Electron Transport Particles Research Articles

High performance perovskite solar cells with functional highly porous TiO2 thin films constructed in ambient air

In the present work we report the synthesis of highly meso- and macro-porous thin TiO2 films as efficient scaffolds for improved performance of heterojunction solid state perovskite solar cells made in Fambient air. TiO2 films were prepared using sol–gel process and Pluronic P-123 block copolymer as organic template while they were formed on conductive glass substrates by spin-coating method. The films were employed to the construction of very efficient perovskite solar cells made at ambient conditions where CH3NH3PbI3−xClx mixed halide perovskite was used as light harvester and P3HT polymer as hole conductor. The very rough and highly porous TiO2 films proved to be an excellent host material for perovskite growth. The structural properties of the TiO2 electron transport layer, thickness, particle size and porosity, strongly affected the overall conversion efficiency. The optimal structure and materials composition exhibited a notably high current density Jsc of 23.8mA/cm2, Voc of 0.995V and fill factor of 0.58. These solar cells prepared under ambient conditions yielded an average power conversion efficiency of 13.7% among the best ever recorded with P3HT polymer as hole conducting material.

Inhibition of brain mitochondrial respiration by dopamine: involvement of H(2)O(2) and hydroxyl radicals but not glutathione-protein-mixed disulfides.

Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mm DA for 15 min at 30 degrees C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H(2) O(2) ), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H(2) O(2) (100 microm) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mm) had no effect on O(2) consumption. Exposure of mitochondria to 1 mm GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4- and 1.5-fold increases in the presence of 100 microm H(2) O(2) or 0.5 mm DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane-bound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H(2) O(2) showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC(50) approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H(2) O(2). Co-administration of desferrioxamine with H(2) O(2) had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freeze-thawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H(2) O(2) and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.

Involvement of mitochondria and other free radical sources in normal and abnormal fetal development.

Shepard and Mackler have documented quantitative increases in mitochondrial cristae between gestational days 10 and 14 in rats accompanied by decreased glucose utilization and increased NADH oxidase activity. Findings show a shift from glycolytic to oxidative metabolism starting at around the time of implantation. Exposure to many substances that cause transient uteroplacental hypoperfusion, including cocaine, phenytoin, calcium channel blockers, and nitric oxide synthase (NOS) inhibitors, induce limb and central nervous system (CNS) malformations while sparing the heart. We have reported that isolated electron transport particles prepared from sensitive tissues show reduced NADH oxidase activities compared with insensitive heart. They also have significantly greater superoxide formation in association with significantly reduced superoxide dismutase activities. NOS inhibitors induce severe limb reductions in late gestation. Exposure is associated with hemorrhage and nitrotyrosine (NT) formation shortly after treatment. Hemorrhage, malformations, and NT formation can be significantly reduced by coadministration of PBN, allopurinol, or aminoguanidine. On the basis of these findings, we have proposed a role for the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the genesis of limb reduction defects. It is important to note that limb reduction defects occur in humans ( approximately 0.22/1000) and have been associated with the agents listed above.

Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[ 3H]pyridaben ([ 3H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP +) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP +, stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP + and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.

Combinatorial synthesis of novel and potent inhibitors of NADH:ubiquinone oxidoreductase

Background: NADH:ubiquinone oxidoreductase (complex I) is the first of three large enzyme complexes located in the cell’s inner mitochondrial membrane which form the electron transport chain that carries electrons from NADH to molecular oxygen during oxidative phosphorylation. There is significant interest in developing small molecule inhibitors of this enzyme for use as biological probes, insecticides and potential chemopreventive/chemotherapeutic agents. Herein we describe the application of novel natural product-like libraries to the discovery of a family of potent benzopyran-based inhibitors. Results: Initially a combinatorial library of benzopyrans, modeled after natural products, was synthesized using a solid phase cycloloading strategy. Screening of this diversity oriented library for inhibitory potency against NADH:ubiquinone oxidoreductase activity in vitro using bovine heart electron transport particles provided several lead compounds which were further refined through a series of focused libraries. Conclusions: Using this combinatorial library approach, a family of potent 2,2-dimethylbenzopyran-based inhibitors was developed with IC 50 values in the range of 18–55 nM. Cell-based assays revealed that these inhibitors were rather non-cytotoxic in the MCF-7 cell line; however, they were quite cytostatic in a panel of cancer cell lines suggesting their potential as chemotherapeutic/chemopreventive candidates.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.

New bioactive flavonoids and stilbenes in cubé resin insecticide.

Fractionation of cubé resin from Lonchocarpus utilus and L. urucu roots led to the isolation and identification of 11 minor flavonoids and stilbenes containing the gem-dimethylpyran moiety or a dihydrodiol derivative thereof. The eight new compounds were as follows: the isoflavonoid cis-4'',5''-dihydro-4'',5' '-dihydroxylonchocarpusone (2); four (2S)-6-(gamma, gamma-dimethylallyl)-6'',6''-dimethylpyran[2'',3'':7, 8]flavanones with substituents of 5-hydroxy-3',4'-dimethoxy (3), 5, 3'-dihydroxy-4'-methoxy (4), 5,4'-dihydroxy-3'-methoxy (5), and 3', 4'-dimethoxy (6); and three 6'',6''-dimethylpyran[2'',3'':3', 4']stilbenes with 4-hydroxy-5'-methoxy (9), 3,5'-dimethoxy-4-hydroxy (10) and 3,4,5-trimethoxy (11) substitution patterns. Structure-activity relationships for inhibition of NADH:ubiquinone oxidoreductase activity (bovine heart electron transport particles) and phorbol ester-induced ornithine decarboxylase activity (cultured MCF-7 cells) generally parallel those for cytotoxicity (MCF-7 and Hepa 1clc7 cells).

Anticancer action of cubé insecticide: correlation for rotenoid constituents between inhibition of NADH:ubiquinone oxidoreductase and induced ornithine decarboxylase activities.

Rotenone and rotenoid-containing botanicals, important insecticides and fish poisons, are reported to have anticancer activity in rats and mice. The toxic action of rotenone is attributed to inhibition of NADH:ubiquinone oxidoreductase activity and the purported cancer chemopreventive effect of deguelin analogs has been associated with inhibition of phorbol ester-induced ornithine decarboxylase (ODC) activity. This study defines a possible relationship between these two types of activity important in evaluating the toxicology of rotenoid pesticides and the suitability of the anticancer model. Fractionation of cubé resin (the commercial rotenoid pesticide) establishes that the activity in both assays is due primarily to rotenone (IC50 = 0.8-4 nM), secondarily to deguelin, and in small part to rotenolone and tephrosin. In addition, the potency of 29 rotenoids from cubé insecticide for inhibiting NADH:ubiquinone oxidoreductase in vitro assayed with bovine heart electron transport particles satisfactorily predicts their potency in vivo in the induced ODC assay using noncytotoxic rotenoid concentrations with cultured MCF-7 human breast cancer cells (r = 0.86). Clearly the molecular features of rotenoids essential for inhibiting NADH:ubiquinone oxidoreductase are similar to those for blocking ODC induction. This apparent correlation extends to 11 flavonoids and stilbenoids from cubé resin (r = 0.98) and genistein and resveratrol except for lower potency and less selectivity than the rotenoids relative to cytotoxicity. These findings on cubé insecticide constituents and our earlier study comparing rotenone and pyridaben miticide indicate that inhibition of NADH:ubiquinone oxidoreductase activity lowers the level of induced ODC activity leading to the antiproliferative effect and anticancer action.

Photoaffinity radioligand for NADH:ubiquinone oxidoreductase: [S-C3H2](trifluoromethyl)diazirinyl-pyridaben

Pyridaben is a new and very potent insecticide and miticide that acts by inhibiting the activity of NADH:ubiquinone oxidoreductase (the most complex of all the respiratory enzymes). The binding site, presumed to be the same as that of rotenone and fenazaquin, resides at an unknown location within the 43-polypeptide-subunit Complex I. To define the structure of the pyridaben-inhibition site(s), we prepared [S-C3H2](trifluoromethyl)diazirinyl-pyridaben as a photoaffinity probe. Tritium was incorporated by reducing 4-(trifluoromethyl)diazirinylphenylacetyl fluoride to the benzyl alcohol with freshly prepared LiB3H4 at 97% tritium enrichment. The tritium-labeled alcohol was converted to the benzyl bromide derivative and coupled to 2-tert-butyl-4-chloro-5-mercapto-3(2H)-pyridazinone to obtain the photoaffinity probe (56 Ci/mmol) with an IC50 of 3.0 nM for NADH:ubiquinone oxidoreductase activity of bovine heart electron transport particles. [S-C3H2](Trifluoromethyl)diazirinyl-pyridaben is an improved photoaffinity radioligand combining outstanding potency for inhibiting NADH:ubiquinone oxidoreductase activity, high specific activity close to the theoretical value, and a preferred photolabile substituent (known to combine high reactivity and generation of a carbene species at wavelengths not damaging to proteins). © 1998 John Wiley & Sons, Ltd.

Fenazaquin Acaricide Specific Binding Sites in NADH: Ubiquinone Oxidoreductase and Apparently the ATP Synthase Stalk

Fenazaquin [4-(4-tert-butylphenethoxy)quinazoline] is one of several acaricides and insecticides, including rotenone and pyridaben, that are reported to act by inhibiting NADH:ubiquinone oxidoreductase (Complex I) (EC 1.6.99.3) in the range 1–10 nM. [3H]Fenazaquin is used here at 0.8 nMas a radioligand with electron transport particles (ETP) of bovine heart mitochondria to develop a new and rapid filtration assay with 42% specific binding (displaced by 2 μMrotenone). The potency of rotenoids, quinazolines, pyridaben, acetogenins, and several other disrupters of electron transport as inhibitors of NADH:ubiquinone oxidoreductase activity generally correlates with their effectiveness in displacing [3H]fenazaquin from its binding site in ETP (r2= 0.96,n= 18). This correlation extends to the fenazaquin analog with (trifluoromethyl)-diazirinyl replacing thetert-butyl substituent assayed at 6 nM,indicating that it also acts at a high-affinity site in Complex I. This diazirinyl analog of fenazaquin with tritium at theO-methylene position (4.7 Ci/mmol), on incubation at 435 nMwith ETP then irradiation at 350 nm, results in photoactivatable, irreversible specific binding which is fully protectable with rotenone. The site of labeling was examined by sequential isolation of the only band of specifically labeled protein(s) (which appeared at 22–24 kDa on SDS–PAGE),in situtryptic digestion of the protein(s) in the band, fractionation of the generated peptides by microbore HPLC, and amino acid sequence analysis. The single labeled HPLC peak of peptides in the tryptic digest was derived from two proteins, both components of ATP synthase, identified as subunit d and the oligomycin sensitivity conferral protein. An unusually low recovery of tryptophan at residue 12 of subunit d of specifically labeled ETP versus control ETP leads to the tentative proposal that labeling occurs at that site. Fenazaquin (and presumably rotenone as well) therefore has at least two specific binding sites in ETP, the anticipated high–affinity site in NADH:ubiquinone oxidoreductase and apparently a low-affinity site of unknown function, photoaffinity labeled in the stalk region of ATP synthase.

Studies of mitochondria in oxidative embryotoxicity.

While the limb bud and brain of the rat develop abnormally in response to transient uteroplacental hypoperfusion during late gestation, the heart appears to be protected. These malformations have been associated with the generation of reactive oxygen species (ROS). Studies were designed to examine superoxide generation by mitochondrial electron transport particles (ETP) from adult and conceptal tissues and to investigate characteristics that could be responsible for heightened concentrations of ROS in sensitive tissues. Parameters investigated included NADH oxidase and cytochrome c oxidase activities, cytochrome content, and superoxide dismutase activity. NADH oxidase activities were significantly lower in sensitive tissues that also developed the highest concentrations of superoxide. Because ETP from adult CNS also had low NADH oxidase activity but did not show increased concentrations of superoxide, inhibition of electron transport did not adequately account for increased ROS concentrations. The reduced NADH oxidase activity of sensitive tissues could not be caused by inhibition at the cytochrome c oxidase region since this latter activity equaled or exceeded the former in all instances. No significant differences were found in the cytochrome contents of different tissues. There was significantly less superoxide dismutase activity in homogenates prepared from either of the two sensitive conceptual tissues compared with those from insensitive conceptual or adult tissues. These studies confirm the presence of heightened concentrations of superoxide anion radical in ETP from teratogenically sensitive tissues and suggest that these concentrations may result primarily from decreased activity of superoxide dismutase(s) in those tissues. Superoxide anion radical could therefore be available to participate in the generation of the more toxic oxidant species such as the hydroxyl radical.

Inhibition of complex I by hydrophobic analogues of N-methyl-4-phenylpyridinium (MPP+) and the use of an ion-selective electrode to measure their accumulation by mitochondria and electron-transport particles

N-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, kills dopaminergic neurons after its accumulation in mitochondria where it inhibits Complex I of the respiratory chain. MPP+ inhibits respiration by binding to both a hydrophobic and a hydrophilic site on Complex I and this inhibition is increased by the lipophilic tetraphenylboron anion (TPB-) which facilitates movement of MPP+ through membranes and its penetration to the hydrophobic binding site on Complex I. To investigate the inhibition of respiration by MPP(+)-like compounds, we have measured simultaneously NADH-linked mitochondrial respiration and the uptake and accumulation of the N-benzyl-4-styrylpyridinium and N-ethyl-4-styrylpyridinium cations in mitochondria using ion-selective electrodes. The data provide direct evidence that TPB- increases the inhibition not by increasing matrix concentration but by facilitating access to the inhibitory sites on Complex I. We have also compared the rates of uptake of MPP+ analogues of varied lipophilicity by the inner membrane and the development of inhibition of NADH oxidation, using an inverted mitochondrial inner membrane preparation and appropriate ion-selective electrodes. These experiments demonstrated that the amount of MPP+ analogue bound to the inner membrane greatly exceeded the quantity required for complete inhibition of NADH oxidation. Moreover, binding to the membrane occurred much more rapidly than the development of inhibition with all MPP+ analogues tested. This suggests that the attainment of a correct orientation of these compounds within the membrane and the binding site may be a rate-limiting step in the development of inhibition.

Effect of methyl methacrylate on mitochondrial function and structure

1. 1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria. 2. 2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria. 3. 3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3. 4. 4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents. 5. 5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity. 6. 6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I. 7. 7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

Studies on the Characterization of the Inhibitory Mechanism of 4′‐Alkylated 1‐Methyl‐4‐Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles

1-Methyl-4-phenylpyridinium (MPP+), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4'-alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 microM and from 1.6 to 3,300 microM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4'-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 microM in mitochondria and from 1.7 to 142 microM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an approximately 600-fold, versus only a 36-fold, increase in the IC50 of MPP+ versus 4'-pentyl-MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4'-decyl-MPP+ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivation of NADH dehydrogenase (complex I) inhibited by 1-methyl-4-(4'-alkylphenyl)pyridinium analogues: a clue to the nature of the inhibition site.

Expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, following oxidation to 1-methyl-4-phenylpyridinium ion (MPP+), is believed to involve inhibition of mitochondrial electron transport from NADH dehydrogenase (complex I) to ubiquinone. MPP+ and its analogues have been shown to block electron transport at or near the same site as two powerful inhibitors of mitochondrial respiration, rotenone and piericidin A. All three types of inhibitors combine at two sites on NADH dehydrogenase, a hydrophilic and hydrophobic one, and occupancy of both sites is required for complete inhibition. Tetraphenylboron anion (TPB-) in catalytic amounts is known to increase the effectiveness of positively charged MPP+ analogues in blocking mitochondrial respiration. A part of this effect involves facilitation of the entry of MPP+ congeners into the hydrophobic site by ion pairing, as has been demonstrated in studies with submitochondrial particles (electron transport particles). This communication documents the fact that TPB-, when present in molar excess over the MPP+ analogues, reverses the inhibition. This seems to involve again strong ion pairing, removal of the inhibitory analogue from one to the two binding sites, and concentration of the inhibitor in the membrane, so that only the hydrophobic binding site remains occupied, resulting in lowering of the inhibition to 30-40%.

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1-ATPase beta subunit.

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1-induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron-transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity. IF1 antibodies have been used to show that IF1 can bind not only to the beta subunit of F1-ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339-1344] but also to a protein present in the inner-mitochondrial membrane. The cross-linking of IF1 to this membrane protein gave a product of M(r) 15000-16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross-linked product was obtained by using a cleavable cross-linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent M(r) 5000-6000. The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross-linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X-100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory-binding site at the beta subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate M(r) 5000-6000.

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.

Inhibition of mitochondrial respiration by neutral, monocationic, and dicationic bis-pyridines related to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium cation (MPP +)

The cytotoxic effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP +) is believed to be associated with a compromise in cellular energy arising as a consequence of its persistent inhibition of mitochondrial respiration. MPP + is a rather weak inhibitor of electron transport, but it undergoes passive accumulation inside actively respiring mitochondria in response to the transmembrane electrochemical potential gradient. In order to test the prediction that dicationic analogs of MPP + might be concentrated to a much greater extent and thereby exert especially potent inhibition of respiration on the intact organelle, we synthesized four differently spaced bis-pyridines, each in neutral, monocationic, and dicationic forms, and evaluated their inhibitory activities in intact mitochondria and in electron transport particles (ETP). Compared to the neutrals, the monocations and especially the dications exhibit reduced inhibition in ETP, but the inhibition in mitochondria is enhanced selectively for the cationic inhibitors presumably on account of their accumulation in the mitochondrial matrix. This enhancement is limited by the relatively poor ability of the cationic bis-pyridines to enter mitochondria, as judged from experiments which evaluated the rate of onset of inhibition (without preincubation), in the absence and presence of tetraphenylborate (TPB −). The dications appear to be transported less well than the monocations, and only the most lipophilic dication exhibited a substantially greater accumulation-dependent enhancement of inhibitory activity on mitochondria than did the corresponding monocation. The compounds studied here constitute a novel class of respiratory chain probes which may be useful for a variety of studies on mitochondria.

Inhibition of mitochondrial respiration by analogues of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium: Structural requirements for accumulation-dependent enhanced inhibitory potency on intact mitochondria

Analogues of 1-methyl-4-phenylpyridinium (MPP +), the neurotoxic metabolite of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, were evaluated for inhibition of respiration in intact mitochondria (M w) and in electron transport particles (ETP). MPP + exhibits relatively weak inhibitory activity in ETP, but potent inhibition in M w occurs on account of its energy-dependent accumulation inside mitochondria. The permeant anion tetraphenylborate potentiates the inhibition in both M w and ETP. Replacement of the 4-phenyl ring of MPP + by a variety of aromatic and nonaromatic rings, and of the N-methylpyridinium group by other cationic aromatic heterocycles, preserves the inhibitory patterns seen for MPP +. The general observation of enhanced inhibitory potency in M w for all these permanently charged cations is consistent with our contention that energy-dependent accumulation inside mitochondria represents a passive Nernstian concentration in response to the transmembrane electrochemical gradient. Nonetheless, the magnitude of the inhibitory potentiation seen in M w relative to ETP varies widely with structure. In particular, less lipophilic analogues, especially those bearing a localized, rather than resonance-stabilized, permanent positive charge, exhibit similar inhibitory activity to MPP + in ETP, but the inhibition in M w is not comparably enhanced. For these same analogues, the inhibitory activity in ETP is only weakly potentiated by tetraphenylborate. Since succinate was found to completely reverse the respiratory inhibition in M w induced by all types of MPP + analogues investigated, a common site 1 inhibition appears to be involved; thus the different inhibitory patterns observed must be due to structural factors governing membrane transport and distribution properties.

Assessment of chemical toxicity using mammalian mitochondrial electron transport particles

New spectrophotometric bioassay procedures have been developed for evaluating chemical toxicity, using electron transport particles isolated from bovine heart mitochondria, based on the ability of many toxic chemicals to interfere with the integrated function of electron transport enzymes. The sensitivity of the mitochondrial assay is compared to published sensitivities of other in vivo and in vitro toxicity testing methods. Regression analysis of logarithmically transformed toxicity values for 42 chemicals, including 8 pesticides, 5 drugs, 6 metals, 8 alcohols, 5 respiratory inhibitors, 4 phenols, and 2 phthalates, indicates excellent correlation between the sensitivity of the new assays and the sensitivity of mammalian cytotoxicity studies (r2 = 0.86). Data from aquatic exposure toxicity tests conducted in fish are also highly correlated with the mitochondrial assay results (r2 = 0.79). However, correlation of data from these methods with median lethal dose studies conducted in rats is not as good because of the inability of in vitro and aquatic exposure analyses to account for the gastrointestinal absorption, hepatic metabolism, and excretion processes which modify toxic responses following oral administration.