Mary S. Albury

Function of the alternative oxidase: is it still a scavenger?

The alternative oxidase is a respiratory chain protein found in all higher plants, fungi, non-fermentative yeasts and trypanosomes. Its primary structure suggests that it is a new member of the di-iron carboxylate protein family. Recent sequence analysis indicates an evolutionary relationship between primitive members of this protein family and the alternative oxidase, suggesting that its early function was to scavenge di-oxygen. However, modelling of plant growth kinetics suggests a different function.

Exploring the molecular nature of alternative oxidase regulation and catalysis.

Plant mitochondria contain a non‐protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post‐translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein‐derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.

Further insights into the structure of the alternative oxidase: from plants to parasites

The AOX (alternative oxidase) is a non-protonmotive ubiquinol-oxygen oxidoreductase that couples the oxidation of ubiquinol with the complete reduction of water. Although it has long been recognized that it is ubiquitous among the plant kingdom, it has only recently become apparent that it is also widely found in other organisms including some human parasites. In this paper, we review experimental studies that have contributed to our current understanding of its structure, with particular reference to the catalytic site. Furthermore, we propose a model for the ubiquinol-binding site which identifies a hydrophobic pocket, between helices II and III, leading from a proposed membrane-binding domain to the catalytic domain.

Towards a structural elucidation of the alternative oxidase in plants

In addition to the conventional cytochrome c oxidase, mitochondria of all plants studied to date contain a second cyanide-resistant terminal oxidase or alternative oxidase (AOX). The AOX is located in the inner mitochondrial membrane and branches from the cytochrome pathway at the level of the quinone pool. It is non-protonmotive and couples the oxidation of ubiquinone to the reduction of oxygen to water. For many years, the AOX was considered to be confined to plants, fungi and a small number of protists. Recently, it has become apparent that the AOX occurs in wide range of organisms including prokaryotes and a moderate number of animal species. In this paper, we provide an overview of general features and current knowledge available about the AOX with emphasis on structure, the active site and quinone-binding site. Characterisation of the AOX has advanced considerably over recent years with information emerging about the role of the protein, regulatory regions and functional sites. The large number of sequences available is now enabling us to obtain a clearer picture of evolutionary origins and diversity.

Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues in addition to conserved cysteines in α‐keto acid activation

Activity of the plant mitochondrial alternative oxidase (AOX) can be regulated by organic acids, notably pyruvate. To date, only two well‐conserved cysteine residues have been implicated in this process. We report the functional expression of two AOX isozymes (Sauromatum guttatum Sg‐AOX and Arabidopsis thaliana At‐AOX1a) in Schizosaccharomyces pombe. Comparison of the response of these two isozymes to pyruvate in isolated yeast mitochondria and disrupted mitochondrial membranes reveals that in contrast to At‐AOX1a, Sg‐AOX activity is insensitive to pyruvate and appears to be in a constitutively active state. As both of these isozymes conserve the two cysteines, we propose that such contrasting behaviour must be a direct result of differences in their amino acid sequence and have subsequently identified novel candidate residues.

A Highly Conserved Glutamate Residue (Glu-270) Is Essential for Plant Alternative Oxidase Activity*

We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase inSchizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062–17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed byS. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.

Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein.

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.

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

The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter. Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S. pombe, which is sensitive to inhibition by octyl-gallate. Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria. Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form. Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity. The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein. To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S. pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity.

Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe.

We have investigated the extent to which functional expression of the plant alternative oxidase (fromSauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h−1 while growth yield is lowered by 20% (from 1.14 × 108 to 9.12 × 107 cells ml−1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.

Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis.

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. To investigate the role of highly conserved amino acid residues in catalysis we have expressed site-directed mutants of Cys-172, Thr-179, Trp-206, Tyr-253, and Tyr-299 in AOX in the yeast Schizosaccharomyces pombe. Assessment of AOX activity in isolated yeast mitochondria reveals that mutagenesis of Trp-206 to phenylalanine or tyrosine abolishes activity, in contrast to that observed with either Tyr-253 or 299 both mutants of which retained activity. None of the mutants exhibited sensitivity to Q-like inhibitors that differed significantly from the wild type AOX. Interestingly, however, mutagenesis of Thr-179 or Cys-172 (a residue implicated in AOX regulation by alpha-keto acids) to alanine not only resulted in a decrease of maximum AOX activity but also caused a significant increase in the enzymes affinity for oxygen (4- and 2-fold, respectively). These results provide important new insights in the mechanism of AOX catalysis and regulation by pyruvate.