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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 O ₂ reduction.

Mitosomes are highly reduced forms of mitochondria which have lost two of the ‘defining’ features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite’s life cycle progression. Using a recently published methodology for synchronising T . hominis infection of mammalian cell lines, we demonstrated specific inhibition of T . hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T . hominis- infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T . hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi , further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T . hominis infecting mammalian host cells. In summary, our results reveal that T . hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T . hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (K m and V max ) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose–response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

Mitochondria are potential targets responsible for some drug- and xenobiotic-induced organ toxicities. However, molecular mechanisms of drug-induced mitochondrial toxicities are mostly unknown. Here, multiple in vitro assays were used to investigate the effects of 22 psychotropic drugs on mitochondrial function. The acute extracellular flux assay identified inhibitors of the electron transport chain (ETC), i.e., aripiprazole, phenytoin, and fluoxetine, an uncoupler (reserpine), substrate inhibitors (quetiapine, carbamazepine, buspirone, and tianeptine), and cytotoxic compounds (chlorpromazine and valproic acid) in HepG2 cells. Using permeabilized HepG2 cells revealed minimum effective concentrations of 66.3, 6730, 44.5, and 72.1 µM for the inhibition of complex-I-linked respiration for quetiapine, valproic acid, buspirone, and fluoxetine, respectively. Assessing complex-II-linked respiration in isolated rat liver mitochondria revealed haloperidol is an ETC inhibitor, chlorpromazine is an uncoupler in basal respiration and an ETC inhibitor under uncoupled respiration (IC50 = 135 µM), while olanzapine causes a mild dissipation of the membrane potential at 50 µM. This research elucidates some mechanisms of drug toxicity and provides some insight into their safety profile for clinical drug decisions.

Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone ( ), a natural compound produced by , and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure-activity relationship study and have identified functional groups of that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of and its potent derivatives , , and were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine biosynthesis pathway. We also provide the first evidence that and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment.

The alternative oxidase (AOX) is widespread in plants, fungi, and some protozoa. While the general structure of the AOX remains consistent, its overall activity, sources of kinetic activation and their sensitivity to inhibitors varies between species. In this study, the recombinant Trypanosoma brucei AOX (rTAO) and Arabidopsis thaliana AOX1A (rAtAOX1A) were expressed in the Escherichia coli Δ hemA mutant FN102, and the kinetic parameters of purified AOXs were compared. Results showed that rTAO possessed the highest V max and K m for quinol-1, while much lower V max and K m were observed in the rAtAOX1A. The catalytic efficiency ( k cat / K m ) of rTAO was higher than that of rAtAOX1A. The rTAO also displayed a higher oxygen affinity compared to rAtAOX1A. It should be noted that rAtAOX1a was sensitive to α-keto acids while rTAO was not. Nevertheless, only pyruvate and glyoxylate can fully activate Arabidopsis AOX. In addition, rTAO and rAtAOX1A showed different sensitivity to AOX inhibitors, with ascofuranone (AF) being the best inhibitor against rTAO, while colletochlorin B (CB) appeared to be the most effective inhibitor against rAtAOX1A. Octylgallate (OG) and salicylhydroxamic acid (SHAM) are less effective than the other inhibitors against protist and plant AOX. A Caver analysis indicated that the rTAO and rAtAOX1A differ with respect to the mixture of polar residues lining the hydrophobic cavity, which may account for the observed difference in kinetic and inhibitor sensitivities. The data obtained in this study are not only beneficial for our understanding of the variation in the kinetics of AOX within protozoa and plants but also contribute to the guidance for the future development of phytopathogenic fungicides.

Aminoglycoside antibiotics are widely prescribed to treat a variety of serious bacterial infections. They are extremely useful clinical tools, but have adverse side effects such as oto- and nephrotoxicity. Once inside a cell they are thought to cause mitochondrial dysfunction, subsequently leading to apoptotic cell death due to an increase in reactive oxygen species (ROS) production. Here we present evidence of a direct effect of gentamicin (the most commonly prescribed aminoglycoside) on the respiratory activities of isolated rat liver and kidney mitochondria. We show that gentamicin stimulates state 4 and inhibits state 3u respiratory rates, thereby reducing the respiratory control ratio (RCR) whilst simultaneously causing a collapse of the mitochondrial membrane potential (MtMP). We propose that gentamicin behaves as an uncoupler of the electron transport chain (ETC) - a hypothesis supported by our evidence that it reduces the production of mitochondrial ROS (MtROS). We also show that gentamicin collapses the MtMP in the sensory hair cells (HCs) of organotypic mouse cochlear cultures.

Proteomic analysis of any biological system by two-dimensional gel electrophoresis (2-DE) requires high resolution and high reproducibility. The results presented here demonstrate the reproducible and accurate separation of rice (Oryza sativa L.) proteins using improved procedures for high resolution 2-DE, which were adapted for the separation of rice lamina proteins. Validation of this system was achieved by measuring the effects of sample preparation and biological variation on the coefficient of variation (CV) for replicate spots. The majority of experimental variation was shown to be introduced by the 2-DE technique (CV 0.26). Analysis of biological variation indicated that approximately 93–95% of spots were within a CV of 0.7. This provided a threshold value from which valid differences in expression between experimental groups could be screened. This system was then utilized for the proteomic analysis of short- and long-term salt-stress-responsive proteins in the rice leaf lamina. Analysis resulted in the separation of approximately 2500 protein species of which 32 were observed to be significantly regulated by salinity; so far 11 of these proteins have been identified by tandem mass spectrometry. An increase in eight proteins, including RuBisCO activase and ferritin, occurred by 24 h of exposure to sodium chloride (50 mM) and continued to increase during the following 6 d. Only one protein, a putative phosphoglycerate kinase, was found to increase in expression within 24 h and did not increase over a longer period of exposure to salt. There were also proteins that showed no change 24 h after exposure to salt, but had increased (superoxide dismutase) or decreased (S-adenosyl-L-methionine synthetase) after 7 d salt treatment.

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 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 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 in established IBS environments.

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.