The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
James N. Siedow - One of the best experts on this subject based on the ideXlab platform.
-
The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco
Plant physiology, 1997Co-Authors: Adrian M. Lennon, Urs Neuenschwander, Miquel Ribas-carbo, Larry Giles, John Ryals, James N. SiedowAbstract:Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the Alternative Oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of Alternative Oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and Alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative Oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the Alternative Oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of Alternative Oxidase protein, suggesting that steady-state Alternative Oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in Alternative Oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the Alternative pathway in virus-infected plants was comparable to that in uninfected controls.
-
Changes in the redox state of the Alternative Oxidase regulatory sulfhydryl/disulfide system during mitochondrial isolation: implications for inferences of activity in vivo
Plant Science, 1997Co-Authors: Ann L. Umbach, James N. SiedowAbstract:Abstract The cyanide-resistant Alternative Oxidase of plants exists as a homodimer in the inner mitochondrial membrane. When the subunits of the dimer are connected by an intermolecular disulfide bond, the enzyme is relatively inactive. When this bond is reduced to its constituent sulfhydryls, the enzyme becomes more active and can be further activated by α-keto acids. We have attempted to correlate the proportions of oxidized and reduced Alternative Oxidase with the physiological state of the plant by using immunoblots to visualize the ratio of Alternative Oxidase species present. However, during the process of mitochondrial isolation from Sauromatum guttatum and Glycine max, the Alternative Oxidase underwent oxidation of the intermolecular sulfhydryl/disulfide system with the result that the proportion of oxidized and reduced Alternative Oxidase species in isolated mitochondria differed from that of the starting plant material. The presence of the sulfhydryl reagents, N-ethylmaleimide and iodoacetate, during mitochondrial isolation prevented Alternative Oxidase sulfhydryl/disulfide oxidation, but also led to reduction of the oxidized protein species. Thus, it appears not possible to determine the redox state of the Alternative Oxidase sulfhydryl/disulfide system in vivo. Further, due to the spontaneous formation of the oxidized Alternative Oxidase species during isolation, Alternative Oxidase activity will be less in isolated mitochondria than in the plant tissue from which the mitochondria were derived.
-
A structural model of the Alternative Oxidase of plant mitochondria.
Biochemical Society transactions, 1995Co-Authors: Anthony L. Moore, Ann L. Umbach, James N. SiedowAbstract:The Alternative Oxidase branches from the main mitochondrial respiratory chain at the level of the ubiquinone pool, it i s non-protonmotive and an integral membrane protein with its active site located on the matrix side of the inner membrane [ I]. Activity of the Alternative Oxidase strongly correlates with the appearance of three proteins with Mr of 35, 36 and 37 k D in thermogenic tissues and 35 kD in non-thermogenic tissues [2]. Monoclonal antibodies cross-react not only with proteins from aroid (thermogenic) species but also with proteins from widely divergent plant species such as tobacco, pea and potato in addition to fungi and trypanosomes suggesting that not only is cyanide-resistance widespread amongst the plant kingdom but also highly conserved [ I ] . The cDNA encoding the precursor of the Alternative Oxidase protein has been isolated and characterised from a number of sources including Sauromatum , Arabidopsis , soybean, tobacco, mango and the yeast Hansenula anomala [see 21. Expression of the Arabidopsis Alternative Oxidase cDNA in E. coli (haem A deficient) is sufficient to support growth in the presence of cyanide [3]. Amino-acid sequence comparison reveals a high degree of homology amongst all species particularly within the two putative membranespanning helical regions and the C-terminal region. Among the plant sequences, only two of the three Cys residues are conserved and comparison with the yeast sequence reveals that only one of these residues is likely to be important for the functioning of the Oxidase [4]. Umbach and Siedow found that the Alternative Oxidase protein exists as a dimer in the mitochondrial membrane which call occur in a disulphide-linked, less active state or in a more active state when the disulphide bond has been reduced. It has been suggested that the reversible nature of the intermolecular disulphide linkage may provide another mechanism, in addition to that observed with organic acids, for regulating enzyme activity in vivo [5]. The ability of the Alternative Oxidase to reduce oxygen to water suggests that the active site of the Oxidase should contain a coupled transition metal center [I]. Previous metal analyses of partially purified Alternative Oxidase preparations have been inconclusive; Fe, Cu and Mn have each been reported in varying amounts. Attempts to characterise the Alternative Oxidase using spectroscopic features have also similarly met with little success [I]. Extensive analyses of isolated plant mitochondria poised in a variety of redox states using electron paramagnetic resonance (EPR) spectroscopy displayed neither any resonances unique to plant mitochondria nor ones whose behaviour were indicative of a specific association with the Alternative pathway. A partially purified Alternative Oxidase preparation from Symplocarpus foeridus spadices also showed no indication of EPR resonances in either the resting (oxidised) or the dithionite-reduced states . Interestingly, the Symplocarpus preparation also had no optical absorbance above 350 nm (Umbach, A.L. and Siedow, J.N., unpublished results), which is unusual for a protein that presumably contains a metal centre. The lack of any optical absorption above 350 nm in Alternative Oxidase preparations plus the capability to reduce 0 2 to H 2 0 is reminiscent of the properties of soluble methane monooxygcnase [6]. Soluble methane mono-oxygenase is a member of the Fe-0-Fe class of metalloproteins [6], the active site structure of which contains a single 0x0-bridged binuclear iron centre with structural features comparable to that found in hemerythrin and thc R 2 subunit of ribonucleotide reductase. In all three of these enzymes. the oxidiscd proteins contain antiferromagnetically coupled high-spin Fe(II1) atoms. In the fully reduced state, the two iron atoms are in the Fe(I1) redox state and remain coupled. As a result, these binuclear iron proteins show no standard EPR signal in either the oxidised or fully reduced states. The three-dimensional structures of three 0x0-bridged binuclear iron proteins (hemerythrin, the R2 subunit of ribonucleotide reductase and the soluble subunit of methane mono-oxygenase) are currently known [see 61, and they show a common set of structural features. While the exact liganding side chains vary among the different proteins, in every case the iron centre is buried within a scaffold that consists of four long (30-35 residues) a-helices organised as a four-helix bundle. With methane mono-oxygenase and ribonucleotide reductase, two of the four helices in the bundle contain the sequence Glu-X-X-His, which is a characteristic of 0x0-bridged diiron proteins [6]. Each of the histidines and one of the two carboxylates serve as monodentate ligands to the iron atoms in the cluster, while the second carboxylate acts as a hidentate ligand, bridging the two iron atoms. The two remaining protein ligands to the iron atoms are provided by carboxylate residues, one in each of the additional two helices of the four-helical bundle. The liganding histidines are further stabilized within the overall structure by hydrogen bonding to the side chain of the residue immediately Nterminal to the glutamate residue within the opposing Glu-X-XHis sequence. An additional feature of the methane monooxygenase and ribonucleotide reductase active sites is the presence of a hydrophobic cavity formed adjacent to the iron center by a series of conserved residues within the four-helix bundle Analysis of the known Alternative Oxidase proteins indicated that the published higher plant sequences contain two conserved sequences, Glu-GluA-I-His and Asp-Glu-A-H-His, in the carboxy-terminal hydrophilic domain of the protein, beginning just beyond the second membrane-spanning helical region. Both sets of sequences appear in regions showing a high probability for formation of a helices. These two motifs correspond to those found in the 0x0-bridged binuclear iron cluster of methane mono-oxygenase, with the single conservative substitution of a glutamate for an aspartate residue ncxt to the liganding glutamate in the more N-terminal of the two sequences, Using the Sauromutum amino acid sequence, and starting at Glu268, it i s possible to generate a model containing four successive helices of 10 to 11 residues each. In this scheme, the two Glu-XX-His iron binding motifs are located on helices 1 and 4, which are oriented in anti-paralleled fashion, analogous to helices C and F in methane mono-oxygenase. The second helix in this series contains a conserved carboxylate residue (Asp-28 1 in Sauromatum but Glu in most other plants), oriented toward the amino terminus of the helix which would position it to act as a bidentate ligand to iron atom Fel (analogous to Glu-I14 in helix B of methane mono-oxygenase). Missing from this model is a conserved aspartate or glutamate residue in helix 3 that could serve as the analogue to Glu-209 in helix E of methane monooxygenaw and, hence, the final carboxylate ligand to iron atom Fe2. There no obvious Alternative conserved residue in helix 3 that might serve in this capacity. However, in ribonucleotide reductase, Asp-84 in helix B, which is the analogue to Glu-I 14 in methane monooxygenase, acts as a bidentate ligand to Fel. If Glu-319 in helix 4 were to doubly co-ordinate to iron atom Fe2 in the Alternative Oxidase active site in a fashion analogous to that of Asp-84 in ribonucleotide reductase, that would eliminate the need for the additional carboxylate ligand. In conclusion, by identifying conserved residues in the plant Alternative Oxidase amino acid sequence, it is possible to construct a structural model incorporating an 0x0-bridged dinuclear iron centre held within a four helix bundle, analogous to the iron centre seen within methane mono-oxygenase and ribonucleotide reductase 1. Moore, A.L. & Siedow, J. N. (1991) Biochim. Biophys. Acta
-
Regulation of the Alternative Oxidase in Plants and Fungi.
Functional Plant Biology, 1995Co-Authors: David A. Day, James N. Siedow, James Whelan, A. H. Millar, Joseph T. WiskichAbstract:The Alternative Oxidase of the inner mitochondria1 membrane catalyses cyanide-insensitive respiration in plants and fungi. The molecular biology and regulation of this Oxidase have been intensively studied over the past 10 years. Genes have been isolated, expression has been investigated and novel mechanisms for the regulation of activity have been discovered. This paper reviews these recent advances, focusing on the regulation of gene expression and activation by protein modification and organic acids, and possible roles of the Alternative Oxidase are discussed.
-
Structure-function relationships of the Alternative Oxidase of plant mitochondria: A model of the active site
Journal of bioenergetics and biomembranes, 1995Co-Authors: Anthony L. Moore, Ann L. Umbach, James N. SiedowAbstract:A major characteristic of plant mitochondria is the presence of a cyanide-insensitive Alternative Oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the Oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the Alternative Oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the Alternative Oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the Alternative Oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.
David A. Day - One of the best experts on this subject based on the ideXlab platform.
-
Alternative Oxidase is positive for plant performance
Trends in Plant Science, 2018Co-Authors: Jennifer Selinski, David A. Day, Renate Scheibe, James WhelanAbstract:The Alternative pathway of mitochondrial electron transport, which terminates in the Alternative Oxidase (AOX), uncouples oxidation of substrate from mitochondrial ATP production, yet plant performance is improved under adverse growth conditions. AOX is regulated at different levels. Identification of regulatory transcription factors shows that Arabidopsis thaliana AOX1a is under strong transcriptional suppression. At the protein level, the primary structure is not optimised for activity. Maximal activity requires the presence of various metabolites, such as tricarboxylic acid-cycle intermediates that act in an isoform-specific manner. In this opinion article we propose that the regulatory mechanisms that keep AOX activity suppressed, at both the gene and protein level, are positive for plant performance due to the flexible short- and long-term fine-tuning.
-
molecular distinction between Alternative Oxidase from monocots and dicots
Plant Physiology, 2002Co-Authors: Michael J Considine, David A. Day, James Whelan, Ruth Holtzapffel, Harvey A MillarAbstract:The Alternative Oxidase (Aox) is encoded in two discrete gene subfamilies in higher plants. Aox1 is most widely known for its induction by stress stimuli in many tissues and is present in both monocot and eudicot plant species. Aox2, on the other hand, is usually constitutive or developmentally
-
An Alternative Oxidase monoclonal antibody recognises a highly conserved sequence among Alternative Oxidase subunits
FEBS letters, 1999Co-Authors: Patrick M. Finnegan, Anthea R. Wooding, David A. DayAbstract:The Alternative Oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the Alternative Oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean Alternative Oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the Alternative Oxidase. The recognition sequence is highly conserved among all Alternative Oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the Alternative Oxidase.
-
The multiple Alternative Oxidase proteins of soybean
Functional Plant Biology, 1999Co-Authors: Marcel Tanudji, Patrick M. Finnegan, David A. Day, Ira Djajanegara, Daniel O. Daley, T.c. Mccabe, James WhelanAbstract:The identity of the multiple Alternative Oxidase bands detected in various soybean tissues was investigated to determine if any modification that can alter the mobility on SDS-PAGE of the Alternative Oxidase occurs after mitochondrial import other than removal of the presequence. Comparison of the mature, in vitro imported products of AOX1, AOX2 and AOX3 in soybean cotyledons and rat liver mitochondria indicated that they had an identical apparent molecular mass to their in vitro expressed mature forms. This suggests that no modification specific to plant Alternative Oxidase altering the mobility on SDS-PAGE, took place. Changing the –2 and/or –3 Arg residue resulted in the inhibition of the generation of this mature form, suggesting that processing was most likely by the general mitochondrial processing peptidase. Comparison of the in vitro expressed mature forms to that detected by immunoblots of soybean tissues, required the induction of AOX1. Treatment of soybean cultured cells with antimycin A resulted in the induction of an additional band cross-reacting to monoclonal antibodies against the Alternative Oxidase. Comparison of the in vitro expressed mature forms to the Alternative Oxidase detected by western blotting indicated that they were identical in apparent molecular mass. These results indicated that no modification other than presequence removal, which alters mobility on SDS-PAGE, was required to generate the mature functional Alternative Oxidase proteins.
-
Nitric oxide inhibits the cytochrome Oxidase but not the Alternative Oxidase of plant mitochondria
FEBS Letters, 1996Co-Authors: A. Harvey Millar, David A. DayAbstract:Abstract Oxygen consumption via the cytochrome pathway in isolated soybean ( Glycine max [L.] Merr.) cotyledon mitochondria was inhibited by nitric oxide (NO) while respiration via the cyanide-insensitive Alternative Oxidase was not significantly affected. Inhibition of cytochrome pathway activity was rapidly reversible upon depletion of the added NO. NO production was also detected in solutions of NaNO 2 plus ascorbate and the extent of cytochrome pathway inhibition was dependent on the NO 2 − concentration. Little inhibition of Alternative pathway respiration was observed under similar conditions. The Alternative Oxidase may play a role in nitric oxide tolerance in higher plants and in organisms such as trypanosomes which contain a plant-like Alternative Oxidase.
James Whelan - One of the best experts on this subject based on the ideXlab platform.
-
Alternative Oxidase is positive for plant performance
Trends in Plant Science, 2018Co-Authors: Jennifer Selinski, David A. Day, Renate Scheibe, James WhelanAbstract:The Alternative pathway of mitochondrial electron transport, which terminates in the Alternative Oxidase (AOX), uncouples oxidation of substrate from mitochondrial ATP production, yet plant performance is improved under adverse growth conditions. AOX is regulated at different levels. Identification of regulatory transcription factors shows that Arabidopsis thaliana AOX1a is under strong transcriptional suppression. At the protein level, the primary structure is not optimised for activity. Maximal activity requires the presence of various metabolites, such as tricarboxylic acid-cycle intermediates that act in an isoform-specific manner. In this opinion article we propose that the regulatory mechanisms that keep AOX activity suppressed, at both the gene and protein level, are positive for plant performance due to the flexible short- and long-term fine-tuning.
-
molecular distinction between Alternative Oxidase from monocots and dicots
Plant Physiology, 2002Co-Authors: Michael J Considine, David A. Day, James Whelan, Ruth Holtzapffel, Harvey A MillarAbstract:The Alternative Oxidase (Aox) is encoded in two discrete gene subfamilies in higher plants. Aox1 is most widely known for its induction by stress stimuli in many tissues and is present in both monocot and eudicot plant species. Aox2, on the other hand, is usually constitutive or developmentally
-
Signals Required for the Import and Processing of the Alternative Oxidase into Mitochondria
The Journal of biological chemistry, 1999Co-Authors: Marcel Tanudji, Elzbieta Glaser, Sara Sjöling, James WhelanAbstract:The critical residues involved in targeting and processing of the soybean Alternative Oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean ...
-
The multiple Alternative Oxidase proteins of soybean
Functional Plant Biology, 1999Co-Authors: Marcel Tanudji, Patrick M. Finnegan, David A. Day, Ira Djajanegara, Daniel O. Daley, T.c. Mccabe, James WhelanAbstract:The identity of the multiple Alternative Oxidase bands detected in various soybean tissues was investigated to determine if any modification that can alter the mobility on SDS-PAGE of the Alternative Oxidase occurs after mitochondrial import other than removal of the presequence. Comparison of the mature, in vitro imported products of AOX1, AOX2 and AOX3 in soybean cotyledons and rat liver mitochondria indicated that they had an identical apparent molecular mass to their in vitro expressed mature forms. This suggests that no modification specific to plant Alternative Oxidase altering the mobility on SDS-PAGE, took place. Changing the –2 and/or –3 Arg residue resulted in the inhibition of the generation of this mature form, suggesting that processing was most likely by the general mitochondrial processing peptidase. Comparison of the in vitro expressed mature forms to that detected by immunoblots of soybean tissues, required the induction of AOX1. Treatment of soybean cultured cells with antimycin A resulted in the induction of an additional band cross-reacting to monoclonal antibodies against the Alternative Oxidase. Comparison of the in vitro expressed mature forms to the Alternative Oxidase detected by western blotting indicated that they were identical in apparent molecular mass. These results indicated that no modification other than presequence removal, which alters mobility on SDS-PAGE, was required to generate the mature functional Alternative Oxidase proteins.
-
Cloning of an additional cDNA for the Alternative Oxidase in tobacco
Plant physiology, 1995Co-Authors: James Whelan, M.k. Smith, Marcel Meijer, Murray R. Badger, G. D. Price, David A. DayAbstract:The Alternative Oxidase is a cyanide-insensitive terminal Oxidase found in a variety of organisms although it is best characterized in plants. It branches from the Cyt chain at the level of ubiquinone and does not pump protons and is thus non-energy-conserving. In thermogenic floral appendages it plays a clear role in the volatilization of compounds to attract insects for pollination, but its role in nonthermogenic plants is unclear (Moore and Siedow, 1991). Induction of the Alternative Oxidase at the gene level has been characterized in a number of studies with compounds that inhibit the Cyt chain. Additionally, aging of potato slices, treatment with ethylene in fruits and storage tissue, cold treatment in tobacco (Nicofiana fobacum) and wheat, and salicylic acid treatment of Sauromatum gut ta tum have a11 been shown to induce the Alternative Oxidase (Day et al., 1995). At the biochemical level allosteric stimulation by pyruvate and the oxidation reduction state of the protein have been shown to be important determinants of activity (Millar et al., 1993; Umbach and Siedow, 1993). We have isolated and sequenced a cDNA clone from tobacco for the Alternative Oxidase. The predicted protein shows high identity with Alternative Oxidase from other species (Table I). However, when it was compared with the recently published sequence from tobacco (Vanlerberghe and McIntosh 1994), it showed significant differences, having 93 and 95% identity at the nucleic acid and protein levels, respectively (Vanlerberghe and McIntosh, 1994). The gene sequenced in this study is 209 bp shorter at the 5‘ end compared to that of Vanlerberghe and McIntosh (1994); additionally, there are 47 base pair differences in the open reading frame, which translate into 14 differences in amino acids. At the 3’ end there are 13 base pairs different between the two clones. The differences between these two tobacco clones may be accounted for by varietal differences between the two studies, since we used cv SRl versus Bright Yellow, which was used by Vanlerberghe and McIntosh (1994). Alternatively, the differences in sequence may indicate a second gene for the Alternative Oxidase in tobacco. This possibility is supported by the following: (a) N. tabacum
Anthony L. Moore - One of the best experts on this subject based on the ideXlab platform.
-
Data_Sheet_1_The Human Gut Colonizer Blastocystis Respires Using Complex II and Alternative Oxidase to Buffer Transient Oxygen Fluctuations in the Gut.PDF
2018Co-Authors: Anastasios D. Tsaousis, Anthony L. Moore, Karleigh A. Hamblin, Catherine R. Elliott, Luke Young, Alicia Rosell-hidalgo, Campbell W. Gourlay, Mark Van Der GiezenAbstract:Blastocystis is the most common eukaryotic microbe in the human gut. It is linked to irritable bowel syndrome (IBS), but its role in disease has been contested considering its widespread nature. This organism is well-adapted to its anoxic niche and lacks typical eukaryotic features, such as a cytochrome-driven mitochondrial electron transport. Although generally considered a strict or obligate anaerobe, its genome encodes an Alternative Oxidase. Alternative Oxidases are energetically wasteful enzymes as they are non-protonmotive and energy is liberated in heat, but they are considered to be involved in oxidative stress protective mechanisms. Our results demonstrate that the Blastocystis cells themselves respire oxygen via this Alternative Oxidase thereby casting doubt on its strict anaerobic nature. Inhibition experiments using Alternative Oxidase and Complex II specific inhibitors clearly demonstrate their role in cellular respiration. We postulate that the Alternative Oxidase in Blastocystis is used to buffer transient oxygen fluctuations in the gut and that it likely is a common colonizer of the human gut and not causally involved in IBS. Additionally the Alternative Oxidase could act as a protective mechanism in a dysbiotic gut and thereby explain the absence of Blastocystis in established IBS environments.
-
Unraveling the heater: new insights into the structure of the Alternative Oxidase.
Annual review of plant biology, 2013Co-Authors: Anthony L. Moore, Kiyoshi Kita, Luke Young, Tomoo Shiba, Shigeharu Harada, Kikukatsu ItoAbstract:The Alternative Oxidase is a membrane-bound ubiquinol Oxidase found in the majority of plants as well as many fungi and protists, including pathogenic organisms such as Trypanosoma brucei. It catalyzes a cyanide- and antimycin-A-resistant oxidation of ubiquinol and the reduction of oxygen to water, short-circuiting the mitochondrial electron-transport chain prior to proton translocation by complexes III and IV, thereby dramatically reducing ATP formation. In plants, it plays a key role in cellular metabolism, thermogenesis, and energy homeostasis and is generally considered to be a major stress-induced protein. We describe recent advances in our understanding of this protein's structure following the recent successful crystallization of the Alternative Oxidase from T. brucei. We focus on the nature of the active site and ubiquinol-binding channels and propose a mechanism for the reduction of oxygen to water based on these structural insights. We also consider the regulation of activity at the posttranslat...
-
Hypothetical scheme of function of the Alternative Oxidase in the microsporidian cell.
2013Co-Authors: Bryony A. P. Williams, Lena Burri, Catherine Elliot, Yasutoshi Kido, Kiyoshi Kita, Anthony L. Moore, Patrick J. KeelingAbstract:Microsporidian cells are known to contain glycolytic enzymes, though no obvious mechanism exists for reoxidising NADH to NAD+. The glycerol-3-phosphate shuttle is encoded in many microsporidian genomes. If this shuttle is coupled to an Alternative Oxidase protein in the mitosome, it could potentially represent a mechanism for regenerating NAD+.
-
Compelling EPR evidence that the Alternative Oxidase is a diiron carboxylate protein
Biochimica et biophysica acta, 2008Co-Authors: Anthony L. Moore, Kiyoshi Kita, Mary S. Albury, Paul G. Crichton, Charles Affourtit, Jane E. Carré, Peter HeathcoteAbstract:The Alternative Oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g = 16 in both purified Alternative Oxidase protein (g = 16.9), isolated mitochondrial membranes (g = 16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g = 16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.
-
The over-expression, purification and crystallisation of the Alternative Oxidase
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008Co-Authors: Catherine Elliott, Mary S. Albury, Momi Iwata, Anthony L. MooreAbstract:The Alternative Oxidase (AOX) is an integral monotopic membrane protein which branches from respiratory chain at the point of the Q-pool in the mitochondria of all flowers, some fungi, and some protists such as the human parasite Trypanosoma brucei. The aim of this project is threefold: to establish an over-expression and purification protocol for recombinant Sauromatum guttatum Alternative Oxidase (SgrAOX); to use expressed SgrAOX for structural analysis such as crystallography; and finally to use in silico methods to model the Alternative Oxidase protein. Of these three, only the first and last have been attempted previously, with varying success. The second, namely structural analysis, has never been attempted with SgrAOX. In order to achieve the aims of this project, primarily laboratory-based protein production were used, in conjunction with downstream analysis using structural biology techniques. The in silico modelling was carried out using a wide range of algorithms freely available on the World Wide Web. Results of this project are: the determination of an over-expression system and purification protocols in two E.coli strains, producing enough protein to use for the second objective detailed above. While no crystal structure has been obtained, significant steps toward identifying a protocol for rAOX crystallisation have been made. Results from structural analysis support modelling predictions and give novel insights into the thermostability of the protein. New and detailed homology models have been created and critically evaluated, with a very recent crystal structure from our collaborators providing a unique set of data for model evaluation. The outcome of this project has contributed towards the determination of conditions under which SgrAOX protein may form crystals, and therefore bringing the acquisition of a SgrAOX protein structure closer.
Ann L. Umbach - One of the best experts on this subject based on the ideXlab platform.
-
Prokaryotic origins for the mitochondrial Alternative Oxidase and plastid terminal Oxidase nuclear genes
FEBS letters, 2003Co-Authors: Patrick M. Finnegan, Ann L. Umbach, Jacqueline A. WilceAbstract:The mitochondrial Alternative Oxidase is a diiron carboxylate quinol Oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal Oxidase is distantly related to Alternative Oxidase and is most likely also a Dox protein. Database searches revealed that the α-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.
-
Changes in the redox state of the Alternative Oxidase regulatory sulfhydryl/disulfide system during mitochondrial isolation: implications for inferences of activity in vivo
Plant Science, 1997Co-Authors: Ann L. Umbach, James N. SiedowAbstract:Abstract The cyanide-resistant Alternative Oxidase of plants exists as a homodimer in the inner mitochondrial membrane. When the subunits of the dimer are connected by an intermolecular disulfide bond, the enzyme is relatively inactive. When this bond is reduced to its constituent sulfhydryls, the enzyme becomes more active and can be further activated by α-keto acids. We have attempted to correlate the proportions of oxidized and reduced Alternative Oxidase with the physiological state of the plant by using immunoblots to visualize the ratio of Alternative Oxidase species present. However, during the process of mitochondrial isolation from Sauromatum guttatum and Glycine max, the Alternative Oxidase underwent oxidation of the intermolecular sulfhydryl/disulfide system with the result that the proportion of oxidized and reduced Alternative Oxidase species in isolated mitochondria differed from that of the starting plant material. The presence of the sulfhydryl reagents, N-ethylmaleimide and iodoacetate, during mitochondrial isolation prevented Alternative Oxidase sulfhydryl/disulfide oxidation, but also led to reduction of the oxidized protein species. Thus, it appears not possible to determine the redox state of the Alternative Oxidase sulfhydryl/disulfide system in vivo. Further, due to the spontaneous formation of the oxidized Alternative Oxidase species during isolation, Alternative Oxidase activity will be less in isolated mitochondria than in the plant tissue from which the mitochondria were derived.
-
A structural model of the Alternative Oxidase of plant mitochondria.
Biochemical Society transactions, 1995Co-Authors: Anthony L. Moore, Ann L. Umbach, James N. SiedowAbstract:The Alternative Oxidase branches from the main mitochondrial respiratory chain at the level of the ubiquinone pool, it i s non-protonmotive and an integral membrane protein with its active site located on the matrix side of the inner membrane [ I]. Activity of the Alternative Oxidase strongly correlates with the appearance of three proteins with Mr of 35, 36 and 37 k D in thermogenic tissues and 35 kD in non-thermogenic tissues [2]. Monoclonal antibodies cross-react not only with proteins from aroid (thermogenic) species but also with proteins from widely divergent plant species such as tobacco, pea and potato in addition to fungi and trypanosomes suggesting that not only is cyanide-resistance widespread amongst the plant kingdom but also highly conserved [ I ] . The cDNA encoding the precursor of the Alternative Oxidase protein has been isolated and characterised from a number of sources including Sauromatum , Arabidopsis , soybean, tobacco, mango and the yeast Hansenula anomala [see 21. Expression of the Arabidopsis Alternative Oxidase cDNA in E. coli (haem A deficient) is sufficient to support growth in the presence of cyanide [3]. Amino-acid sequence comparison reveals a high degree of homology amongst all species particularly within the two putative membranespanning helical regions and the C-terminal region. Among the plant sequences, only two of the three Cys residues are conserved and comparison with the yeast sequence reveals that only one of these residues is likely to be important for the functioning of the Oxidase [4]. Umbach and Siedow found that the Alternative Oxidase protein exists as a dimer in the mitochondrial membrane which call occur in a disulphide-linked, less active state or in a more active state when the disulphide bond has been reduced. It has been suggested that the reversible nature of the intermolecular disulphide linkage may provide another mechanism, in addition to that observed with organic acids, for regulating enzyme activity in vivo [5]. The ability of the Alternative Oxidase to reduce oxygen to water suggests that the active site of the Oxidase should contain a coupled transition metal center [I]. Previous metal analyses of partially purified Alternative Oxidase preparations have been inconclusive; Fe, Cu and Mn have each been reported in varying amounts. Attempts to characterise the Alternative Oxidase using spectroscopic features have also similarly met with little success [I]. Extensive analyses of isolated plant mitochondria poised in a variety of redox states using electron paramagnetic resonance (EPR) spectroscopy displayed neither any resonances unique to plant mitochondria nor ones whose behaviour were indicative of a specific association with the Alternative pathway. A partially purified Alternative Oxidase preparation from Symplocarpus foeridus spadices also showed no indication of EPR resonances in either the resting (oxidised) or the dithionite-reduced states . Interestingly, the Symplocarpus preparation also had no optical absorbance above 350 nm (Umbach, A.L. and Siedow, J.N., unpublished results), which is unusual for a protein that presumably contains a metal centre. The lack of any optical absorption above 350 nm in Alternative Oxidase preparations plus the capability to reduce 0 2 to H 2 0 is reminiscent of the properties of soluble methane monooxygcnase [6]. Soluble methane mono-oxygenase is a member of the Fe-0-Fe class of metalloproteins [6], the active site structure of which contains a single 0x0-bridged binuclear iron centre with structural features comparable to that found in hemerythrin and thc R 2 subunit of ribonucleotide reductase. In all three of these enzymes. the oxidiscd proteins contain antiferromagnetically coupled high-spin Fe(II1) atoms. In the fully reduced state, the two iron atoms are in the Fe(I1) redox state and remain coupled. As a result, these binuclear iron proteins show no standard EPR signal in either the oxidised or fully reduced states. The three-dimensional structures of three 0x0-bridged binuclear iron proteins (hemerythrin, the R2 subunit of ribonucleotide reductase and the soluble subunit of methane mono-oxygenase) are currently known [see 61, and they show a common set of structural features. While the exact liganding side chains vary among the different proteins, in every case the iron centre is buried within a scaffold that consists of four long (30-35 residues) a-helices organised as a four-helix bundle. With methane mono-oxygenase and ribonucleotide reductase, two of the four helices in the bundle contain the sequence Glu-X-X-His, which is a characteristic of 0x0-bridged diiron proteins [6]. Each of the histidines and one of the two carboxylates serve as monodentate ligands to the iron atoms in the cluster, while the second carboxylate acts as a hidentate ligand, bridging the two iron atoms. The two remaining protein ligands to the iron atoms are provided by carboxylate residues, one in each of the additional two helices of the four-helical bundle. The liganding histidines are further stabilized within the overall structure by hydrogen bonding to the side chain of the residue immediately Nterminal to the glutamate residue within the opposing Glu-X-XHis sequence. An additional feature of the methane monooxygenase and ribonucleotide reductase active sites is the presence of a hydrophobic cavity formed adjacent to the iron center by a series of conserved residues within the four-helix bundle Analysis of the known Alternative Oxidase proteins indicated that the published higher plant sequences contain two conserved sequences, Glu-GluA-I-His and Asp-Glu-A-H-His, in the carboxy-terminal hydrophilic domain of the protein, beginning just beyond the second membrane-spanning helical region. Both sets of sequences appear in regions showing a high probability for formation of a helices. These two motifs correspond to those found in the 0x0-bridged binuclear iron cluster of methane mono-oxygenase, with the single conservative substitution of a glutamate for an aspartate residue ncxt to the liganding glutamate in the more N-terminal of the two sequences, Using the Sauromutum amino acid sequence, and starting at Glu268, it i s possible to generate a model containing four successive helices of 10 to 11 residues each. In this scheme, the two Glu-XX-His iron binding motifs are located on helices 1 and 4, which are oriented in anti-paralleled fashion, analogous to helices C and F in methane mono-oxygenase. The second helix in this series contains a conserved carboxylate residue (Asp-28 1 in Sauromatum but Glu in most other plants), oriented toward the amino terminus of the helix which would position it to act as a bidentate ligand to iron atom Fel (analogous to Glu-I14 in helix B of methane mono-oxygenase). Missing from this model is a conserved aspartate or glutamate residue in helix 3 that could serve as the analogue to Glu-209 in helix E of methane monooxygenaw and, hence, the final carboxylate ligand to iron atom Fe2. There no obvious Alternative conserved residue in helix 3 that might serve in this capacity. However, in ribonucleotide reductase, Asp-84 in helix B, which is the analogue to Glu-I 14 in methane monooxygenase, acts as a bidentate ligand to Fel. If Glu-319 in helix 4 were to doubly co-ordinate to iron atom Fe2 in the Alternative Oxidase active site in a fashion analogous to that of Asp-84 in ribonucleotide reductase, that would eliminate the need for the additional carboxylate ligand. In conclusion, by identifying conserved residues in the plant Alternative Oxidase amino acid sequence, it is possible to construct a structural model incorporating an 0x0-bridged dinuclear iron centre held within a four helix bundle, analogous to the iron centre seen within methane mono-oxygenase and ribonucleotide reductase 1. Moore, A.L. & Siedow, J. N. (1991) Biochim. Biophys. Acta
-
Structure-function relationships of the Alternative Oxidase of plant mitochondria: A model of the active site
Journal of bioenergetics and biomembranes, 1995Co-Authors: Anthony L. Moore, Ann L. Umbach, James N. SiedowAbstract:A major characteristic of plant mitochondria is the presence of a cyanide-insensitive Alternative Oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the Oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the Alternative Oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the Alternative Oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the Alternative Oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.
-
regulation of Alternative Oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria
FEBS Letters, 1994Co-Authors: Ann L. Umbach, Joseph T. Wiskich, James N. SiedowAbstract:Two factors known to regulate plant mitochondrial cyanide-resistant Alternative Oxidase activity, pyruvate and the redox status of the enzyme's intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated Alternative Oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced Alternative Oxidase protein, a wide range of enzyme activity is possible in vivo.