Targeting the Plant Alternative Oxidase Protein to   Mitochondria Confers Cyanide-insensitive Respiration

Albury, Mary S.; Dudley, P; Watts, Felicity Z.; Moore, Anthony L.

doi:10.1074/jbc.271.29.17062

Cited by 39 publications

(42 citation statements)

References 42 publications

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“…2A reveals that the respiratory activity exhibited by mitochondria containing the wild-type AOX is partially resistant to antimycin A (ϳ18% of the NADH-dependent rate). This antimycin-resistant respiratory rate is inhibited by the addition of octyl-gallate, demonstrating that it is due to AOX activity, confirming earlier observations made in nonpurified mitochondria (11,12). Fig.…”

Section: Fig 2 Shows Typical Respiratory Traces and A Western Blotsupporting

confidence: 79%

“…Although both models classify the AOX as a member of the di-iron carboxylate family of proteins, the major difference between them concerns the topology of the enzyme within the inner mitochondrial membrane. In this paper, we have presented data obtained from studies in which an array of sitedirected AOX mutants was expressed in S. pombe, a functional expression system that is highly suited to study AOX activity in situ (11,12,26). Importantly, our results provide the first experimental information to distinguish between the two structural models and, furthermore, provide insights into the possible roles of two highly conserved tyrosine residues.…”

Section: Discussionmentioning

confidence: 96%

“…Gel Electrophoresis and Western Blotting-Separation of proteins on SDS-polyacrylamide gels, transfer to nitrocellulose membranes, and probing with monoclonal antibodies specific for the AOX (AOA antibodies (23)) was carried out as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…In this system, the AOX is functionally targeted to the mitochondria and as such confers antimycin-resistant mitochondrial respiration upon S. pombe cells (11). Using the system, we have previously shown that mutating Glu-270 to alanine abolishes this antimycin-resistant respiratory activity, suggesting that this residue is important for AOX activity (12).…”

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confidence: 99%

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Structure of the Plant Alternative Oxidase

Albury

Affourtit

Crichton

et al. 2002

Journal of Biological Chemistry

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All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool. In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe. Mitochondrial respiratory analysis reveals that S. pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity. Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center. We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively. These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX. The alternative oxidase (AOX)1 is a ubiquinol:oxygen oxidoreductase that catalyzes the four electron reduction of oxygen to water (for recent reviews, see Refs. 1-4). This terminal oxidase branches from the cytochrome pathway at the level of the ubiquinone pool and is non-protonmotive. The AOX is resistant to inhibitors of the cytochrome pathway such as cyanide and antimycin A but inhibited by a number of compounds including hydroxamic acids (e.g. salicylhydroxamic acid) and n-alkyl-gallates.Two structural models of the AOX currently exist. The first model proposed by Siedow et al. (5,6) was based on relatively few AOX sequences and classified the AOX as a member of the di-iron family of proteins that also includes the R2 subunit of ribonucleotide reductase and the hydroxylase component of methane monooxygenase. Based on hydropathy analysis, the AOX was predicted to contain two transmembrane helices that are connected by a helix located in the intermembrane space (6) (Fig. 1A). Since this model was proposed, further AOX sequences were identified, resulting in the proposal by Andersson and Nordlund (7) of an alternative structural model (Fig. 1B). Although this second model also classifies the AOX as a di-iron protein, it differs in the precise ligation sphere of the di-iron center (7). For instance, one of the C-terminal Glu-X-X-His motifs identified by Siedow et al. (5,6), containing Glu-270, appeared not to be fully conserved in the newly identified sequences and consequently seemed unlikely to play a role in ligating iron. Instead, Andersson and Nordlund used a third Glu-X-X-His motif (that contains Glu-217, which is located in the intermembrane space according to the Siedow et al. model) to coordinate the iron atoms. Since such a choice implies that the transmembrane helices can no longer be retained, Andersson and Nordlund (7) proposed that the AOX is an interfacial rather than a transmembrane protein.Recently, the IMMUTANS (...

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Section: Fig 2 Shows Typical Respiratory Traces and A Western Blotsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structure of the Plant Alternative Oxidase

Albury

Affourtit

Crichton

et al. 2002

Journal of Biological Chemistry

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“…Previous work from our laboratory has resulted in the functional expression of the plant AOX in Schizosaccharomyces pombe (28) that, importantly, has allowed site-directed mutagenesis studies into the potential structure of the active site of the oxidase (29). Currently, this system is being exploited to express AOX clones with mutations in amino acid residues that are potentially involved in Q-binding (M.S.…”

Section: Resultsmentioning

confidence: 99%

Maesaquinone: A Novel Inhibitor of Plant Mitochondrial Respiratory Enzymes That React with Ubiquinone

2000

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SummaryThe effect of maesaquinone, 2-(14-nonadecenyl)-3,6-dihydroxy-5-methyl-1,4-benzoquinone, on plant mitochondrial respiration has been investigated. In mitochondria isolated from thermogenic Arum maculatum spadices, this compound inhibits both cytochrome and alternative pathway activities. Kinetic analyses reveal that this inhibition is the result of potent effects of maesaquinone on the alternative oxidase (ID 50 < 0.3 µ M) and complex III (ID 50 < 5 µ M). Succinate dehydrogenase and external NADH dehydrogenase are also inhibited, albeit to a lesser extent (» 30% at 1 µ M). These data suggest that maesaquinone speci cally affects the interaction of the respective enzymes with ubiquinone. IUBMB Life, 49: 533 -537, 2000

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The active site of the plant alternative oxidase: structural and mechanistic considerations

et al. 2000

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In the present review we seek to provide an up‐to‐date view on the molecular nature of the active site of the plant alternative oxidase which has been postulated to comprise of a binuclear iron centre. A three‐dimensional model of the catalytic centre of the oxidase is presented which is based on the active site structure of the free radical component of ribonucleotide reductase and methane monooxygenase. The model indicates that a highly conserved carboxylate (Glu‐270) occupies a central position within the proposed di‐iron centre as it co‐ordinates both iron atoms. The expression of an alternative oxidase protein in Schizosaccharomyces pombe in which Glu‐270 was mutated to asparagine yields an inactive protein. The implications of this in relation to the structural model of the active site of the oxidase suggests that Glu‐270 is essential for catalytic alternative oxidase activity. A kinetic mechanism is suggested which accounts for the full reduction of dioxygen to water catalysed by a single di‐iron centre. © 2000 Society of Chemical Industry

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Targeting the Plant Alternative Oxidase Protein to Mitochondria Confers Cyanide-insensitive Respiration

Cited by 39 publications

References 42 publications

Structure of the Plant Alternative Oxidase

Structure of the Plant Alternative Oxidase

Maesaquinone: A Novel Inhibitor of Plant Mitochondrial Respiratory Enzymes That React with Ubiquinone

The active site of the plant alternative oxidase: structural and mechanistic considerations

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