Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4-/- mouse.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4-/- mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.

Investigation of hydrated channels and proton pathways in a high-resolution cryo-EM structure of mammalian complex I.

Respiratory complex I, a key enzyme in mammalian metabolism, captures the energy released by reduction of ubiquinone by NADH to drive protons across the inner mitochondrial membrane, generating the proton-motive force for ATP synthesis. Despite remarkable advances in structural knowledge of this complicated membrane-bound enzyme, its mechanism of catalysis remains controversial. In particular, how ubiquinone reduction is coupled to proton pumping and the pathways and mechanisms of proton translocation are contested. We present a 2.4-Å resolution cryo-EM structure of complex I from mouse heart mitochondria in the closed, active (ready-to-go) resting state, with 2945 water molecules modeled. By analyzing the networks of charged and polar residues and water molecules present, we evaluate candidate pathways for proton transfer through the enzyme, for the chemical protons for ubiquinone reduction, and for the protons transported across the membrane. Last, we compare our data to the predictions of extant mechanistic models, and identify key questions to answer in future work to test them.

Cryo-EM structures of mitochondrial respiratory complex I from Drosophila melanogaster.

Respiratory complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from NADH oxidation by ubiquinone to drive protons across an energy-transducing membrane. Drosophila melanogaster is a candidate model organism for complex I due to its high evolutionary conservation with the mammalian enzyme, well-developed genetic toolkit, and complex physiology for studies in specific cell types and tissues. Here, we isolate complex I from Drosophila and determine its structure, revealing a 43-subunit assembly with high structural homology to its 45-subunit mammalian counterpart, including a hitherto unknown homologue to subunit NDUFA3. The major conformational state of the Drosophila enzyme is the mammalian-type 'ready-to-go' active resting state, with a fully ordered and enclosed ubiquinone-binding site, but a subtly altered global conformation related to changes in subunit ND6. The mammalian-type 'deactive' pronounced resting state is not observed: in two minor states, the ubiquinone-binding site is unchanged, but a deactive-type π-bulge is present in ND6-TMH3. Our detailed structural knowledge of Drosophila complex I provides a foundation for new approaches to disentangle mechanisms of complex I catalysis and regulation in bioenergetics and physiology.

Experimental evaluation of the influence of combined particle size pretreatment and Fe3O4 additive on fuel yields of Arachis Hypogea shells.

A smart energy recovery process can achieve maximum energy recovery from organic wastes. Pretreatment of feedstock is essential to biogas and methane yields during the anaerobic digestion process. This work combined particle size reduction with Fe3O4 nanoparticles to investigate their influence on biogas and methane yields from anaerobic digestion of Arachis hypogea shells. Twenty milligrams per litre of Fe3O4 nanoparticles was implemented with 2, 4, 6 and 8 mm particle sizes and a single treatment of Fe3O4 for 35 days. The treatments were compared with each other and were discovered to significantly (p < 0.05) enhance biogas yield by 37.40%, 50.10%, 54.40%, 51.40% and 35.50% compared with control, respectively. Specific biogas yield recorded was 966.2, 1406, 1552.7, 1317.4, 766.2 and 413 mL g-1 volatile solid. This study showed the combination of Fe3O4 with 6 mm particle size of Arachis hypogea shells produced the optimum biogas and methane yields. The addition of Fe3O4 to particle sizes below 6 mm resulted in over-accumulation of volatile fatty acids and lowered the gas yield. This can be applied on an industrial scale.

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I.

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure-activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.

Modelling the effects of particle size pretreatment method on biogas yield of groundnut shells.

Optimising biogas yields from anaerobic digestion of organic wastes is significant to maximum energy recovery in the biodigestion process and has become an important topic of interest. Substrate particle size is an important process parameter in biogas production, and it precedes other pretreatments methods for the majority of the lignocellulose materials. Optimisation of biogas yield using Response Surface Methodology (RSM) was done, and temperature, hydraulic retention time and particle size were considered variables to develop the predictive models. Pretreatment of groundnut shells was investigated using particle size reduction of mechanical pretreatment methods. After pretreatment, 30 samples were digested in a batch digester at mesophilic temperature. The experimental results showed that the temperature, hydraulic retention time and particle size had significant effects of interaction (p < 0.05). The optimum experimental and predicted yields are: 44.70 and 42.92 (lNkgoDM) organic dry matter biogas yield, 20.80 and 19.09 (lN/kgFM) fresh mass biogas yield, 24.00 and 22.68 (lNCH4oDM) organic dry methane yield and 12.30 and 15.59 (lNCH4FM) fresh mass methane yield, respectively. The R2 recorded for the four yield components were 0.6268, 0.5875, 0.6109 and 0.5547. These values seem to be lower and a sign of the average fit of the model. Biogas production from groundnut shells was significantly improved with statistical optimisation and the pretreatment method.

Optimization of biogas yield from lignocellulosic materials with different pretreatment methods: a review.

Population increase and industrialization has resulted in high energy demand and consumptions, and presently, fossil fuels are the major source of staple energy, supplying 80% of the entire consumption. This has contributed immensely to the greenhouse gas emission and leading to global warming, and as a result of this, there is a tremendous urgency to investigate and improve fresh and renewable energy sources worldwide. One of such renewable energy sources is biogas that is generated by anaerobic fermentation that uses different wastes such as agricultural residues, animal manure, and other organic wastes. During anaerobic digestion, hydrolysis of substrates is regarded as the most crucial stage in the process of biogas generation. However, this process is not always efficient because of the domineering stableness of substrates to enzymatic or bacteria assaults, but substrates' pretreatment before biogas production will enhance biogas production. The principal objective of pretreatments is to ease the accessibility of the enzymes to the lignin, cellulose, and hemicellulose which leads to degradation of the substrates. Hence, the use of pretreatment for catalysis of lignocellulose substrates is beneficial for the production of cost-efficient and eco-friendly process. In this review, we discussed different pretreatment technologies of hydrolysis and their restrictions. The review has shown that different pretreatments have varying effects on lignin, cellulose, and hemicellulose degradation and biogas yield of different substrate and the choice of pretreatment technique will devolve on the intending final products of the process.

Chlorine inhalation induces acute chest syndrome in humanized sickle cell mouse model and ameliorated by postexposure hemopexin.

Triggering factors of Acute Chest Syndrome (ACS) is a leading cause of death in patients with Sickle Cell Disease (SCD) and targeted therapies are limited. Chlorine (Cl2) inhalation happens frequently, but its role as a potential trigger of ACS has not been determined. In this study, we hypothesized that Cl2 exposure resembling that in the vicinity of industrial accidents induces acute hemolysis with acute lung injury, reminiscent of ACS in humanized SCD mice. When exposed to Cl2 (500 ppm for 30 min), 64% of SCD mice succumbed within 6 h while none of the control mice expressing normal human hemoglobin died (p<0.01). Surviving SCD mice had evidence of acute hemolysis, respiratory acidosis, acute lung injury, and high concentrations of chlorinated palmitic and stearic acids (p<0.05) in their plasmas and RBCs compared to controls. Treatment with a single intraperitoneal dose of human hemopexin 30 min after Cl2 inhalation reduced mortality to around 15% (p<0.01) with reduced hemolysis (decreased RBCs fragility (p<0.001) and returned plasma heme to normal levels (p<0.0001)), improved oxygenation (p<0.0001) and reduced acute lung injury scores (p<0.0001). RBCs from SCD mice had significant levels of carbonylation (which predisposes RBCs to hemolysis) 6 h post-Cl2 exposure which were absent in RBCs of mice treated with hemopexin. To understand the mechanisms leading to carbonylation, we incubated RBCs from SCD mice with chlorinated lipids and identified sickling and increased hemolysis compared to RBCs obtained from control mice and treated similarly. Our study indicates that Cl2 inhalation induces ACS in SCD mice via induction of acute hemolysis, and that post exposure administration of hemopexin reduces mortality and lung injury. Our data suggest that SCD patients are vulnerable in Cl2 exposure incidents and that hemopexin is a potential therapeutic agent.

Electrochemical corrosion behavior of copper in graphene-based thermal fluid with different surfactants.

This study investigates the effect of different surfactant-dispersed graphene nanofluid on the electrochemical behavior of copper. This study was achieved by measuring the open circuit potential and potentiodynamic polarization of copper in the nanofluids at room temperature. The test media includes surfactant-free graphene nanofluid and graphene nanofluid dispersed using four different surfactants, which are sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, Gum Arabic, and Tween 80. The surface characterization and elemental composition of the copper sample before and after the corrosion tests were determined using a scanning electron microscope coupled with energy-dispersive X-ray spectroscopy. The phase formation after corrosion was also evaluated by measuring X-ray diffraction. The quantity of copper dissolved in the test media was evaluated using an inductively coupled plasma mass spectrometry (ICP-MS). The open-circuit potential measurements revealed that the current free corrosion potential of copper in the different surfactant-aided graphene nanofluids are different. The electrochemical corrosion potential, Tafel slopes, and corrosion rates revealed the better corrosion performance of copper in the nanofluid of different surfactants in the increasing order GA, SDS, Tween 80, and SDBS. Copper in GA-based graphene nanofluid was found to have the lowest corrosion rate while that of SDBS has the highest corrosion rate. However, the ICP-MS result revealed a discrepancy in the corrosion behavior and quantity of copper dissolved in the different test media. This could be attributed to the dissimilar dissolution-redeposition rate of copper in different media.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Mammalian Respiratory Complex I Through the Lens of Cryo-EM.

Single-particle electron cryomicroscopy (cryo-EM) has led to a revolution in structural work on mammalian respiratory complex I. Complex I (mitochondrial NADH:ubiquinone oxidoreductase), a membrane-bound redox-driven proton pump, is one of the largest and most complicated enzymes in the mammalian cell. Rapid progress, following the first 5-Å resolution data on bovine complex I in 2014, has led to a model for mouse complex I at 3.3-Å resolution that contains 96% of the 8,518 residues and to the identification of different particle classes, some of which are assigned to biochemically defined states. Factors that helped improve resolution, including improvements to biochemistry, cryo-EM grid preparation, data collection strategy, and image processing, are discussed. Together with recent structural data from an ancient relative, membrane-bound hydrogenase, cryo-EM on mammalian complex I has provided new insights into the proton-pumping machinery and a foundation for understanding the enzyme's catalytic mechanism.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.

Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase.

BACKGROUND: The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. RESULTS: Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. CONCLUSIONS: Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

Female and male gamete mitochondria are distinct and complementary in transcription, structure, and genome function.

Respiratory electron transport in mitochondria is coupled to ATP synthesis while generating mutagenic oxygen free radicals. Mitochondrial DNA mutation then accumulates with age, and may set a limit to the lifespan of individual, multicellular organisms. Why is this mutation not inherited? Here we demonstrate that female gametes-oocytes-have unusually small and simple mitochondria that are suppressed for DNA transcription, electron transport, and free radical production. By contrast, male gametes-sperm-and somatic cells of both sexes transcribe mitochondrial genes for respiratory electron carriers and produce oxygen free radicals. This germ-line division between mitochondria of sperm and egg is observed in both the vinegar fruitfly and the zebrafish-species spanning a major evolutionary divide within the animal kingdom. We interpret these findings as an evidence that oocyte mitochondria serve primarily as genetic templates, giving rise, irreversibly and in each new generation, to the familiar energy-transducing mitochondria of somatic cells and male gametes. Suppressed mitochondrial metabolism in the female germ line may therefore constitute a mechanism for increasing the fidelity of mitochondrial DNA inheritance.

Energy, ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template.

Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. In eukaryotes, this coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates free radicals, reactive oxygen species (ROS), which are mutagenic. In animals, mutational damage to mitochondrial DNA therefore accumulates within the lifespan of the individual. Fertilization generally requires motility of one gamete, and motility requires ATP. It has been proposed that oxidative phosphorylation is nevertheless absent in the special case of quiescent, template mitochondria, that these remain sequestered in oocytes and female germ lines and that oocyte mitochondrial DNA is thus protected from damage, but evidence to support that view has hitherto been lacking. Here we show that female gametes of Aurelia aurita, the common jellyfish, do not transcribe mitochondrial DNA, lack electron transport, and produce no free radicals. In contrast, male gametes actively transcribe mitochondrial genes for respiratory chain components and produce ROS. Electron microscopy shows that this functional division of labour between sperm and egg is accompanied by contrasting mitochondrial morphology. We suggest that mitochondrial anisogamy underlies division of any animal species into two sexes with complementary roles in sexual reproduction. We predict that quiescent oocyte mitochondria contain DNA as an unexpressed template that avoids mutational accumulation by being transmitted through the female germ line. The active descendants of oocyte mitochondria perform oxidative phosphorylation in somatic cells and in male gametes of each new generation, and the mutations that they accumulated are not inherited. We propose that the avoidance of ROS-dependent mutation is the evolutionary pressure underlying maternal mitochondrial inheritance and the developmental origin of the female germ line.