Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa

1. Neurospora crassa was found to contain two distinct superoxide dismutases. 2. Most of the activity is associated with the cytosolic fraction and was shown to be the Cu/Zn-containing form of the protein. 3. Mitochondria isolated from Neurospora crassa showed two distinct superoxide dismutases: a cyanide-sensitive Cu/Zn-containing protein and a cyanide-insensitive form which probably contains manganese. 4. Localization experiments, using selective marker enzymes and digitonin fractionation, indicated that the cyanide-sensitive form is localized in the intermembrane space, whereas the cyanide-insensitive form is confined to the mitochondrial matrix space. 5. The cytosolic Cu/Zn-containing superoxide dismutase was isolated in high yields and extensively characterized by using e.p.r. spectroscopy, isoelectric focusing and analytical ultracentrifugation. 6. E.p.r. spectroscopy was used to monitor changes in the copper environment of the native protein after the addition of a number of potential inhibitors and after high-pH treatment. 7. Both of the cyanide-sensitive Cu/Zn-containing enzymes (cytosolic and mitochondrial) appeared to have identical properties which in turn were different from the cyanide-insensitive enzyme. 8. It is probable that the cyanide-insensitive enzyme was not previously detected, owing to its low amount (less than 10% of the total activity), greater lability than the cyanide-sensitive enzyme and the necessity of obtaining a mitochondrial-enriched fraction before its isolation.     

The inhibition of plant mitochondrial respiration by the synthetic analog of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)

The quinone analog, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), has been shown to inhibit cyanide-sensitive and cyanide-insensitive respiration in higher plant mitochondria. The inhibition is dependent upon the concentration of mitochondrial protein. The low concentrations of UHDBT required to inhibit the cyanide-sensitive pathway (microM) and the cyanide-insensitive pathway (nM) indicate that UHDBT is acting as a tight-binding inhibitor of ubiquinol oxidation. Inhibition of both pathways was dependent upon pH. It is shown that UHDBT appears to be a less potent inhibitor of cyanide-sensitive NADH oxidation than of cyanide-sensitive succinate oxidation, and that the pH dependence of inhibition of these two pathways differs. The inhibition of NADH and succinate oxidation by the cyanide-insensitive pathway shows similar pH dependences although at a given pH NADH oxidation is more susceptible to inhibition than succinate oxidation.     

Involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica

The involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica has been established on the following basis: (1) endogenous metal chelation by either benzyl- or salicylhydroxamic acid, EDTA or nitrilotriacetate prevented the biogenesis of the cyanide-insensitive respiratory pathway in S. lipolytica. (2) Addition of Fe(III) during the biogenesis increased both the rate of the appearance of the alternative respiratory pathway and its extent. Neither Fe(II), nor Co(II), Cu(II), Al(III), La(III), Mn(II) or Mg(II) could substitute for Fe(III). (3) The biogenesis of the alternative respiratory pathway could be dissociated into two steps: (a) a first one, slow, cycloheximide-sensitive, temperature-dependent, iron-independent, leading to cells still fully cyanide-sensitive, presumably involving the de novo biosynthesis of an inactive protein moiety and (b) a second step, fast, iron-dependent, temperature-independent, cycloheximide-insensitive, leading to cells with a cyanide-insensitive respiration, presumably the activation by iron of the inactive precursor.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanide-sensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 microM and 20 microM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Cyanide-insensitive respiration of Candida lipolytica

Whole cells of the yeast Candida lipolytica exhibited a high, cyanide-sensitive endogenous respiration which became completely cyanide-insensitive under certain physiological circumstances namely (1) in the stationary phase of growth and (2) upon aeration in the resting state. This cannot be due to a change in permeability of the cell wall as the respiration of protoplasts showed the same (in)sensitivity to cyanide as the cells from which they were obtained.The cyanide-insensitive respiration of C. lipolytica was located in the mitochondria and coexisted with the normal respiratory chain, as the mitochondria isolated from cyanide-insensitive cells exhibited at the same time a cyanidesensitive respiration of ascorbate and N,N,N,N-tetramethyl-p-phenylenediamine and a cyanide-insensitive respiration of succinate.The alternate respiratory pathway was sensitive to benzyl- and salicylhydroxamic acids. In this respect it resembles the alternate mitochondrial pathway described in the literature for various plants.The cyanide-insensitive respiration did not appear in the resting state when the cells were aerated in the presence of cycloheximide nor at 0 C instead of at room temperature. These facts suggest some form of induction involving new protein synthesis. The induction process depends on the presence of molecular oxygen as the cyanide-insensitive endogenous respiration did not appear during agitation of yeast cells in the resting state if the gaseous atmosphere lacked oxygen.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Effect of chloramphenicol on the electron systems in Ustilago cynodontis.

The mycelial cells of Ustilago cynodontis possess at least two electron transport systems: a cyanide-sensitive cytochrome pathway, which represents the major route for electron transport, and an alternative cyanide-insensitive pathway, inhibited by salicylhydroxamic acid. In the presence of chloramphenicol in the culture medium, mycelial cells respire only by the alternatuve chain. The stable induced yeast-like cells, obtained by prolonged chloramphenicol treatment of the mycelial cells, respire as the untreated mycelial cells; this result indicates that the phenotypic change induced by chloramphenicol is not related to a modification of the respiratory system.     

Effect of tobacco mosaic virus infection of levels of soluble superoxide dismutase (SOD) in nicotiana tabacum and nicotiana glutinosa leaves

The activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was evaluated on Nicotiana tabacum and Nicotiana glutinosa leaf tissue after Tobacco Mosaic Virus (TMV) infection. Significant increase in extracted SOD appeared to be directly related to the appearance of necrotic and systemic symptoms in hypersensitive (N. glutinosa and N. tabacum cv. Havana 425) and susceptible (N. tabacum cv. Bright BC 60) leaves, respectively. SOD activity did not change significantly during the replication of TMV in the inoculated susceptible leaves up to 4 days after inoculation. Both cyanide-insensitive (2 days after inoculation) and sensitive (3–4 days after inoculation) enzymes increased during the expression of the hypersensitivity. Only cyanide-sensitive enzyme increased in systematically infected leaves. SOD and peroxidase increased simultaneously and the enhancement of peroxidase was higher than that of SOD. The values of peroxidase greatly exceeded that of SOD only in the hypersensitive leaves during local lesion differentiation. In N. tabacum leaves 4 clear SOD bands were separated by polyacrylamide gel electrophoresis: 3 cyanide-sensitive (Cu,Zn enzyme) and 1 cyanide-insensitive, while N. glutinosa had 3 bands: 2 cyanide-sensitive and 1 cyanide-insensitive. The cyanide-insensitive band, both in N. tabacum and N. glutinosa, was sensitive to H₂O₂ and insensitive to chloroform-ethanol treatment and thus supposed to be Fe enzyme. The infection did not induce change in the electrophoretic pattern of SOD enzymes.In summary, our results indicate that the pathogenic alteration caused by TMV infection both in the compatible and in the incompatible combinations are characterized by an induction of SOD activity, particularly cyanide-sensitive Cu,Zn-SOD. The connection between the induction of SOD and a possible activation of O₂⁻ production in the hypersensitive tissue following TMV infection is discussed.     

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

The effect of respiratory inhibitors during growth and development of Dictyostelium discoideum

Two major pathways of electron transport to oxygen were identified in intact cells of Dictyostelium discoideum, a cyanide-sensitive pathway and a cyanide-insensitive, salicylhydroxamic-acid-sensitive pathway. The extent to which each pathway contributed to the total respiratory activity was shown to change during exponential growth and throughout development. During exponential growth both pathways appear to be utilized to varying degrees dependent on culture age, during late exponential growth the activity of the salicylhydroxamic-acid-sensitive pathway would seem to be almost totally lost. During development the cyanide-sensitive pathway appears to be dominant up to the aggregation stage, but both pathways are active in pseudoplasmodial cells. It is also suggested that the presence of iron in the growth medium may be essential directly or indirectly, for the maintained activity of the salicylhydroxamic-acid-sensitive pathway late in exponential growth.     

Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate

Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide-sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.

The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate-insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns.

     

Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus.

Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide-insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha-ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger.     

Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen.

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.     

Suppression by Exogenous Phospholipid of Cyanide-Insensitive Respiration of Submitochondrial Particles from Sweet Potato Root Tissue

Submitochondrial particles from sweet potato root tissue retainedthe respiratory characteristics of the intact mitochondria withrespect to the sensitivity to cyanide and salicylhydroxamicacid. The activities of total, cyanide-insensitive, and salicylhydroxamate-sensitiverespiration of the submitochondrial particles yielded from adefinite weight of tissue slices incubated under aerobic conditions,particularly in ethylenecontaining air, were higher than thosefrom the same weight of intact tissue. The less phospholipidthe submitochondrial particles contained relative to protein,the higher the activities of cyanide-insensitive and salicylhydroxamate-sensitiverespiration tended to be relative to total respiratory activity.When the submitochondrial particles were incubated with phospholipidliposomes, the activities of cyanide-insensitive and salicylhydroxamate-sensitive,but not cyanide-sensitive, respiration became extremely low.All phospholipids showed this effect. Such incubation of thesubmitochondrial particles with phospholipid liposomes yieldedlighter particles, indicating close association of exogenouslyadded phospholipid with the particles. Phospholipid moleculesseemed to enter the membrane of the particles. We propose thatphospholipid deficiency in the mitochondrial inner membranefacilitates operation of the cyanide-insensitive electron transportpath. (Received March 30, 1984; Accepted June 15, 1984)     

Respiratory pathways in Agaricus bisporus and Scytalidium thermophilum

Abstract The respiratory pathways of Agaricus bisporus and Scytalidium thermophilum were studied. A. bisporus appeared to possess both a cyanide-sensitive and a cyanide-insensitive respiration while in S. thermophilum the cyande-insensitive respiration was absent. Growth experiments showed the ecological advantage for A. bisporus under conditions where cytochrome mediated respiration is inhibited.     

Identification of the quinone species in cyanide-sensitive and cyanide-insensitive mitochondria of Moniliella tomentosa.

Moniliella tomentosa was investigated for the presence of different quinones that might be involved in the cyanide-sensitive and/or cyanide-insensitive electron-transport pathways. The naturally occurring quinone in Moniliella tomentosa was found to be ubiquinone-45. Other quinone species could not be detected. The concentration of ubiquinone-45 in mitochondria is not related to the presence or absence of the alternative oxidase activity.     

2,6-Dichloro-phenol indophenol prevents switch-over of electrons between the cyanide-sensitive and -insensitive pathway of the mitochondrial electron transport chain in the presence of inhibitors

To evolve a simple oxygen electrode-based method to estimate alternative respiration, one needs to develop a procedure to prevent switch-over of electrons to either pathway upon inhibition by cyanide or salicylhydroxamic acid. It was hypothesized that the inclusion of appropriate electron acceptor, possessing redox potential close to one of the electron transport carriers in between ubiquinone (branch point) and cytochrome a-a3, should be able to stop switch-over of electrons to either pathway by working as an electron sink. To test the hypothesis, 2,6-dichloro-phenol indophenol (DCPIP; redox potential +0.217 V), an artificial electron acceptor having a redox potential quite similar to the site near cytochrome c1 (redox potential +0.22 V) on the cyanide-sensitive pathway, was used with isolated mitochondria and leaf discs in the absence and presence of inhibitors (potassium cyanide, antimycin A, and salicylhydroxamic acid). Polarographic data confirmed electron acceptance by DCPIP only from the inhibited (by cyanide or salicylhydroxamic acid) mitochondrial electron transport chain, hence preventing switch-over of electrons between the cyanide-sensitive and cyanide-insensitive pathway of respiration. Results with antimycin A and reduction status of DCPIP further confirmed electron acceptance by DCPIP from the mitochondrial electron transport chain. Possible implications of the results have been discussed.     

Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals.