Flow microcalorimetry of a respiration-deficient mutant of Saccharomyces cerevisiae

In aerobic batch cultures in mineral medium with glucose of a respiration-deficient mutant of Saccharomyces cerevisiae, growth parameters were estimated and the heat evolved was measured by a flow microcalorimeter. A growth enthalpy of – 163.6 joule per mole of glucose consumed was measured. Under anaerobic conditions, the value was – 134.6 joule, closer to the expected for alcoholic fermentation alone. The difference was found to be due to cyanide-resistant respiration under aerobic conditions.     

A mutation in cytochrome oxidase subunit 2 restores respiration of the mutant pet ts1402

The yeast PET1402/OXA1 gene encoding a 44.8-kDa protein is required for mitochondrial biogenesis. Substitution of Leu240 to serine in the protein results in an accumulation of the precursor form of the mitochondrially encoded subunit 2 of cytochrome oxidase (Cox2) and temperature-sensitive respiration. This temperature sensitivity can be suppressed by a mutation in the cox2 gene changing Ala189 of the Cox2 protein to proline. In the cox2-ts1402 double mutant respiration is restored without removal of the Cox2 pre-sequence. The suppression suggests an interaction of the Pet1402 protein with the cytochrome oxidase complex. Antibodies raised against the predicted C-terminus and the tagged N-terminus of the Pet1402 protein reacted with a 37-kDa polypeptide. This protein, present in the mitochondrial fraction, is localized within the inner membrane. The difference in size can be explained by the removal of the predicted mitochondrial-targeting sequence from the Pet1402 protein. The mitochondrial localization of the protein points to a direct interaction with the cytochrome oxidase complex. Received: 4 December 1996 / 26 January 1997     

Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L

A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5′-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger. Received: 10 August / 13 October 1998     

Sporulation in mitochondrial OXI3 mutants of Saccharomyces cerevisiae

Summary By use of a set of mitochondrial oxi3 mutants (mit ^–, defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level of respiration (below l% of the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation of these results with the genetic map of the OXI3 region reveals that one group of mutants, mapping in the central part of the OXI3 region, is capable of sporulation.     

Oxidative stress and ageing in the fungus Podospora anserina.

The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally.     

Characterization of a respiratory-deficient mutant of Pachysolen tannophilus

Summary A pleiotropic, respiration-deficient mutant was isolated from the petite negative yeast Pachysolen tannophilus after UV mutagenesis. The mutant is unable to utilize xylose, arabinose, galactose or glycerol, and shows no detectable respiration when grown on glucose. Cytochrome c oxidase, xylose reductase and xylitol dehydrogenase activities are lacking. Mitochondrial ultrastructre is altered. The results support the hypothesis that functioning mitochondria are necessary for xylose utilization in this organism.     

Bioenergetic parameters of Acremonium chrysogenum

Bioenergetic parameters of two isogenic strains of Acremonium chrysogenum markedly differing in their capacity for biosynthesis of cephalosporin C were studied. It was shown that in the high productive strain the system of oxidative phosphorylation played the dominant role in the cellular energy supply (the index of the substrate consumption efficiency amounted to 0.82). In both strains terminal oxidation of the reduction equivalents mainly occurred via the respiration chain with cytochrome oxidase as the terminal component. In the low productive strain an alternative cyanide resistant pathway was observed, in addition to the classic cytochrome chain. A procedure suitable for the isolation of stable protoplasts from the mycelium of A. chrysogenum was developed. It was proved to be useful in the isolation of functionally active mitochondria possessing theoretical phosphorylation efficiency and high phosphate acceptor respiration control. It was concluded that there are three oxidative phosphorylation sites in the respiration chain of the high productive strain of A. chrysogenum.     

Comparative Use of 2,7-dichlorofluorescin diacetate,dihydrorhodamine 123, and hydroethidine to study oxidative metabolism in phagocytic cells

Tests carried out with model systems to assess the sensitivity of 2,7-dichlorofluorescin diacetate, dihydrorhodamine 123, and hydroethidine to the reactive oxygen species generated in these systems showed that all three fluorochromes are sensitive to hydroxyl and superoxide radicals and to hypochlorite ions and less sensitive to hydrogen peroxide. They do not react with singlet oxygen. Hydroethidine reacts with superoxide radicals in the 1∶1 molar ratio. The relative proportions of reactive oxygen species generated extra- and intracellularly were estimated for phorbol myristate acetate-stimulated peritoneal and alveolar rat macrophages and for polymorphonuclear leukocytes from the blood of healthy donors. In activated leukocytes of different types, intracellular generation accounted for approximately 20% of the total reactive oxygen species produced. Intracellular production of these species was found to show considerable cyanide sensitivity, which indicates that cyanide-resistant NADPH oxidase located in the plasma membrane is not implicated in the generation of reactive oxygen species. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 4, pp. 361–365, April, 1995     

Biochemical, genetic and ultrastructural defects in a mitochondrial mutant (ER-3) of Neurospora crassa with senescence phenotype

The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant ER-3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. Cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33% greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33%) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant ER-3 possesses a defect in the terminal portion of the mitochondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial DNA populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in Neurospora mutant ER-3.     

Presence of an endogenous superoxide dismutase activity in three rodent malaria species

Superoxide dismutase (SOD) was investigated in three species of rodent malaria (Plasmodium berghei, P. yoelii andP. vinckei). The isoelectric points (pI) of isozymes found in purified parasites were identical. SOD activities detected by isoelectrofocusing at pl 5.0, 5.6, and 6.4 were cyanide-sensitive and could be considered as having been adopted by the parasites from the host red blood cell. The three rodent malaria parasites also contained a cyanide-resistant, hydrogen peroxide-sensitive SOD activity not found in the host red blood cell. It is therefore concluded that the three rodent malaria parasites possess an endogenous SOD. Two bands of endogenous SOD were found at pl 6.2 and 6.8 for the three species, and one additional band was detected at pl 5.7 forP. berghei andP. vinckei. This first report in rodentPlasmodium of a cyanide-resistant, hydrogen peroxide-sensitive SOD suggests that these parasites may be capable of at least partly resisting activated oxygen species using an endogenous SOD.     

Ischaemia-reperfusion induced alterations of mitochondrial function in hypertrophied rat heart

The impact of in vivo ischaemia and ischaemia-reperfusion (I-R) on mitochondrial respiratory function was investigated in hypertrophied (HP) hearts with aortic constriction compared with control hearts using an open-chest rat surgical model. Moreover, mitochondrial susceptibility to superoxide radicals (O₂P^-) in vitro was examined in HP and control hearts with or without I-R. With the site I substrates pyruvate-malate, mitochondrial state 4 (basal) respiration and the respiratory control index (RCI) were not affected by either ischaemia alone or I-R in both HP and control hearts. State 3 (ADP-stimulated) respiration was increased with I-R in control hearts, but showed a reduction after I-R in the HP hearts. Exposure of mitochondria to O₂P^- (20 nm hypoxanthine in the presence of 0.13 unit mL^-1 xanthine oxidase) significantly increased state 4 respiration, whereas state 3 respiration and RCI were decreased in all treatment groups. I-R hearts in both HP and control showed greater increases in state 4 respiration with O₂P^- than either sham or ischaemic hearts. HP hearts exhibited a significantly lesser extent of inhibition in state 3 respiration and RCI by O₂P^- compared with control hearts. These changes in mitochondrial respiratory properties were not observed with the site II substrate succinate. Myocardial reduced vs. oxidized glutathione ratio was significantly decreased after I-R in both control and HP hearts. Malondialdehyde content showed an increase with I-R, but the increase was significant only in control hearts. These data indicate that short-term in vivo I-R does not impair heart mitochondrial respiratory function, but renders the organelles more vulnerable to imposed oxidative stress. Mitochondria from the HP hearts are more resistant to free radical damage under normal and ischaemic conditions; however, this advantage is severely compromised after reperfusion.     

Characterization of mouse nuclear and mitochondrial mutants with increased resistance to cytochromeb inhibitors

A series of mouse lines with increased resistance to respiratory inhibitors which block electron transport through the protonmotive cytochrome bof complex III have been isolated in this laboratory. We describe here the isolation of a mutant with increased resistance to HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) whose phenotype is due to a nuclear mutation. At the cellular level, there is a severe reduction in respiration with the residual oxygen consumption being resistant to inhibitors of both ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase. At the mitochondrial level, there was a severe derangement in NADH oxidase activity. Electron transport through the succinate oxidase span of the respiratory chain and its coupling to oxidative phosphorylation are also reduced in this nuclear mutant but not to the same extent. It is concluded that the primary defect in the mutant lies within a nuclear gene encoding a component of complex I (NADH-ubiquinol oxidoreductase). In addition, further biochemical characterization of the mitochondrially inherited inhibitor-resistant mutants has demonstrated that they also show significant reductions in the efficiency of energy transduction and in the rate of cytochrome belectron transport.     

Two nuclearly inherited loci conferring increased diuron resistance to NADH oxidase in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, diuron blocks the respiration pathway at the level of the bc₁ complex. Nuclear diuron-resistant mutations which confer in vitro resistance to mitochondrial NADH oxidase have been identified. Five mutations were found to be clustered at two distinct nuclear loci, DIU3 and DIU4. The distance between the two loci was estimated to be about 36.7 cM. These loci do not appear to be centromere-linked and did not show a linkage to any of the genes coding for bc₁ complex subunits. DIU3 and DIU4 loci might, therefore, code for other components of the respiratory chain.Research associate to the National Fund for Scientific Research, Belgium     

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.     

Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl

Several agricultural fungicides related to the antifungal strobilurins act as inhibitors of respiration by binding to mitochondrial cytochrome b. Two types of laboratory mutants resisting higher doses of the strobilurin-related inhibitor kresoxim-methyl were characterized for Venturia inaequalis, the causal agent of apple scab. Selection of mutagenized conidia by kresoxim-methyl yielded mutants altered in the expression of alternative respiration during the stage of conidia germination. Cytochrome b sequences were not affected in the respective mutants. Selection of conidia on media containing the alternative oxidase inhibitor salicylhydroxamic acid in addition to kresoxim-methyl yielded a highly resistant mutant distinguished by a G143A exchange in cytochrome b. The status of mitochondrial cytochrome b genes remained heteroplasmic, and mitochondria containing wild-type cytochrome b returned to high frequencies during cultivation on inhibitor-free medium. However, continuation of the selection process led to a more pronounced replacement of sensitive by mutated mitochondria. The G143A mutation of cytochrome b causing resistance of V. inaequalis to a strobilurin-related inhibitor has been reported previously for mouse mitochondria; and a permanent G143A exchange rendering naturally resistant mitochondria has been reported for the strobilurin-producing basidiomycete Mycena galopoda and for the sea urchin Paracentrotus lividus. At the corresponding position, alanine was also present in chloroplast cytochrome b ₆ exhibiting low binding of strobilurin-related inhibitors. The mutation of cytochrome b reported here for V. inaequalis describes the first example of a mutation in filamentous ascomycetes and is part of an assessment of resistance risks inherent to strobilurin fungicides. Received: 20 March 2000 / Accepted: 26 May 2000     

Mechanisms of immunotoxic effects of acrylonitrile

Cholinesterase reactivator dipyroxime and hydrogen cyanide antidote anticyan partially restore cell and humoral immune response in mice after acute acrylonitrile poisoning (0.5 LD₅₀). When used in combination these drugs virtually completely prevented the suppression of immune reactions. The main mechanism of immunotoxic effect of acrylonitrile is mediated through inhibition of T lymphocyte esterases and a₃ component of cytochromec oxidase of immunocyte mitochondrial respiration enzymes, which is important for prevention and treatment of immune disturbances caused by this toxin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 5, pp. 547–549, May, 2000     

Bacterial Terminal Oxidases

Abstract

Oxidases, as such, regardless of their source, represent a diverse and complex series of enzymes. What they have in common is the ability to react with molecular oxygen, activate it chemically (in a manner which is still not understood), and utilize the “activated atoms of oxygen” primarily as electron acceptors. Should the “activated oxygen atoms” be used directly for oxygenating substrate molecules, such as hydrocarbons, then according to the conventions used today, the enzyme is termed an oxygenase rather than an oxidase. The subject of oxygenases is far too complex to be reviewed in any detailed treatment of oxidases. All oxidases serve as electron acceptors for specific dehydrogenation reactions that are carried out by the multitude of dehydrogenases that are found in tissues as well as in bacteria. The major end product that results from the oxidase reaction is either H₂O or H₂O₂. The oxidases can be (1) simple flavoprotein-containing enzymes, such as the glucose oxidase or the D- and L-amino acid oxidases, (2) metalloflavoprotein-containing enzymes, such as the xanthine oxidase which in addition to flavin contains both molybdenum and nonheme iron, (3) the heme-containing oxidases which essentially are free of flavoprotein yet carry out a peroxidase-oxidase type of reaction, and finally (4) the heme-containing and flavoprotein-free cytochrome (or terminal) oxidase molecule which may contain as many as two heme “a” components (multiple heme iron), copper protein, and the active form of which requires an enzyme complex containing some phospholipid.^{1 4 5} It is this latter group, the cytochrome or terminal oxidases, that will be considered in this review, particularly what is known about them in bacterial systems. The cytochrome or terminal oxidases are usually membrane-bound entities, play a major role in electron transport, and are very important in the bioenergetic mechanism of aerobic cells that allow for respiration.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Expression of the Aspergillus niger glucose oxidase gene in A. niger,A. nidulans and Saccharomyces cerevisiae

Summary We report the cloning of the Aspergillus niger glucose oxidase gene and its use to elevate glucose oxidase productivity in A. niger by increasing the gene dosage. In addition, the gene has been introduced into A. nidulans where it provides the novel capacity to produce glucose oxidase. A plasmid, in which DNA encoding the mature form of glucose oxidase was preceded by a Saccharomyces cerevisiae secretion signal, effected high-level production of extracellular glucose oxidase in this yeast.