I and II by cloning, overexpression and in vitro reconstitution. The superoxide-generation site of the cytochrome bc1 complex is located at reduced bL and Q*-. The reaction is membrane potential-, and cytochrome c-dependent.     

Intermonomer electron transfer in the bc1 complex dimer is controlled by the energized state and by impaired electron transfer between low and high potential hemes

The cytochrome bc(1) complex (commonly called Complex III) is the central enzyme of respiratory and photosynthetic electron transfer chains. X-ray structures have revealed the bc(1) complex to be a dimer, and show that the distance between low potential (b(L)) and high potential (b(H)) hemes, is similar to the distance between low potential hemes in different monomers. This suggests that electron transfer between monomers should occur at the level of the b(L) hemes. Here, we show that although the rate constant for b(L)-->b(L) electron transfer is substantial, it is slow compared to the forward rate from b(L) to b(H), and the intermonomer transfer only occurs after equilibration within the first monomer. The effective rate of intermonomer transfer is about 2-orders of magnitude slower than the direct intermonomer electron transfer.     

Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.     

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (ΔΨ) and pH difference (ΔpH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K⁺ diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K⁺ permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with ΔΨ formed by valinomycin-K⁺ did not significantly alter 9-AA fluorescence, but unexpectedly dissipated ΔΨ. The ΔΨ preformed by valinomycin-K⁺ decreased gradually following the addition of increasing concentrations of anacardic acid. The ΔΨ dissipation rate was dependent on the pre-existing magnitude of ΔΨ, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of ΔpH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by ΔΨ, and a ‘proton carrier’ that dissipates the transmembrane proton gradient formed.     

The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex

Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.     

Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Structure and function of the mitochondrial bc1 complex. Properties of the complex in temperature-sensitive cor1 mutants

The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit. All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection. Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C. The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit. The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants. Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro. The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol. The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min. It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway. The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex.     

Resolution of the circular dichroism spectra of the mitochondrial cytochrome bc1 complex

The circular dichroic spectrum of the mitochondrial cytochrome bc1 complex isolated from bovine heart has been resolved into the contributions from the prosthetic groups: cytochrome c1, the 'Rieske' iron-sulphur centre and the two b cytochromes. It is apparent that firstly, the circular dichroism (CD) properties of cytochrome c1 within the bc1 complex differ from those found in the isolated cytochrome c1 and secondly, both the oxidized and reduced b cytochromes exhibit an intense spectrum of bilobic shape, with the wavelengths of the cross-over points closely corresponding to those of the maxima in the optical absorbance spectra. These latter CD features are discussed in relation to the proposed structure of cytochrome b.