Anthony L. Moore

The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria

In addition to possessing multiple NAD(P)H dehydrogenases, most plant mitochondria contain a cyanide- and antimycin-insensitive alternative terminal oxidase. Although the general characteristics of this terminal oxidase have been known for a considerable number of years, the mechanism by which it is regulated is unclear and until recently there has been relatively little information on its exact nature. In the past 5 years, however, the application of molecular and novel voltametric techniques has advanced our understanding of this oxidase considerably. In this article, we review briefly current understanding on the structure and function of the multiple NADH dehydrogenases and consider, in detail, the nature and regulation of the alternative oxidase. We derive a kinetic model for electron transfer through the ubiquinone pool based on a proposed model for the reduction of the oxidase by quinol and show how this can account for deviations from Q-pool behaviour. We review information on the attempts to isolate and characterise the oxidase and finally consider the molecular aspects of the expression of the alternative oxidase.

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.

The active site of the cyanide-resistant oxidase from plant mitochondria contains a binuclear iron center.

The cyanide‐resistant, alternative oxidase of plant mitochondria catalyzes the four‐electron reduction of oxygen to water, but the nature of the catalytic center associated with this oxidase has yet to be elucidated. We have identified conserved amino acids, including two copies of the iron‐binding motif Glu‐X‐X‐His, in the carboxy‐terminal hydrophilic domain of the alternative oxidase that suggest the presence of a hydroxo‐bridged binuclear iron center, analogous to that found in the enzyme methane monooxygenase. Using the known three‐dimensional structures of other binuclear iron proteins, we have developed a structural model for the proposed catalytic site of the alternative oxidase based on these amino acid sequence similarities.

Regulation of alternative pathway activity in plant mitochondria: Nonlinear relationship between electron flux and the redox poise of the quinone pool

The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.

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.

Structure-function relationships of the alternative oxidase of plant mitochondria: A model of the active site

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.

Structure of the trypanosome cyanide-insensitive alternative oxidase

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. 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, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.

Measurement of the redox state of the ubiquinone pool in plant mitochondria

We have investigated the dependence of the respiratory rate on the redox poise of the quinone pool in isolated turnip and pea leaf mitochondria. A linear relationship has been found between these two parameters during succinate oxidation under both state 3 and 4 conditions. When succinate is oxidised by the alternative oxidase the dependence of oxygen uptake on the steady‐state reduction level of quinone is markedly non‐linear. These results are discussed within the frame‐work of a homogeneous quinone pool.

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.

Regulation of thermogenesis in flowering Araceae: The role of the alternative oxidase

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.