Beyond Sham and Cyanide: Opportunities for Studying the Alternative Oxidase in Plant Respiration Using Oxygen Isotope Discrimination.

Robinson, SA; Ribas‐Carbó, Miquel; Yakir, Dan; Giles, Larry; Reuveni, Y; Berry, JA

doi:10.1071/pp9950487

Cited by 58 publications

(75 citation statements)

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“…For the alternative oxidase of soybean root mitochondria, although pyruvate and iodoacetate both stimulate the enzyme, neither pyruvate nor iodoacetate protect the enzyme from inhibition by NEM, 3 suggesting a possible additional site of NEM reactivity in the root enzyme. Soybean root alternative oxidase is known to differ from that of green cotyledons with respect to oxygen isotope fractionation (47). This, together with the discovery of three alternative oxidase genes in soybean (8), suggests that the inhibition of soybean root alternative oxidase activity in the presence of pyruvate or iodoacetate by NEM may be the result of its action on a different gene product from that (or those) expressed in cotyledons.…”

Section: ␣-Keto Acid Activation Of the Cyanide-resistant Oxidasementioning

confidence: 97%

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Umbach

Siedow

1996

Journal of Biological Chemistry

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The cyanide-resistant alternative oxidase of plant mitochondria is known to be activated by ␣-keto acids, such as pyruvate, and by the reduction of a disulfide bond that bridges the two subunits of the enzyme homodimer. When the regulatory cysteines are oxidized, the inactivated enzyme is much less responsive to pyruvate than when these groups are reduced. When soybean cotyledon mitochondria were isolated in the presence of iodoacetate or N-ethylmaleimide, the intermolecular disulfide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternative oxidase activity, but iodoacetate was found to stimulate activity much like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocked inhibition of the enzyme by N-ethylmaleimide, indicating that all three compounds acted at the same sulfhydryl group on the alternative oxidase protein. The site of pyruvate and iodoacetate action was shown to be a different sulfhydryl than that involved in the redox-active regulatory disulfide bond, because iodoacetate bound to the alternative oxidase at the activating site even when the redox-active regulatory sulfhydryls were oxidized. Given the nature of the covalent adduct formed by the reaction of iodoacetate with sulfhydryls, the activation of the alternative oxidase by ␣-keto acids appears to involve the formation of a thiohemiacetal.Plant mitochondria possess a terminal cyanide-resistant alternative oxidase in the electron transport system of the inner membrane (1, 2). This oxidase reduces oxygen to water with electrons derived directly from the ubiquinone pool. Because no protonmotive force is generated during this reaction, the alternative pathway appears to be energetically wasteful (1, 2). cDNA sequences have now been reported for the nuclear-encoded alternative oxidase from several plants, two fungi, and a protozoan (2-7).1 The mature protein contains 280 -290 amino acids and migrates as a 32-35-kDa protein on SDS-PAGE gels as revealed by immunoblotting. Hydropathy analysis has led to a model in which the alternative oxidase protein is anchored to the mitochondrial inner membrane by two membrane-spanning ␣-helices, with large hydrophilic domains approximately 100 amino acids in length flanking each membrane-spanning region and extending into the mitochondrial matrix (9). In the carboxyl-terminal hydrophilic domain, just beyond the second membrane-spanning region, is a set of conserved amino acid motifs found in the family of coupled binuclear iron proteins to which methane monooxygenase belongs (9, 10). Cross-linking studies have indicated that the plant alternative oxidase exists in the membrane as a homodimer (11).As a result of a search for the role of the alternative oxidase in plant metabolism, several regulators of its activity have been identified. The amounts of alternative oxidase protein in the membrane (e.g. Refs. 12-14) and the extent of ubiquinone pool reduction (...

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Section: ␣-Keto Acid Activation Of the Cyanide-resistant Oxidasementioning

confidence: 97%

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Umbach

Siedow

1996

Journal of Biological Chemistry

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“…This method is based on the fact that AOX and COX discriminate against the isotope 18 O to different extents. Both gasphase (for use with whole tissue) and aqueous-phase (for use with mitochondria or cell culture) systems connected online to a gas chromatography-mass spectrometry unit have been used for this purpose (Robinson et al 1995). The sample is placed in a leaktight reaction vessel from which gas samples can be withdrawn at regular intervals as oxygen is consumed by respiration.…”

Section: Subunit Structure: Monomeric Versus Dimericmentioning

confidence: 99%

Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase

McDonald¹

2008

Functional Plant Biol.

114

106

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Abstract. Alternative oxidase (AOX) is a terminal quinol oxidase located in the respiratory electron transport chain that catalyses the oxidation of quinol and the reduction of oxygen to water. However, unlike the cytochrome c oxidase respiratory pathway, the AOX pathway moves fewer protons across the inner mitochondrial membrane to generate a proton motive force that can be used to synthesise ATP. The energy passed to AOX is dissipated as heat. This appears to be very wasteful from an energetic perspective and it is likely that AOX fulfils some physiological function(s) that makes up for its apparent energetic shortcomings. An examination of the known taxonomic distribution of AOX and the specific organisms in which AOX has been studied has been used to explore themes pertaining to AOX function and regulation. A comparative approach was used to examine AOX function as it relates to the biochemical function of the enzyme as a quinol oxidase and associated topics, such as enzyme structure, catalysis and transcriptional expression and post-translational regulation. Hypotheses that have been put forward about the physiological function(s) of AOX were explored in light of some recent discoveries made with regard to species that contain AOX. Fruitful areas of research for the AOX community in the future have been highlighted.

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“…Surface-dried leaf samples were placed in the cuvette and left with the inlet vent open for 3-5 min with air mixing to allow for air and isotopic equilibration between the inside and the outside of the tissue. Oxygen extraction and isotope analysis were carried out as described in Robinson et al (1995) and . At regular time intervals an air sample was taken into a 100 mL loop and directed into the helium flow of the gas chromatography-mass spectrometry unit.…”

Section: Respiratory Measurements and Oxygen Isotope Analysismentioning

confidence: 99%

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

et al. 2001

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In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

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Beyond Sham and Cyanide: Opportunities for Studying the Alternative Oxidase in Plant Respiration Using Oxygen Isotope Discrimination.

Cited by 58 publications

References 0 publications

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

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