Luke Young

Unraveling the Heater: New Insights into the Structure of the Alternative Oxidase

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 proteins 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 posttranslational and retrograde levels and highlight challenges for future research.

The alternative oxidases: simple oxidoreductase proteins with complex functions

The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water.

Structural insights into the alternative oxidases: are all oxidases made equal?

The alternative oxidases (AOXs) are ubiquinol-oxidoreductases that are members of the diiron carboxylate superfamily. They are not only ubiquitously distributed within the plant kingdom but also found in increasing numbers within the fungal, protist, animal and prokaryotic kingdoms. Although functions of AOXs are highly diverse in general, they tend to play key roles in thermogenesis, stress tolerance (through the management of radical oxygen species) and the maintenance of mitochondrial and cellular energy homeostasis. The best structurally characterised AOX is from Trypanosoma brucei In this review, we compare the structure of AOXs, created using homology modelling, from many important species in an attempt to explain differences in activity and sensitivity to AOX inhibitors. We discuss the implications of these findings not only for future structure-based drug design but also for the design of novel AOXs for gene therapy.

Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOXs catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Structure and Mechanism of Action of the Alternative Quinol Oxidases

In addition to heme-copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide- and antimycin-insensitive alternative oxidase (AOX). It is a di-iron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol, 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. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, until recently structural features of any AOX were unknown. In this review 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 describe a mechanism for the reduction of oxygen to water based on these structural insights.

Purification and characterisation of recombinant DNA encoding the alternative oxidase from Sauromatum guttatum.

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. Structural and functional characterisation of this important but enigmatic plant diiron protein has been hampered by an inability to obtain sufficient native protein from plant sources. In the present study recombinant SgAOX (rSgAOX), overexpressed in a ΔhemA-deficient Escherichia coli strain (FN102), was solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. The kinetics of ubiquinol-1 oxidation by purified rSgAOX showed typical Michaelis-Menten kinetics (K(m) of 332 μM and Vmax of 30 μmol(-1) min(-1) mg(-1)), a turnover number 20 μmol s(-1) and a remarkable stability. The enzyme was potently inhibited not only by conventional inhibitors such as SHAM and n-propyl gallate but also by the potent TAO inhibitors ascofuranone, an ascofuranone-derivative colletochlorin B and the cytochrome bc1 inhibitor ascochlorin. Circular dichroism studies revealed that AOX was approximately 50% α-helical and furthermore such studies revealed that rSgAOX and rTAO partially retained the helical absorbance signal even at 90 °C (58% and 64% respectively) indicating a high conformational stability. It is anticipated that highly purified and active AOX and its mutants will facilitate investigations into the structure and reaction mechanisms of AOXs through the provision of large amounts of purified protein for crystallography and contribute to further progress of the study on this important plant terminal oxidase.

The human gut colonizer Blastocystis respires using Complex II and alternative oxidase to buffer transient oxygen fluctuations in the gut

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.

Expression and Crystallization of the Plant Alternative Oxidase

The alternative oxidase (AOX) is an integral monotopic membrane protein located on the inner surface of the inner mitochondrial membrane. Branching from the traditional respiratory chain at the quinone pool, AOX is responsible for cyanide-resistant respiration in plants and fungi, heat generation in thermogenic plants, and survival of parasites, such as Trypanosoma brucei, in the human host. A recently solved AOX structure provides insight into its active site, thereby facilitating rational phytopathogenic and antiparasitic drug design. Here, we describe expression of recombinant AOX using two different expression systems. Purification protocols for the production of highly pure and stable AOX protein in sufficient quantities to facilitate further kinetic, biophysical, and structural analyses are also described.