Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O2 as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O2 concentration for saturation than does cytochrome c oxidase. 4. The apparent Km for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4–10 times higher when measured in submitochondrial particles. The apparent Km for NADH is roughly the same for both oxidases. 5. The apparent Km values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent Km for AMP, acting as a stimulator of the alternative oxidase, is the same (25μm) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.

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Functional analysis reveals effects of tobacco alternative oxidase gene (NtAOX1a) on regulation of defence responses against abiotic and biotic stresses

Bioscience Reports ◽

10.1042/bsr20080133 ◽

2009 ◽

Vol 29 (6) ◽

pp. 375-383 ◽

Cited By ~ 6

Author(s):

Yi Zhang ◽

Dongmei Xi ◽

Jian Wang ◽

Dongfang Zhu ◽

Xingqi Guo

Keyword(s):

Oxidative Stress ◽

Low Temperature ◽

Alternative Oxidase ◽

Alternative Pathway ◽

Respiratory Pathway ◽

Oxidase Gene ◽

Biotic Stresses ◽

Alternative Respiratory Pathway ◽

Cytochrome Pathway ◽

Defence Responses

Mitochondrial AOX (alternative oxidase) is the terminal oxidase of the CN (cyanide)-resistant alternative respiratory pathway in plants. To investigate the role of the tobacco AOX gene (NtAOX1a) (where Nt is Nicotiana tabacum) under deleterious conditions which could induce ROS (reactive oxygen species) accumulation, we generated and characterized a number of independent transgenic tobacco (N. tabacum) lines with altered NtAOX1a gene expression and AP (alternative pathway) capacity. AOX efficiently inhibited the production of low-temperature-induced H2O2 and might be a major enzyme for scavenging H2O2 at low temperature. Furthermore, NtAOX1a may act as a regulator of KCN-induced resistance to TMV (tobacco mosaic virus) through the regulation of H2O2. Notably, a moderate accumulation of H2O2 under the control of NtAOX1a was crucial in viral resistance. Analysis of seed germination indicated an important role for NtAOX1a in germination under H2O2-induced oxidative stress when the CP (cytochrome pathway) was inhibited. These results demonstrate that NtAOX1a is necessary for plants to survive low temperature, pathogen attack and oxidative stress by scavenging ROS under these adverse conditions when the CP is restricted.

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Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L

Biochemical Journal ◽

10.1042/bj3170313 ◽

1996 ◽

Vol 317 (1) ◽

pp. 313-319 ◽

Cited By ~ 19

Author(s):

Graeme R. LEACH ◽

Klaas KRAB ◽

David G. WHITEHOUSE ◽

Anthony L. MOORE

Keyword(s):

Respiratory Rate ◽

Succinate Dehydrogenase ◽

Oxidase Activity ◽

Alternative Oxidase ◽

Alternative Pathway ◽

Respiratory Control ◽

Stage Of Development ◽

Activation Level ◽

Cytochrome Pathway ◽

Arum Maculatum

The dependence of the rate of oxygen uptake upon the ubiquinone (Q)-pool reduction level in mitochondria isolated during the development of thermogenesis of Arum maculatum spadices has been investigated. At the α-stage of development, the respiratory rate was linearly dependent upon the reduction level of the Q-pool (Qr) both under state-3 and -4 conditions. Progression through the β/γ to the Δ-stage resulted in a non-linear dependence of the state-4 rate on Qr. In the Δ-stage of development, both state-3 and -4 respiratory rates were linearly dependent upon Qr due to a shift in the engagement of the alternative oxidase to lower levels of Qr. Western blot analysis revealed that increased alternative oxidase activity could be correlated with expression of a 35 kDa protein. Respiratory control was only observed with mitochondria in the α-stage of development. At the β/γ-stage of development, the addition of ADP resulted in a significant oxidation of the Q-pool which was accompanied by a decrease in the respiratory rate. This was due either to decreased contribution of the alternative pathway to the overall respiratory rate under state 3 or by deactivation of succinate dehydrogenase activity by ADP. Cold-storage of the spadices at the β-stage of development led to increased activity of both the cytochrome pathway and succinate dehydrogenase, without any change in alternative oxidase activity. Results are discussed in terms of how changes in the activation level of the alternative oxidase and succinate dehydrogenase influence the activity and engagement of the quinol-oxidizing pathways during the development of thermogenesis in A. maculatum.

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Effect of growth at low temperature on the alternative pathway respiration in Acanthamoeba castellanii mitochondria.

Acta Biochimica Polonica ◽

10.18388/abp.2001_3907 ◽

2001 ◽

Vol 48 (3) ◽

pp. 729-737 ◽

Cited By ~ 15

Author(s):

W Jarmuszkiewicz ◽

O Fraczyk ◽

L Hryniewiecka

Keyword(s):

Low Temperature ◽

Alternative Oxidase ◽

Alternative Pathway ◽

Plant Mitochondria ◽

Physiological Role ◽

Acanthamoeba Castellanii ◽

Control Culture ◽

Cytochrome Pathway ◽

Coupling Parameters ◽

Unicellular Organisms

Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6 degrees C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28 degrees C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.

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The cytochrome oxidase inhibitor azide also inhibits the alternative pathway of Neurospora crassa

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o84-019 ◽

1984 ◽

Vol 62 (2-3) ◽

pp. 129-136 ◽

Cited By ~ 2

Author(s):

Peter J. Bridge ◽

Helmut Bertrand

Keyword(s):

Neurospora Crassa ◽

Cytochrome Oxidase ◽

Alternative Oxidase ◽

Alternative Pathway ◽

Oxidase Inhibitor ◽

The Novel ◽

Respiratory Pathway ◽

Maximal Inhibition ◽

Cytochrome Pathway ◽

Mutant Strains

Mutant strains of Neurospora crassa were used to investigate the effects of the inhibitor azide upon respiration mediated by either the cyanide-insensitive alternative mitochondrial respiratory pathway or the "standard" cyanide-sensitive cytochrome pathway. The use of appropriate cytochrome-oxidase-deficient and alternative-oxidase-deficient mutants permitted respiration to be restricted to the pathway of choice. In addition to its known effect upon cytochrome oxidase, azide was found to inhibit the alternative pathway of Neurospora with half-maximal inhibition occurring at 1.3 mM azide. This finding indicates that the activities of at least some of the "novel" azide-sensitive oxidases that have been discovered recently in organisms with inducible or constitutive cyanide-insensitive respiratory systems might be attributable to the "standard" salicylhydroxamate-sensitive mitochondrial alternative pathway.

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Pyruvate transport by thermogenic-tissue mitochondria

Biochemical Journal ◽

10.1042/bj2470441 ◽

1987 ◽

Vol 247 (2) ◽

pp. 441-447 ◽

Cited By ~ 11

Author(s):

M O Proudlove ◽

R B Beechey ◽

A L Moore

Keyword(s):

Alternative Oxidase ◽

Competitive Inhibition ◽

Feedback Inhibition ◽

Total Activity ◽

Mitochondrial Inner Membrane ◽

Pyruvate Oxidation ◽

Cytochrome Pathway ◽

Arum Maculatum ◽

Long Chain ◽

Sauromatum Guttatum

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

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