Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000muM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.     

Inactivation of vesicular stomatitis virus by photosensitization following incubation with a pyrene-fatty acid

Vesicular stomatitis virus (VSV) was incubated with pyrene dodecanoic acid (P12), a fluorescent derivative of a medium-chain length fatty acid, and subjected to irradiation with a long wavelength ultra-violet light source at 366 nm (UVA). This inactivated the virus, resulting in a drastic decrease of its infectivity. The virus inactivation was proportional to the concentration of the pyrene-fatty acid, the length of exposure of the virus to P12 and the irradiation dose.     

Multiple sites of inhibition of mitochondrial electron transport by local anesthetics

Local anesthetics and alcohols were found to inhibit mitochondrial electron transport at several points along the chain. The anesthetics employed were the tertiary amines procaine, tetracaine, dibucaine, and chlorpromazine, and the alcohols were n-butanol, n-pentanol, n-hexanol, and benzyl alcohol. Uncoupled sonic submitochondrial particles from beef heart and rat liver were studied. We report the following: (1) All of the anesthetics were found to inhibit each of the segments of the electron transport chain assayed; these included cytochrome c oxidase, durohydroquinone oxidase, succinate oxidase, NADH oxidase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, and NADH-cytochrome c oxidoreductase. (2) NADH oxidase and NADH-cytochrome c oxidoreductase required the lowest concentrations of anesthetic for inhibition, and cytochrome c oxidase required the highest concentrations. (3) We conclude that there are several points along the chain at which inhibition occurs, the most sensitive being in the region of Complex I (NADH dehydrogenase). (4) Beef heart submitochondrial particles are less sensitive to inhibition than are rat liver particles. (5) Low concentrations of several of the anesthetics gave enhancement of electron transport activity, whereas higher concentrations of the same agents caused inhibition. (6) The concentrations of anesthetics (alcohol and tertiary amine) which gave 50% inhibition of NADH oxidase were lower than the reported concentrations required for blockage of frog sciatic nerve.     

Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Molecular localization of a redox-modulated process regulating plant mitochondrial electron transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.     

Effective photocleavage of DNA by pheophorbide a.

Remarkably effective photocleavage of plasmid DNA with pheophorbide a, as a visible light sensitizer, has been found for the first time. DNA is photocleaved even in the absence of oxygen. The novel oxygen-independent scission is ascribed to specific photodynamic function of the ring V in pheophorbide a.     

Oxygen tolerance and coupling of mitochondrial electron transport

Oxygen is critical to aerobic metabolism, but excessive oxygen (hyperoxia) causes cell injury and death. An oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O2, termed "HeLa-80," was derived from wild-type HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. Surprisingly, antioxidant defenses and susceptibility to oxidant-mediated killing do not differ between these two strains of HeLa cells. However, under both 20 and 80% O2, intracellular reactive oxygen species (ROS) production is significantly (approximately 2-fold) less in HeLa-80 cells. In both cell lines the source of ROS is evidently mitochondrial. Although HeLa-80 cells consume oxygen at the same rate as HeLa-20 cells, they consume less glucose and produce less lactic acid. Most importantly, the oxygen-tolerant HeLa-80 cells have significantly higher cytochrome c oxidase activity (approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. Indeed, preferential inhibition of cytochrome c oxidase by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a in cytochrome c oxidase) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells. Thus, it appears that the remarkable oxygen tolerance of these cells derives from tighter coupling of the electron transport chain.     

Damage to mitochondrial electron transport and energy coupling by visible light

The effect of treating mitochondria with visible light above 400 nm on electron transport and coupled reactions was examined. The temporal sequence of changes was: stimulation of respiration coupled to ATP synthesis, a decline in ATP synthesis, inactivation of respiration, increased ATPase activity and, later, loss of the membrane potential. Loss of respiration was principally due to inactivation of dehydrogenases. Of the components of dehydrogenase systems, flavins and quinones were most susceptible to illumination, the iron-sulfur centers were remarkably resistant to being damaged. Succinate dehydrogenase was inactivated before choline and NADH dehydrogenase. Redox reactions of cytochromes and cytochrome c oxidase activity were unaffected.Inactivation was O₂-dependent and prevented by anaerobiosis or the presence of substrates for the dehydrogenases. Light in the range 400–500 nm was most effective and the presence of free flavins greatly enhanced inactivation of all of the above mitochondrial activities. This suggests that visible light mediates a flavin-photosensitized reaction that initiates damage involving participation of an activated species of oxygen in the damage propagation.     

Specific inhibition by macrocyclic polyethers of mitochondrial electron transport at site I

The macrocyclic polyethers dibenzo-18-crown-6 (XXVIII) and dicyclohexyl-18-crown-6 (XXXI) inhibit the valinomycin-mediated K⁺ accumulation energized by glutamate, -ketoglutarate, malate plus pyruvate or isocitrate but not that promoted by succinate, ascorbate plus TMPD or ATP. The polyethers inhibit the oxidation of the former group of substrates without preventing either the oxidation of succinate or ascorbate plus TMPD or the hydrolysis of ATP.The substrate oxidation inhibited by the macrocyclic polyethers is relieved in intact mitochondria by increasing the concentration of K⁺ in the medium. It is also completely reverted by supplementing the medium with valinomycin, Cs⁺ and phosphate, or else by the addition of vitamin K₃.In submitochondrial sonic particles the macrocyclic polyethers inhibit the oxidation of NADH as well as the ATP-driven reversal of electron flow at the site I of the electron transport chain. They also block the oxidation of NADH in non-phosphorylating Keilin-Hartree particles as well as in Hatefi's NADH-coenzyme Q reductase. The polyethers do not inhibit electron transport in mitochondria from the yeast which lack the first coupling site.The inhibition of electron transport by the polyethers do not require of the addition of alkali metal cations such as K⁺ in intact mitochondria or other membrane preparations.It is established that the macrocyclic polyethers XXVIII and XXXI, already characterized as mobile carrier molecules for K⁺ in model lipid membranes, inhibit electron transport at site I of the electron transport chain from mitochondrial membranes.It is suggested that the ability of the polyethers to coordinate alkali metal cations in aqueous versus lipid environments, but not K⁺ transportper se, is related to their rotenone-like induced inhibition of electron flow in mitochondrial membranes.Supported in part by a Grant from the Research Corporation.     

Triosephosphates modulate leaf mitochondrial phosphorylation by inhibition and uncoupling of electron transport

The effect of TP (triosephosphates:glyceraldehyde-3 phosphate, GAP, +dihydroxyacetone phosphate, DHAP) on respiration, phosphorylation and matrix ATP/ADP ratios of isolated oat mesophyll mitochondria was investigated. With both malate and NADH, a 50% inhibition of state 3-phosphorylation was induced by about 15 to 20 millimolar GAP and 30 to 40 millimolar DHAP. However, the nature of the inhibition appeared to be different with the two respiratory substrates. In the presence of NADH, TP did not inhibit the rate of state 3 (addition of ADP) O₂ consumption. In fact, depending on concentration, TP gradually increased the rates measured without ADP towards those seen under state 3, acting as uncouplers. When malate was the substrate for respiration, state 3 rates were decreased. The effect was comparable to that of rotenone and could be abolished by the addition of NADH. These observations indicate a dual action of TP: inhibition of electron transport around site I and uncoupling. In any case, the intramitochondrial ATP/ADP ratio decreased upon addition of TP. The effective TP concentrations as well as the changes in mitochondrial ATP/ADP ratios were comparable to results on changes of compartmental pool sizes of adenylates and other metabolites during dark/light transition of oat mesophyll protoplasts (R. Hampp, M. Goller, H. Füllgraf, and I. Eberle 1985 Plant Cell Physiol 24: 99). The possible role of TP in the regulation of mitochondrial respiration in the light, as well as modes of interference, are discussed.     

Lateral diffusion as a rate-limiting step in ubiquinone-mediated mitochondrial electron transport

Data are presented which indicate that the diffusion-based collisions of ubiquinone with its redox partners in the mitochondrial inner membrane are a rate-limiting step for maximum (uncoupled) rates of succinate-linked electron transport. Data were obtained from experimental analysis of a comparison of the apparent activation energies of lateral diffusion rates, collision frequencies, and electron transport rates in native and protein-diluted (phospholipid-enriched) inner membranes. Diffusion coefficients for Complex III (ubiquinol:cytochrome c oxidoreductase) and ubiquinone redox components were determined as a function of temperature using fluorescence recovery after photobleaching, and collision frequencies of appropriate redox partners were subsequently calculated. The data reveal that 1) the apparent activation energies for both diffusion and electron transport were highest in the native inner membrane and decreased with decreasing protein density, 2) the apparent activation energy for the diffusion step of ubiquinone made up the most significant portion of the activation energy for the overall kinetic activity, i.e. electron transport steps plus the diffusion steps, 3) the apparent activation energies for both diffusion and electron transport decreased in a proportionate manner as the membrane protein density was decreased, and 4) Arrhenius plots of the ratio of experimental electron transport productive collisions (turnovers) to calculated theoretically predicted, diffusion-based collisions for ubiquinone with its redox partners had little or no temperature dependence, indicating that as temperature increases, increases in electron transport rate are accounted for by the increases in diffusion-based collisions. These data support the Random Collision Model of mitochondrial electron transport in which the rates of diffusion and appropriate concentrations of redox components limit the maximum rates of electron transport in the inner membrane.     

Inactivation of mitochondrial ATPase by ultraviolet light

The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation.     

Immobilized mitochondrial electron transport particle for NADH determination

Nonphosphorylating electron transport particles (ETP) prepared from beef heart mitochondrion were immobilized in agar gel. The immobilized ETP showed an oxidase activity to both NADH and succinate. The immobilized ETP was reusable. An electrochemical device for the determination of either NADH or succinate was assembled consisting of the membrane-bound ETP and an oxygen probe. The response to succinate was specifically inhibited by the addition of malonate.