Drive ATP Synthesis Research Articles

Inter-organ differences in redox imbalance and apoptosis depict metabolic resilience in migratory redheaded buntings

Migration, a bird’s metabolic apex, depends primarily on the liver and muscle for fuel mobilization and endurance flight. In migratory redheaded buntings, adaptive increase in mitochondrial membrane (MM) proton gradient to drive ATP synthesis, measured by MM potential (MMP+) and reactive oxygen species (ROS) response, have been well characterized in the blood but not in the muscle or liver. We assessed MMP+, ROS, and apoptosis in the liver and pectoralis muscle of photosensitive nonmigratory (nMig.) male redheaded buntings photoinduced to migratory (Mig.) states. Relative expression levels of genes associated with energy (ACADM, PEPCK, GOT2, GLUT1, and CS), ROS modulation (SIRT1), mitochondrial free-radical scavengers (SOD1, PRX4, NOS2, GPX1, and GPX4), anti-apoptotic genes (NF-κβ), apoptotic (CASP7), and tissue damage using histology, during migration were assessed. The MMP+ decreased and the ROS concentration increased, due to the metabolic load on liver and pectoralis muscle tissues during Mig. However, percentage of apoptotic cells increased in liver but decreased in muscle, which is of functional significance to migratory passerines. During Mig., in muscle, SIRT1 increased, while an increase in anti-apoptotic NF-κβ aided immune pathway-mediated antioxidant activity and guarded against muscle oxidative damage during migration. Inter-organ differences in metabolism add to our current understanding of metabolic flexibility that supports successful migration in buntings.

Potential Role of the Anammoxosome in the Adaptation of Anammox Bacteria to Salinity Stress.

The underlying adaptative mechanisms of anammox bacteria to salt stress are still unclear. The potential role of the anammoxosome in modulating material and energy metabolism in response to salinity stress was investigated in this study. The results showed that anammox bacteria increased membrane fluidity and decreased mechanical properties by shortening the ladderane fatty acid chain length of anammoxosome in response to salinity shock, which led to the breakdown of the proton motive force driving ATP synthesis and retarded energy metabolism activity. Afterward, the fatty acid chain length and membrane properties were recovered to enhance the energy metabolic activity. The relative transmission electron microscopy (TEM) area proportion of anammoxosome decreased from 55.9 to 38.9% under salinity stress. The 3D imaging of the anammox bacteria based on Synchrotron soft X-ray tomography showed that the reduction in the relative volume proportion of the anammoxosome and the concave surfaces was induced by salinity stress, which led to the lower energy expenditure of the material transportation and provided more binding sites for enzymes. Therefore, anammox bacteria can modulate nitrogen and energy metabolism by changing the membrane properties and morphology of the anammoxosome in response to salinity stress. This study broadens the response mechanism of anammox bacteria to salinity stress.

Bio-inspired High-Performance Artificial Ion Pump Mediated by Subnanoscale Dehydration Hydration Effects.

The energy conversion in plant chloroplast is carried out by pumping protons into the thylakoid for driving ATP synthesis. Inspired by ion active transport in living organisms, we attempted to design an artificial ion pump induced by subnanoconfinement effects. This ionic device uses two polarity functional nanoporous films as ion-selective valves at both ends and UiO-66 metal-organic framework-filled microchannels as ion storage cavities. In the charging process, ions could be pumped into the central cavities by nanovalves, which produced an ion gradient 10 to 100 times higher than the bulk, and were trapped within the subnanocages by dehydration. In the discharging process, the enriched ions were rehydrated and slowly released by the surface charge of the nanovalves, producing a sustainable ion current. The ion storage efficiency of this nanofluidic device could be improved to 60.3%, and the release time of ion current was also prolonged by 1 order of magnitude. This work combines the active and passive transport of ions to realize fast storage and slow release of ionic current, which provides an ion gradient-mediated novel energy conversion strategy.

The exceptional form and function of the giant bacterium Ca. Epulopiscium viviparus revolves around its sodium motive force.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

Heme-copper and Heme O2-derived synthetic (bioinorganic) chemistry toward an understanding of cytochrome c oxidase dioxygen chemistry

Cytochrome c oxidase (CcO), also widely known as mitochondrial electron-transport-chain complex IV, is a multi-subunit transmembrane protein responsible for catalyzing the last step of the electron transport chain, dioxygen reduction to water, which is essential to the establishment and maintenance of the membrane proton gradient that drives ATP synthesis. Although many intermediates in the CcO catalytic cycle have been spectroscopically and/or computationally authenticated, the specifics regarding the IP intermediate, hypothesized to be a heme-Cu (hydro)peroxo species whose O−O bond homolysis is supported by a hydrogen-bonding network of water molecules, are largely obscured by the fast kinetics of the A (FeIII–O2•–/CuI/Tyr) → PM (FeIV=O/CuII–OH/Tyr•) step. In this review, we have focused on the recent advancements in the design, development, and characterization of synthetic heme-peroxo‑copper model complexes, which can circumvent the abovementioned limitation, for the investigation of the formation of IP and its O−O cleavage chemistry. Novel findings regarding (a) proton and electron transfer (PT/ET) processes, together with their contributions to exogenous phenol induced O−O cleavage, (b) the stereo-electronic tunability of the secondary coordination sphere (especially hydrogen-bonding) on the geometric and spin state alteration of the heme-peroxo‑copper unit, and (c) a plausible mechanism for the Tyr-His cofactor biogenesis, are discussed in great detail. Additionally, since the ferric-superoxide and the ferryl-oxo (Compound II) species are critically involved in the CcO catalytic cycle, this review also highlights a few fundamental aspects of these heme-only (i.e., without copper) species, including the structural and reactivity influences of electron-donating trans-axial ligands and Lewis acid-promoted H-bonding.

Pyrroloquinoline quinone drives ATP synthesis in vitro and in vivo and provides retinal ganglion cell neuroprotection

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.

Extracellular electron transfer drives ATP synthesis for nitrogen fixation by Pseudomonas stutzeri

Biological nitrogen fixation is a key step in the reduction of N2 to available nitrogen in the global nitrogen cycle. Pseudomonas stutzeri A1501 is an electroactive diazotroph and previous studies have shown that its nitrogen fixation performance is better in a micro-oxygen environment than in an oxygen-free environment. In this study, a bioelectrochemical system (nitrogen fixation in an anode chamber) was set up to explore whether extracellular electrodes can replace oxygen in acting as electron acceptors to drive ATP synthesis for nitrogen fixation by P. stutzeri under oxygen-free conditions. Nitrogenase activity, extracellular NH4+ production, increase of total nitrogen, 15N/14N atom ratio and the genes related to nitrogen fixation by P. stutzeri in the anodic bioelectrochemical group under oxygen-free conditions were at least 1.64 times higher than the corresponding values without electron output to the anode. The planktonic cells in the anode chamber were responsible for most of the electron output via an electron shuttle–electron transfer pathway. The transmembrane proton motive force produced by the transfer of electrons from the intracellular environment to the anode drives ATP synthesis to meet the high energy demand of the nitrogen fixation reaction in the absence of O2. These findings provide a basis for optimization of the nitrogen fixing performance of P. stutzeri in an oxygen-free environment.

MitoQ as an antenatal antioxidant treatment improves markers of lung maturation in healthy and hypoxic pregnancy.

Chronic fetal hypoxaemia is a common pregnancy complication that increases the risk of infants experiencing respiratory complications at birth. In turn, chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in animal models of hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. However, whether antenatal antioxidant therapy confers any benefit on lung development in complicated pregnancies has not yet been investigated. Here, we tested the hypothesis that maternal antenatal treatment with MitoQ will protect the developing lung in hypoxic pregnancy in sheep, a species with similar fetal lung developmental milestones as humans. Maternal treatment with MitoQ during late gestation promoted fetal pulmonary surfactant maturation and an increase in the expression of lung mitochondrial complexes III and V independent of oxygenation. Maternal treatment with MitoQ in hypoxic pregnancy also increased the expression of genes regulating liquid reabsorption in the fetal lung. These data support the hypothesis tested and suggest that MitoQ as an antenatal targeted antioxidant treatment may improve lung maturation in the late gestation fetus. KEY POINTS: Chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. MitoQ is a targeted antioxidant that uses the cell and the mitochondrial membrane potential to accumulate within the mitochondria. Treatment of healthy or hypoxic pregnancy with MitoQ, increases the expression of key molecules involved in surfactant maturation, lung liquid reabsorption and in mitochondrial proteins driving ATP synthesis in the fetal sheep lung. There were no detrimental effects of MitoQ treatment alone on the molecular components measured in the present study, suggesting that maternal antioxidant treatment has no effect on other components of normal maturation of the surfactant system.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

The Role of TCA Cycle Enzymes in Plants.

As one of the iconic pathways in plant metabolism, the tricarboxylic acid (TCA) cycle is commonly thought to not only be responsible for the oxidization of respiratory substrate to drive ATP synthesis but also provide carbon skeletons to anabolic processes and contribute to carbon-nitrogen interaction and biotic stress responses. The functions of the TCA cycle enzymes are characterized by a saturation transgenesis approach, whereby the constituent expression of proteins is knocked out or reduced in order to investigate their functionin vivo. The alteration of TCA cycle enzyme expression results in changed plant growth and photosynthesis under controlled conditions. Moreover, improvements in plant performance and postharvest properties are reported by overexpression of either endogenous forms or heterologous genes of a number of the enzymes. Given the importance of the TCA cycle in plant metabolism regulation, here, the function of each enzyme and its roles in different tissues are discussed. This article additionally highlights the recent finding that the plant TCA cycle, like that of mammals and microbes, dynamically assembles functional substrate channels or metabolons and discusses the implications of this finding to the current understanding of the metabolic regulation of the plant TCA cycle.

TELP theory: Elucidating the major observations of Rieger et al. 2021 in mitochondria

The transmembrane-electrostatically localized protons (TELP) theory may represent a complementary development to Mitchell's chemiosmotic theory. The combination of the two together can now excellently explain the energetics in mitochondria. My calculated transmembrane-attractive force between an excess proton and an excess hydroxide explains how TELP may stay within a 1-nm thin layer at the liquid-membrane interface. Consequently, any pH sensor (sEcGFP) located at least 2–3 nm away from the membrane surface will not be able to see TELP. This feature as predicted from the TELP model was observed exactly in the experiment of Rieger et al., 2021. In contrast to their belief “the Δp at ATP synthase is almost negligible under OXPHOS conditions”, I find, when TELP activity is included in the energy calculations, there is plenty of total protonic Gibbs free energy (ΔGT) well above the physiologically required value of −24.5 kJ mol−1 to drive ATP synthesis through FoF1-ATP synthase.

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.

Discovery of 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as new cytochrome bd oxidase inhibitors for tuberculosis therapy

The cytochrome bcc-aa3 oxidase (Cyt-bcc) of Mycobacterium tuberculosis (Mtb) is a promising anti-tuberculosis target. However, when Cyt-bcc is inhibited, cytochrome bd terminal oxidase (Cyt-bd) can still maintain the activity of the respiratory chain and drive ATP synthesis. Through virtual screening and biological validation, we discovered two FDA-approved drugs, ivacaftor and roquinimex, exhibited moderate binding affinity to Cyt-bd. Structural modifications of them led to 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as potent new Cyt-bd inhibitors. Compound 8d binds to Cyt-bd with a Kd value of 4.17 μM and inhibits the growth of the Cyt-bcc knock-out strain (ΔqcrCAB, Cyt-bd+) with a MIC value of 6.25 μM. The combination of 8d with the Cyt-bcc inhibitor Q203 completely inhibited oxygen consumption of the wild-type strain and the inverted-membrane vesicles expressing M. tuberculosis Cyt-bd (ΔcydAB::MtbCydAB+). Our study provides a promising starting point for the development of novel dual chemotherapies for tuberculosis.

Type-B Energetic Processes and Their Associated Scientific Implications

Recently, our work has identified two thermodynamically distinct types (A and B) of energetic processes naturally occurring on Earth: Type-A energy processes such as the classical heat engines, ATP hydrolysis, and many of the known chemical, electrical, and mechanical processes apparently well follow the second law of thermodynamics; and Type-B energy processes, such as the newly discovered thermotrophic function that isothermally utilizes environmental heat energy to do useful work in driving ATP synthesis, which follows the first law of thermodynamics (conservation of mass and energy), but does not necessarily have to be constrained by the second law, owing to their special asymmetric functions. In mitochondria, their special asymmetric functions associated with Type-B processes comprise: 1) The transmembrane asymmetry of inner mitochondrial membrane structure with the protonic outlets of redox-driven proton-pumping protein complexes protruded away from the membrane surface by about 1-3 nm into the bulk liquid p-phase while the protonic inlet of the F0F1-ATP synthase located rightly at the transmembrane electrostatically localized proton (TELP) layer; and 2) The lateral asymmetry of mitochondrial cristae with an ellipsoidal shape that enhances the density of TELP at the cristae tips where the F0F1-ATP synthase enzymes are located in supporting the TELP-associated thermotrophic function. The identification of Type-B energy processes indicates that there is an entirely new world of physical and energy sciences yet to be fully uncovered. Innovative efforts on Type-B processes to enable isothermally utilizing endless environmental heat energy could help to liberate all peoples from their dependence of fossil fuel energy, thus helping to reduce greenhouse gas CO2 emissions and control climate change toward a sustainable future for the humanity on Earth.

Silencing mouse circular RNA circSlc8a1 by circular antisense cA-circSlc8a1 induces cardiac hepatopathy

Circular RNAs (circRNAs) are a group of non-coding RNAs with a unique circular structure generated by back-splicing. It is acknowledged that circRNAs play critical roles in cardiovascular diseases. However, functional studies of circRNAs were impeded due to lack of effective invivo silencing approaches. Since most circRNAs are produced by protein-coding transcripts, gene editing typically affects the coding activity of the parental genes. In this study, we developed a circular antisense RNA (cA-circSlc8a1) that could silence the highly expressed circRNA circSlc8a1 in the mouse heart but not its parental Slc8a1 linear mRNA. Transgenic cA-circSlc8a1 mice developed congestive heart failure resulting in a significant increase in the body weight secondary to peripheral edema and congestive hepatopathy. To further test the role of circSlc8a1, we generated transgenic mice overexpressing circSlc8a1 and observed a protective effect of circSlc8a1 in a pressure overload model. Mechanistically, we found that circSlc8a1 translocated into mitochondria to drive ATP synthesis. While establishing a transgenic murine model for antisense-mediated circRNA silencing without interfering with the parental linear RNA, our finding revealed the essential role of circSlc8a1 in maintaining heart function and may lay the groundwork of using the circular antisense RNA as a potential gene therapy approach for cardiovascular diseases.

Advances in the Understanding of the Lifecycle of Photosystem II.

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair.

Proteome Expression and Survival Strategies of a Proteorhodopsin-Containing Vibrio Strain under Carbon and Nitrogen Limitation.

ABSTRACTPhotoheterotrophy is a widespread mode of microbial metabolism, notably in the oligotrophic surface ocean, where microbes experience chronic nutrient limitation. One especially widespread form of photoheterotrophy is based on proteorhodopsin (PR), which uses light to generate proton motive force that can drive ATP synthesis, flagellar movement, or nutrient uptake. To clarify the physiological benefits conferred by PR under nutrient stress conditions, we quantified protein-level gene expression of Vibrio campbellii CAIM 519 under both carbon and nitrogen limitation and under both light and dark conditions. Using a novel membrane proteomics strategy, we determined that PR expression is higher under C limitation than N limitation but is not light regulated. Despite expression of PR photosystems, V. campbellii does not exhibit any growth or survival advantages in the light and only a few proteins show significant expression differences between light and dark conditions. While protein-level proteorhodopsin expression in V. campbellii is clearly responsive to nutrient limitation, photoheterotrophy does not appear to play a central role in the survival physiology of this organism under these nutrient stress conditions. C limitation and N limitation, however, result in very different survival responses: under N-limited conditions, viability declines, cultivability is lost rapidly, central carbon flux through the Entner-Doudoroff pathway is increased, and ammonium is assimilated via the GS-GOGAT pathway. In contrast, C limitation drives cell dwarfing with maintenance of viability, as well as utilization of the glyoxylate shunt, glutamate dehydrogenase and anaplerotic C fixation, and a stringent response mediated by the Pho regulon.IMPORTANCE Understanding the nutrient stress responses of proteorhodopsin-bearing microbes like Vibrio campbellii yields insights into microbial contributions to nutrient cycling, lifestyles of emerging pathogens in aquatic environments, and protein-level adaptations implemented during times of nutrient limitation. In addition to its broad taxonomic and geographic prevalence, the physiological role of PR is diverse, so we developed a novel proteomics strategy to quantify its expression at the protein level. We found that proteorhodopsin expression levels in this wild-type photoheterotroph under these experimental conditions, while higher under C than under N limitation, do not afford measurable light-driven growth or survival advantages. Additionally, this work links differential protein expression patterns between C- and N-limited cultures to divergent stationary-phase survival phenotypes.

Quinone binding sites of cyt bc complexes analysed by X-ray crystallography and cryogenic electron microscopy.

Cytochrome (cyt) bc1, bcc and b6f complexes, collectively referred to as cyt bc complexes, are homologous isoprenoid quinol oxidising enzymes present in diverse phylogenetic lineages. Cyt bc1 and bcc complexes are constituents of the electron transport chain (ETC) of cellular respiration, and cyt b6f complex is a component of the photosynthetic ETC. Cyt bc complexes share in general the same Mitchellian Q cycle mechanism, with which they accomplish proton translocation and thus contribute to the generation of proton motive force which drives ATP synthesis. They therefore require a quinol oxidation (Qo) and a quinone reduction (Qi) site. Yet, cyt bc complexes evolved to adapt to specific electrochemical properties of different quinone species and exhibit structural diversity. This review summarises structural information on native quinones and quinone-like inhibitors bound in cyt bc complexes resolved by X-ray crystallography and cryo-EM structures. Although the Qi site architecture of cyt bc1 complex and cyt bcc complex differs considerably, quinone molecules were resolved at the respective Qi sites in very similar distance to haem bH. In contrast, more diverse positions of native quinone molecules were resolved at Qo sites, suggesting multiple quinone binding positions or captured snapshots of trajectories toward the catalytic site. A wide spectrum of inhibitors resolved at Qo or Qi site covers fungicides, antimalarial and antituberculosis medications and drug candidates. The impact of these structures for characterising the Q cycle mechanism, as well as their relevance for the development of medications and agrochemicals are discussed.

Differences in Bioenergetic Metabolism of Obligately Alkaliphilic Bacillaceae Under High pH Depend on the Aeration Conditions.

Alkaliphilic Bacillaceae appear to produce ATP based on the H+-based chemiosmotic theory. However, the bulk-based chemiosmotic theory cannot explain the ATP production in alkaliphilic bacteria because the H+ concentration required for driving ATP synthesis through the ATPase does not occur under the alkaline conditions. Alkaliphilic bacteria produce ATP in an H+-diluted environment by retaining scarce H+ extruded by the respiratory chain on the outer surface of the membrane and increasing the potential of the H+ for ATP production on the outer surface of the membrane using specific mechanisms of ATP production. Under high-aeration conditions, the high ΔΨ (ca. -170 mV) of the obligate alkaliphilic Evansella clarkii retains H+ at the outer surface of the membrane and increases the intensity of the protonmotive force (Δp) per H+ across the membrane. One of the reasons for the production of high ΔΨ is the Donnan potential, which arises owing to the induction of impermeable negative charges in the cytoplasm. The intensity of the potential is further enhanced in the alkaliphiles compared with neutralophiles because of the higher intracellular pH (ca. pH 8.1). However, the high ΔΨ observed under high-aeration conditions decreased (∼ -140 mV) under low-aeration conditions. E. clarkii produced 2.5–6.3-fold higher membrane bound cytochrome c in the content of the cell extract under low-aeration conditions than under high-aeration conditions. The predominant membrane-bound cytochrome c in the outer surface of the membrane possesses an extra Asn-rich segment between the membrane anchor and the main body of protein. This structure may influence the formation of an H+-bond network that accumulates H+ on the outer surface of the membrane. Following accumulation of the H+-bond network producing cytochrome c, E. clarkii constructs an H+ capacitor to overcome the energy limitation of low aeration at high pH conditions. E. clarkii produces more ATP than other neutralophilic bacteria by enhancing the efficacy per H+ in ATP synthesis. In low H+ environments, E. clarkii utilizes H+ efficiently by taking advantage of its high ΔΨ under high-aeration conditions, whereas under low-aeration conditions E. clarkii uses cytochrome c bound on its outer surface of the membrane as an H+ capacitor.

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.