Cytochrome Bc1 Complex Research Articles

Elucidating the Effects of Distal Charges on the Reduction Potential of the Rieske Protein

The Rieske protein plays a key role in both cellular respiration and photosynthesis, transferring electrons through the respective cytochrome bc1 and b6f complexes of each process. It is characterized by a [2Fe‐2S] cluster and is unique in that one iron is ligated by nitrogens from two solvent‐exposed histidine residues while the other is ligated by the sulfurs of two cysteines. In the electron transport chain (ETC), the Rieske protein oxidizes ubiquinol and reduces cytochrome c, transferring electrons between complexes II and IV.Previous work has demonstrated how hydrogen bonds, solvent accessibility of the ligands, and the presence of nearby charged residues can impact the reduction potential of the protein. However, the full extent of this third element, how far from the cluster these charged residues can reside and still impact the reduction potential, has yet to be explored. Two types of mutants have been created to probe the effects of these distal charges. First, mutations removing positively charged residues were predicted to decrease reduction potential. Likewise, mutations removing negatively charged residues were expected to increase reduction potential. Each mutation either resides a unique distance from the cluster or produces a different change in overall charge, demonstrating the impact of each factor both individually and additively. Lastly, the pKa values of the histidine ligands were also expected to be inversely related to reduction potential.Using pH‐dependent UV‐Visible spectrophotometry, the pKa values of these mutants were determined and correlated to these relationships. While the overall trend of pKa values increased as expected for the negative‐charge mutants, confounding elements also appeared, such as a third pKa sigmoid. Furthermore, E146R, a positive‐charge mutant, also demonstrated increased pKa values, opposite to the hypothesized trend. New mutants that implement a neutral to positive substitutions have been developed to probe the effect of making the protein more positive.Support or Funding InformationMurchison Summer Undergraduate Research Fellowship, Welch Foundation

Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure–activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 μM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc1 complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.

New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis.

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02–0.09 μg/mL) and low toxicity (TD50 ≥ 5μg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc1 by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc1 complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc1 complex.

The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani.

Available treatments for Chagas’ disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.

Naphthoquinones isolated from Eleutherine plicata herb: in vitro antimalarial activity and molecular modeling to investigate their binding modes

Plasmodium falciparum is the cause of malaria and has become resistant to the drugs used to treat the disease. Therefore, the development for novel compounds with antimalarial activity has become urgent. Plant species, such as naphthoquinone-rich Eleutherine plicata, may provide novel substances that exhibit antimalarial activity and serve as an alternative for the treatment of this disease. From this plant species, ethanol extracts were obtained, fractionated, and the isolated substances eleutherin, and isoleutherin were characterized by nuclear magnetic resonance. There in vitro activity against Plasmodium falciparum was examined using the traditional Microtest method using the extract, fractions, and isolated molecules. Eleutherin and isoleutherin showed the best activity toward the parasite with IC50 values of 10.45 and 8.70 µg/mL, respectively. Characterization of the binding mode of the compounds with a target enzyme and identification of the molecular interactions were revealed via molecular docking results. Eleutherin and isoeleutherin interacted with highly conserved residues from the binding cavity of the cytochrome bc1 complex, a protein found in mitochondria. Therefore, the eleutherin and isoeleutherin naphthoquinones showed antiplasmodial activity with a similar mechanism to that of atovaquone were able to interact with the cytochrome bc1 complex, and showed promise for antimalarial treatments.

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1lp.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cybp.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1lp.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc1 complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

Differences in Breast Muscle Mitochondrial Respiratory Capacity, Reactive Oxygen Species Generation, and Complex Characteristics between 7-week-old Meat- and Laying-type Chickens.

The skeletal muscle growth rate is a major feature differentiating meat- and laying-type chickens. A large amount of ATP is required during skeletal muscle synthesis, in which mitochondrial energy production capacities play a significant role. Additionally, mitochondria may participate in muscle protein degradation via reactive oxygen species generation. To investigate the differences in mitochondrial energetic characteristics between chickens exhibiting different growth rates, this study evaluated respiratory capacities in response to different types of respiratory substrate, protein abundances, assembly of individual respiratory complexes (I–V) and supercomplexes, and reactive oxygen species generation rates. These characteristics were compared between mitochondria from the breast muscle (M. pectoralis superficialis) of seven-week-old meat- and laying-type male chickens. Blue native polyacrylamide gel electrophoresis analysis revealed that meat-type chickens exhibited a significantly lower protein abundance of complex III (cytochrome bc1 complex), complex V (F0F1 ATP synthase), and total amount of supercomplexes than did laying-type chickens. There were no differences between chicken types in the respiration rate of mitochondria incubated with either pyruvate/malate or succinate, each of which drives complex I- and complex II-linked respiration. Carnitine palmitoyltransferase-1-dependent and -independent respiration during ATP synthesis and carnitine palmitoyltransferase-2 enzymatic activity were significantly lower in meat-type chickens than in layingtype chickens. For mitochondria receiving pyruvate/malate plus succinate, the reactive oxygen species generation rate and its ratio to the oxygen consumed (the percentage of free radical leak) were also significantly lower in meat-type chickens than in laying-type chickens. These results suggested that the mitochondrial energetic capacities of the breast muscle of meat-type chickens could be lower than those of laying-type chickens at seven weeks of age. Furthermore, the lower reactive oxygen species generation rate in meat-type chickens might have implications for rapid muscle development, which is possibly related to their lower muscle protein degradation rates.

Antifungal activity of metyltetraprole against the existing QoI-resistant isolates of various plant pathogenic fungi: Metyltetraprole against QoI-R isolates.

BACKGROUNDMetyltetraprole is a novel quinol oxidation site of Complex III inhibitor (QoI) fungicide that inhibits mitochondrial electron transport at the Qo site of the cytochrome bc1 complex. Previous reports have demonstrated that it is also active against the QoI‐resistant (QoI‐R) isolates of Zymoseptoria tritici and Pyrenophora teres with the mutations G143A and F129L in their cytochrome b gene, respectively. Further studies on cross‐resistance between metyltetraprole and existing QoIs were performed using an increased number of isolates of Z. tritici, P. teres, Ramularia collo‐cygni, Pyrenophora tritici‐repentis, and several other plant pathogenic fungi.RESULTSDifferences in the EC50 values between the wild‐type and QoI‐R isolates with the mutations G143A or F129L were always smaller for metyltetraprole compared to those for the existing QoIs, and they were never greater than five in terms of resistance factor. The 2‐year field experiments showed that the metyltetraprole treatment did not increase the percentage of QoI‐R isolates likely to harbor the G143A mutation in a Z. tritici population.CONCLUSIONThe unique behavior of metyltetraprole against the existing QoI‐R isolates was confirmed for all tested pathogen species. Our results provide important information to establish a fungicide resistance management strategy using metyltetraprole in combination or alternation with other fungicides. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

An exclusive computational insight toward molecular mechanism of MMV007571, a multitarget inhibitor of Plasmodium falciparum

Recently, two Malaria Box molecules namely MMV007571 and MMV020439 well known inhibitors of New Permeability Pathway (NPP) function also showed a secondary phenotype of inhibition of enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) and cytochrome bc1 complex in metabolic profile assays. Intricacies of their binding at the newly identified targets was need of the hour which motivated us to study their binding using molecular docking and dynamics simulations approach. Interestingly, molecular docking results of both MMV007571 and MMV020439 showed good binding affinity toward the Qo site of cytochrome bc1 complex while only MMV007571 illustrated notable binding characterstics for PfDHODH. Molecular Dynamics (MD) simulations when carried out for native-PfDHODH, PfDHODH-MMV007571 and PfDHODH-Genz667348 models (100 ns each) demonstrated the role of inhibitors over the N-terminus domain which experienced conformational transition from an open state (22 Å) to closed state (16 Å) in the protein-inhibitor models. Dynamics also indicated that the loop domain near cofactor flavin mononucleotide (FMN) attained more felxibility which further lead to its poor binding and may contribute to inhibition of the oxidation (catalytic) process. Moreover, the pharmacophoric features of MMV007571 was justified and may serve as a template for the design of novel series of more potent multitarget inhibitors against Plasmodium falciparum.AbbreviationsÅAngstromACTsArtemisinin combination therapiescyt bc1cytochrome bc1 complexhhour(s)KKelvinµMmicromolarMMVMedicine for malaria ventureNLucNanoluciferasenMnanomolarNPPNew permeation pathwayPDBProtein data bankPfDHODHPlasmodium falciparum dihydroorotate dehydrogenasePOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholineRBCRed blood corpusclesRMSDRoot-mean-square deviationSPStandard precisionvdWvan der WaalsXPExtra precisionyDHODHYeast dihydroorotate dehydrogenaseCommunicated by Ramaswamy H. Sarma

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.

A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

AbstractRespiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

A 3.3 Å-Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability.

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc 1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c 1 was found to greatly enhance the interaction between cyt. b and cyt. c 1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death

Rapanone is a natural occurring benzoquinone with several biological effects including unclear cytotoxic mechanisms. Here we addressed if mitochondria are involved in the cytotoxicity of rapanone towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In the HepG2, rapanone (20–40 μM) induced a concentration-dependent mitochondrial membrane potential dissipation, ATP depletion, hydrogen peroxide generation and, phosphatidyl serine externalization; the latter being indicative of apoptosis induction. Rapanone toxicity towards primary rats hepatocytes (IC50 = 35.58 ± 1.50 μM) was lower than that found for HepG2 cells (IC50 = 27.89 ± 0.75 μM). Loading of isolated mitochondria with rapanone (5–20 μM) caused a concentration-dependent inhibition of phosphorylating and uncoupled respirations supported by complex I (glutamate and malate) or the complex II (succinate) substrates, being the latter eliminated by complex IV substrate (TMPD/ascorbate). Rapanone also dissipated mitochondrial membrane potential, depleted ATP content, released Ca2+ from Ca2+-loaded mitochondria, increased ROS generation, cytochrome c release and membrane fluidity. Further analysis demonstrated that rapanone prevented the cytochrome c reduction in the presence of decylbenzilquinol, identifying complex III as the site of its inhibitory action. Computational docking results of rapanone to cytochrome bc1 (Cyt bc1) complex from the human sources found spontaneous thermodynamic processes for the quinone-Qo and Qi binding interactions, supporting the experimental in vitro assays. Collectively, these observations suggest that rapanone impairs mitochondrial respiration by inhibiting electron transport chain at Complex III and promotes mitochondrial dysfunction. This property is potentially involved in rapanone toxicity on cancer cells.

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.

Crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron–sulfur protein subunit

Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiquinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction requires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron-sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron-sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-cycle mechanism of cyt bc1 function.

Azaaurones as Potent Antimycobacterial Agents Active against MDR- and XDR-TB.

Herein we report the screening of a small library of aurones and their isosteric counterparts, azaaurones and N-acetylazaaurones, against Mycobacterium tuberculosis. Aurones were found to be inactive at 20 μm, whereas azaaurones and N-acetylazaaurones emerged as the most potent compounds, with nine derivatives displaying MIC99 values ranging from 0.4 to 2.0 μm. In addition, several N-acetylazaaurones were found to be active against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical M. tuberculosis isolates. The antimycobacterial mechanism of action of these compounds remains to be determined; however, a preliminary mechanistic study confirmed that they do not inhibit the mycobacterial cytochrome bc1 complex. Additionally, microsomal metabolic stability and metabolite identification studies revealed that N-acetylazaaurones are deacetylated to their azaaurone counterparts. Overall, these results demonstrate that azaaurones and their N-acetyl counterparts represent a new entry in the toolbox of chemotypes capable of inhibiting M. tuberculosis growth.

Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue

The widespread use of immobilized metal-affinity chromatography (IMAC) for fast and efficient purification of recombinant proteins has brought potentially toxic transition elements into common laboratory usage. However, there are few studies on the leaching of metal from the affinity resin, such as nickel-nitrilotriacetic acid (Ni-NTA), with possible deleterious impact on the biological activity. This is of particular importance when reducing or chelating eluants stronger than imidazole are used. We present a detailed study of hydroxynaphthol blue (HNB) as an indicator of several divalent metal cations, but with emphasis on Ni2+, clarifying and correcting many errors and ambiguities in the older literature on this dye compound. The assay is simple and sensitive and many metals, notably Ni2+, Zn2+, Cu2+, Pb2+, Fe2+, Co2+, and Al3+, can be readily detected and quantified at concentrations down to 15–50 nM (1–5 ppb) at neutral pH and in most commonly used buffers using spectroscopic equipment available in typical biochemistry research labs. Using this method, we show that significant amounts of Ni2+ (up to 20 mM) are co-purified with a target protein (cytochrome bc1 complex) when histidine is used to elute from Ni-NTA resin.

Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages.

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.

Glucose Metabolism Regulates Mitochondrial Supercomplex Abundance in Murine Heart

BackgroundMitochondrial electron transport chain (ETC) complexes assemble to form supercomplexes, which are thought to optimize ATP production via electron channelling. Supercomplexes appear to be more abundant in organs with a large energy demand, such as the heart. Nevertheless, the mechanisms that regulate supercomplex abundance in the heart remain unclear.ObjectiveThe purpose of this study was to determine how glycolytic activity regulates the abundance and activity of mitochondrial supercomplexes in the adult mouse heart.MethodsMitochondria isolated from adult, male FVB/NJ mouse hearts were solubilized with 2% digitonin. Mitochondrial supercomplexes (>1 MDa) were then resolved by electrophoresis on 3–12% blue native‐polyacrylamide (BN‐PAGE) gradient gels, and the protein constituents of each supercomplex were identified by mass spectrometry and Western blotting. To determine how glycolysis affects mitochondrial supercomplex abundance in the heart, we assessed mitochondrial supercomplexes in cardiac mitochondria isolated from mice that express cardiac‐specific, dominant‐negative (GlycoLo) or dominant‐positive (GlycoHi) forms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase, which constitutively lower or augment glycolytic rate, respectively. The enzymatic activities of supercomplex components were assessed using in‐gel activity assays. Differences in supercomplex abundances and activities were calculated using one‐way ANOVA, followed by Tukey post‐hoc tests.ResultsCardiac mitochondria contained at least six distinct supercomplexes, with each supercomplex comprising NADH dehydrogenase, cytochrome bc1 complex, cytochrome c oxidase, and ATP synthase. In Coomassie‐stained BN‐PAGE gels and in Western blots, mitochondria from GlycoLo hearts showed higher supercomplex abundance than mitochondria from WT and GlycoHi mice. Furthermore, we found that mitochondria from GlycoLo hearts had a higher abundance of NADH dehydrogenase and cytochrome bc1 complex. Furthermore, the enzymatic activities of supercomplex‐associated NADH dehydrogenase and cytochrome c oxidase were higher in GlycoLo hearts than in GlycoHi and WT hearts.ConclusionLow glycolytic activity in the heart increases mitochondrial supercomplex abundance and activity. These findings could provide new insights into the metabolic mechanisms that regulate cardiac health and remodeling.Support or Funding InformationNIH (HL122580, HL130174, ES028268, HL78825, and GM103492) and from the American Diabetes Association (1‐16‐JDF‐041)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.