Proton-coupled electron transfer dynamics in the alternative oxidase.

The alternative oxidase (AOX) is a membrane-bound di-iron enzyme that catalyzes O2-driven quinol oxidation in the respiratory chains of plants, fungi, and several pathogenic protists of biomedical and industrial interest. Yet, despite significant biochemical and structural efforts over the last decades, the catalytic principles of AOX remain poorly understood. We develop here multi-scale quantum and classical molecular simulations in combination with biochemical experiments to address the proton-coupled electron transfer (PCET) reactions responsible for catalysis in AOX from Trypanosoma brucei, the causative agent of sleeping sickness. We show that AOX activates and splits dioxygen via a water-mediated PCET reaction, resulting in a high-valent ferryl/ferric species and tyrosyl radical (Tyr220˙) that drives the oxidation of the quinol via electric field effects. We identify conserved carboxylates (Glu215, Asp100) within a buried cluster of ion-pairs that act as a transient proton-loading site in the quinol oxidation process, and validate their function experimentally with point mutations that result in drastic activity reduction and pK a-shifts. Our findings provide a key mechanistic understanding of the catalytic machinery of AOX, as well as a molecular basis for rational drug design against energy transduction chains of parasites. On a general level, our findings illustrate how redox-triggered conformational changes in ion-paired networks control the catalysis via electric field effects.

The striking differences in the bioenergetics of brain and liver mitochondria are enhanced in mitochondrial disease.

Mitochondrial disorders are hallmarked by the dysfunction of oxidative phosphorylation (OXPHOS) yet are highly heterogeneous at the clinical and genetic levels. Striking tissue-specific pathological manifestations are a poorly understood feature of these conditions, even if the disease-causing genes are ubiquitously expressed. To investigate the functional basis of this phenomenon, we analyzed several OXPHOS-related bioenergetic parameters, including oxygen consumption rates, electron transfer system (ETS)-related coenzyme Q (mtCoQ) redox state and production of reactive oxygen species (ROS) in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by ETS impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to Complex I (CI) or Complex III (CIII) deficiency, respectively. Detailed OXPHOS analysis revealed striking differences between brain and liver mitochondria in the capacity of the different metabolic substrates to fuel the ETS, reduce the ETS-related mtCoQ, and to induce ROS production. In addition, ETS deficiency due to either CI or CIII dysfunction had a much greater impact on the intrinsic bioenergetic parameters of brain compared with liver mitochondria. These findings are discussed in terms of the still rather mysterious tissue-specific manifestations of mitochondrial disease.

Risk Assessment of Psychotropic Drugs on Mitochondrial Function Using In Vitro Assays.

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.

Prediction of drug-induced mitochondrial dysfunction using succinate-cytochrome c reductase activity, QSAR and molecular docking.

There is increasing evidence that links mitochondrial off-target effects with organ toxicities. For this reason, predictive strategies need to be developed to identify mitochondrial dysfunction early in the drug discovery process. In this study, as a major mechanism of mitochondrial toxicity, first, the inhibitory activity of 35 compounds against succinate-cytochrome c reductase (SCR) was investigated. This in vitro study led to the generation of consistent experimental data for a diverse range of compounds, including pharmaceutical drugs and fungicides. Next, molecular docking and protein-ligand interaction fingerprinting (PLIF) analysis were used to identify significant residues and protein-ligand interactions for the Qo site of complex III and Q site of complex II. Finally, this data was used for the development of QSAR models using a regression-based approach to highlight structural and chemical features that might be responsible for SCR inhibition. The statistically validated QSAR models from this work highlighted the importance of low aqueous solubility, low ionisation, fewer 6-membered rings and shorter hydrocarbon alkane chains in the molecular structure for increased inhibition of SCR, hence mitochondrial toxicity. PLIF analysis highlighted two key residues for inhibitory activity of the Qo site of complex III: His 161 as H-bond acceptor and Pro 270 for arene interactions. Currently, there are limited structure-activity models published in the scientific literature for the prediction of mitochondrial toxicity. We believe this study helps shed light on the chemical space for the inhibition of mitochondrial electron transport chain (ETC).

Xenotopic expression of alternative oxidase (AOX) to study mechanisms of mitochondrial disease.

The mitochondrial respiratory chain or electron transport chain (ETC) facilitates redox reactions which ultimately lead to the reduction of oxygen to water (respiration). Energy released by this process is used to establish a proton electrochemical gradient which drives ATP formation (oxidative phosphorylation, OXPHOS). It also plays an important role in vital processes beyond ATP formation and cellular metabolism, such as heat production, redox and ion homeostasis. Dysfunction of the ETC can thus impair cellular and organismal viability and is thought to be the underlying cause of a heterogeneous group of so-called mitochondrial diseases. Plants, yeasts, and many lower organisms, but not insects and vertebrates, possess an enzymatic mechanism that confers resistance to respiratory stress conditions, i.e., the alternative oxidase (AOX). Even in cells that naturally lack AOX, it is autonomously imported into the mitochondrial compartment upon xenotopic expression, where it refolds and becomes catalytically engaged when the cytochrome segment of the ETC is blocked. AOX was therefore proposed as a tool to study disease etiologies. To this end, AOX has been xenotopically expressed in mammalian cells and disease models of the fruit fly and mouse. Surprisingly, AOX showed remarkable rescue effects in some cases, whilst in others it had no effect or even exacerbated a condition. Here we summarize what has been learnt from the use of AOX in various disease models and discuss issues which still need to be addressed in order to understand the role of the ETC in health and disease.

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

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.

Targeting the alternative oxidase (AOX) for human health and food security, a pharmaceutical and agrochemical target or a rescue mechanism?

Some of the most threatening human diseases are due to a blockage of the mitochondrial electron transport chain (ETC). In a variety of plants, fungi, and prokaryotes, there is a naturally evolved mechanism for such threats to viability, namely a bypassing of the blocked portion of the ETC by alternative enzymes of the respiratory chain. One such enzyme is the alternative oxidase (AOX). When AOX is expressed, it enables its host to survive life-threatening conditions or, as in parasites, to evade host defenses. In vertebrates, this mechanism has been lost during evolution. However, we and others have shown that transfer of AOX into the genome of the fruit fly and mouse results in a catalytically engaged AOX. This implies that not only is the AOX a promising target for combating human or agricultural pathogens but also a novel approach to elucidate disease mechanisms or, in several cases, potentially a therapeutic cure for human diseases. In this review, we highlight the varying functions of AOX in their natural hosts and upon xenotopic expression, and discuss the resulting need to develop species-specific AOX inhibitors.

Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State in Isolated Respiring Mitochondria.

The ubiquinone (Q) pool represents a node in the mitochondrial electron transport chain (ETC) onto which the electrons of all respiratory dehydrogenases converge. The redox state of the Q pool correlates closely with the electron flux through the ETC and is thus a parameter of great metabolic value for both the mitochondrial and cellular metabolism. Here, we describe the simultaneous measurement of respiratory rates of isolated mouse heart mitochondria and the redox state of their Q pool using a custom-made combination of a Clark-type oxygen electrode and a Q electrode.

Comparison of the Kinetic Parameters of Alternative Oxidases From Trypanosoma brucei and Arabidopsis thaliana-A Tale of Two Cavities.

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 ΔhemA 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.

In vivo active organometallic-containing antimycotic agents.

Fungal infections represent a global problem, notably for immunocompromised patients in hospital, COVID-19 patient wards and care home settings, and the ever-increasing emergence of multidrug resistant fungal strains is a sword of Damocles hanging over many healthcare systems. Azoles represent the mainstay of antifungal drugs, and their mode of action involves the binding mode of these molecules to the fungal lanosterol 14α-demethylase target enzyme. In this study, we have prepared and characterized four novel organometallic derivatives of the frontline antifungal drug fluconazole (1a-4a). Very importantly, enzyme inhibition and chemogenomic profiling demonstrated that lanosterol 14α-demethylase, as for fluconazole, was the main target of the most active compound of the series, (N-(ferrocenylmethyl)-2-(2,4-difluorophenyl)-2-hydroxy-N-methyl-3-(1H-1,2,4-triazol-1-yl)propan-1-aminium chloride, 2a). Transmission electron microscopy (TEM) studies suggested that 2a induced a loss in cell wall integrity as well as intracellular features ascribable to late apoptosis or necrosis. The impressive activity of 2a was further confirmed on clinical isolates, where antimycotic potency up to 400 times higher than fluconazole was observed. Also, 2a showed activity towards azole-resistant strains. This finding is very interesting since the primary target of 2a is the same as that of fluconazole, emphasizing the role played by the organometallic moiety. In vivo experiments in a mice model of Candida infections revealed that 2a reduced the fungal growth and dissemination but also ameliorated immunopathology, a finding suggesting that 2a is active in vivo with added activity on the host innate immune response.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system.

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 (Km and Vmax) 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.

Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase.

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O2 was a unique side-on type compared to other iron proteins. In order to characterize the O2 binding state of TAO, the O2 binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O2 binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O2 binds very weakly to the TAO iron center (ΔE =6.7 kcal mol-1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· -) as seen in hemerythrin. Coordination of other ligands such as water, Cl-, CN-, CO, N3- and H2O2 was also examined, and H2O2 was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol-1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H2O2 in a dose-dependent and reversible manner.

QSAR and molecular docking for the search of AOX inhibitors: a rational drug discovery approach.

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. Although a number of AOX inhibitors have been discovered, little is still known about the ligand-protein interaction and essential chemical characteristics of compounds required for a potent inhibition. Furthermore, owing to the rapidly growing resistance to existing inhibitors, new compounds with improved potency and pharmacokinetic properties are urgently required. In this study we used two computational approaches, ligand-protein docking and Quantitative Structure-Activity Relationships (QSAR) to investigate binding of AOX inhibitors to the enzyme and the molecular characteristics required for inhibition. Docking studies followed by protein-ligand interaction fingerprint (PLIF) analysis using the AOX enzyme and the mutated analogues revealed the importance of the residues Leu 122, Arg 118 and Thr 219 within the hydrophobic cavity. QSAR analysis, using stepwise regression analysis with experimentally obtained IC50 values as the response variable, resulted in a multiple regression model with a good prediction accuracy. The model highlighted the importance of the presence of hydrogen bonding acceptor groups on specific positions of the aromatic ring of ascofuranone derivatives, acidity of the compounds, and a large linker group on the compounds on the inhibitory effect of AOX.

Degradation of mitochondrial alternative oxidase in the appendices of Arum maculatum.

Cyanide-resistant alternative oxidase (AOX) is a nuclear-encoded quinol oxidase located in the inner mitochondrial membrane. Although the quality control of AOX proteins is expected to have a role in elevated respiration in mitochondria, it remains unclear whether thermogenic plants possess molecular mechanisms for the mitochondrial degradation of AOX. To better understand the mechanism of AOX turnover in mitochondria, we performed a series of in organello AOX degradation assays using mitochondria from various stages of the appendices of Arum maculatum. Our analyses clearly indicated that AOX proteins at certain stages in the appendices are degraded at 30°C, which is close to the maximum appendix temperature observed during thermogenesis. Interestingly, such temperature-dependent protease activities were specifically inhibited by E-64, a cysteine protease inhibitor. Moreover, purification and subsequent nano LC-MS/MS analyses of E-64-sensitive and DCG-04-labeled active mitochondrial protease revealed an ∼30 kDa protein with an identical partial peptide sequence to the cysteine protease 1-like protein from Phoenix dactylifera. Our data collectively suggest that AOX is a potential target for temperature-dependent E-64-sensitive cysteine protease in the appendices of A. maculatum. A possible retrograde signalling cascade mediated by specific degradation of AOX proteins and its physiological significance are discussed.

Kinetic and structural characterisation of the ubiquinol-binding site and oxygen reduction by the trypanosomal alternative oxidase.

The alternative oxidase (AOX) is a monotopic di­iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs. In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in Vmax and catalytic efficiency whilst some also affected the Km for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC50 values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SIGNIFICANCE: The alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.

Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa.

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry.

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET.

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.

Gentamicin Affects the Bioenergetics of Isolated Mitochondria and Collapses the Mitochondrial Membrane Potential in Cochlear Sensory Hair Cells.

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.

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

Insights into the ubiquinol/dioxygen binding and proton relay pathways of the alternative oxidase.

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7 Å, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.