Investigation and determination of CoQ10(H2) and CoQ10(H4) species from black yeast-like fungi and filamentous fungi.

Coenzyme Q (CoQ) or ubiquinone functions as an electron transporter in the electron transport system in both prokaryotes and eukaryotes. The isoprenyl side chain of CoQ is modified in some organisms, especially in fungi, for optimal electron transport performance under various conditions. In this study, we investigated the side chain saturated dihydro CoQ (CoQ10(H2)) in Aureobasidium pullulans EXF-150, Sydowia polyspora NBRC 30562, and naturally isolated Plowrightia sp. A37, all of which are melanized Dothideomycetes species within the Ascomycota, and also in filamentous fungi Aspergillus oryzae and Aspergillus terreus. Plowrightia sp. A37 produced the rarely synthesized tetrahydro type CoQ10(H4), especially in glucose-rich medium, during extended cultivation in contrast to CoQ10(H2) in time-limited cultivation. Using liquid chromatography-mass spectrometry, we identified demethoxyubiquinone-H2 (DMQ(H2)) as an indicative intermediate that suggests that the side chain-saturation of CoQ occurs after the formation of DMQ and not always in the last step as previously considered.

Physiological responses of the invasive blue crabs Callinectes sapidus to salinity variations: Implications for adaptability and invasive success.

This study provides a comprehensive analysis of the eco-physiological responses of the blue crab (Callinectes sapidus) to variations in salinity, shedding light on its adaptability and invasive success in aquatic environments. Gender-specific differences in osmoregulation and Electron Transport System (ETS) activity highlight the importance of considering sex-specific aspects when understanding the physiological responses of invasive species. Females exhibited increased ETS activity at lower salinities, potentially indicative of metabolic stress, while males displayed constant ETS activity across a range of salinities. Osmoregulatory capacity which depended on gender and salinity, was efficient within meso-polyhaline waters but decreased at higher salinities, particularly in males. These findings provide valuable understandings into how C. sapidus specimens in an invaded area responds to salinity changes, important for considerate its distribution through saline pathways during tidal cycle fluctuations. This study shows the importance of interdisciplinary research for effective management of invasive species and conservation of affected aquatic ecosystems.

Effect of magnetic powder (Fe3O4) on heterotrophic-sulfur autotrophic denitrification efficiency and electron transport system activity for marine recirculating aquacultural wastewater treatment.

As an efficient nitrogen removal process, heterotrophic-sulfur autotrophic denitrification (HSAD) has attracted extensive attention in wastewater treatment. However, the effects of magnetic powder (Fe3O4) on the electron transport activity in HSAD process remain unclear. Therefore, in this study, a heterotrophic-sulfur autotrophic denitrification system was established to remove nitrogen from marine recirculating aquacultural wastewater for evaluating the effects of Fe3O4. At the optimal Fe3O4 concentration of 50 mg/L, the nitrogen removal efficiency reached 100% with lower sulfate accumulation, and the start-up time was shortened. The assays of denitrifying enzymes and electron transport system activity showed that Fe3O4 improved the activities of nitrate and nitrite reductases, and increased the efficiency of electron transport. Microbial community analysis revealed that Fe3O4 enriched heterotrophic denitrifier Thauera and sulfur autotrophic denitrifier Canditatus Thiobios, and thus enhanced denitrification efficiencies. This study demonstrated that Fe3O4 is an efficient denitrification accelerator in HSAD for treating marine recirculating aquacultural wastewater.

Enhanced mitochondrial buffering prevents Ca2+ overload in naked mole-rat brain.

Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia-mediated increases in cytosolic Ca2+ are retarded in hypoxia-tolerant naked mole-rat brain. We hypothesized that naked mole-rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole-rat cortical tissue. We report that naked mole-rat brain mitochondria buffer >2-fold more exogenous Ca2+ than mouse brain mitochondria, and that the half-maximal inhibitory concentration (IC50 ) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2-fold higher in naked mole-rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm ), and the IC50 at which Ca2+ decreases Δψm is ∼4-fold higher in naked mole-rat than mouse brain. The ability of naked mole-rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole-rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole-rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia-tolerance of naked mole-rat brain. KEY POINTS: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia-mediated Ca2+ influx into naked mole-rat brain is markedly reduced relative to mice. This is important because naked mole-rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain. We show that in hypoxic naked mole-rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress. This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential-dependant mechanism. The unique ultrastructure of naked mole-rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia-tolerance.

Consistent changes in muscle metabolism underlie dive performance across multiple lineages of diving ducks.

Diving animals must sustain high activity with limited O2 stores to successfully capture prey. Studies suggest that increasing body O2 stores supports breath-hold diving, but less is known about metabolic specializations that underlie underwater locomotion. We measured maximal activities of 10 key enzymes in locomotory muscles (gastrocnemius and pectoralis) to identify biochemical changes associated with diving in pathways of oxidative and substrate-level phosphorylation and compared them across three groups of ducks-the longest diving sea ducks (eight spp.), the mid-tier diving pochards (three spp.) and the non-diving dabblers (five spp.). Relative to dabblers, both diving groups had increased activities of succinate dehydrogenase and cytochrome c oxidase, and sea ducks further showed increases in citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HOAD). Both diving groups had relative decreases in capacity for anaerobic metabolism (lower ratio of lactate dehydrogenase to CS), with sea ducks also showing a greater capacity for oxidative phosphorylation and lipid oxidation (lower ratio of pyruvate kinase to CS, higher ratio of HOAD to hexokinase). These data suggest that the locomotory muscles of diving ducks are specialized for sustaining high rates of aerobic metabolism, emphasizing the importance of body O2 stores for dive performance in these species.

Acute pH alterations do not impact cardiac mitochondrial respiration in naked mole-rats or mice.

Energetically demanding conditions such as hypoxia and exercise favour anaerobic metabolism (glycolysis), which leads to acidification of the cellular milieu from ATP hydrolysis and accumulation of the anaerobic end-product, lactate. Cellular acidification may damage mitochondrial proteins and/or alter the H+ gradient across the mitochondrial inner membrane, which may in turn impact mitochondrial respiration and thus aerobic ATP production. Naked mole-rats are among the most hypoxia-tolerant mammals, and putatively experience intermittent environmental and systemic hypoxia while resting and exercising in their underground burrows. Previous studies in naked mole-rat brain, heart, and skeletal muscle mitochondria have demonstrated adaptations that favour improved efficiency in hypoxic conditions; however, the impact of cellular acidification on mitochondrial function has not been explored. We hypothesized that, relative to hypoxia-intolerant mice, naked mole-rat cardiac mitochondrial respiration is less sensitive to cellular pH changes. To test this, we used high-resolution respirometry to measure mitochondrial respiration by permeabilized cardiac muscle fibres from naked mole-rats and mice exposed in vitro to a pH range from 6.6 to 7.6. Surprisingly, we found that acute pH changes do not impact cardiac mitochondrial respiration or compromise mitochondrial integrity in either species. Our results suggest that acute alterations of cellular pH have minimal impact on cardiac mitochondrial respiration.

What to do with low O2: Redox adaptations in vertebrates native to hypoxic environments.

Reactive oxygen species (ROS) are important cellular signalling molecules but sudden changes in redox balance can be deleterious to cells and lethal to the whole organism. ROS production is inherently linked to environmental oxygen availability and many species live in variable oxygen environments that can range in both severity and duration of hypoxic exposure. Given the importance of redox homeostasis to cell and animal viability, it is not surprising that early studies in species adapted to various hypoxic niches have revealed diverse strategies to limit or mitigate deleterious ROS changes. Although research in this area is in its infancy, patterns are beginning to emerge in the suites of adaptations to different hypoxic environments. This review focuses on redox adaptations (i.e., modifications of ROS production and scavenging, and mitigation of oxidative damage) in hypoxia-tolerant vertebrates across a range of hypoxic environments. In general, evidence suggests that animals adapted to chronic lifelong hypoxia are in homeostasis, and do not encounter major oxidative challenges in their homeostatic environment, whereas animals exposed to seasonal chronic anoxia or hypoxia rapidly downregulate redox balance to match a hypometabolic state and employ robust scavenging pathways during seasonal reoxygenation. Conversely, animals adapted to intermittent hypoxia exposure face the greatest degree of ROS imbalance and likely exhibit enhanced ROS-mitigation strategies. Although some progress has been made, research in this field is patchy and further elucidation of mechanisms that are protective against environmental redox challenges is imperative for a more holistic understanding of how animals survive hypoxic environments.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

Naked mole-rat brain mitochondria tolerate in vitro ischaemia.

Naked mole-rats (NMRs; Heterocephalus glaber) are among the most hypoxia-tolerant mammals. There is evidence that the NMR brain tolerates in vitro hypoxia and NMR brain mitochondria exhibit functional plasticity following in vivo hypoxia; however, if and how these organelles tolerate ischaemia and how ischaemic stress impacts mitochondrial energetics and redox regulation is entirely unknown. We hypothesized that mitochondria fundamentally contribute to in vitro ischaemia resistance in the NMR brain. To test this, we treated NMR and CD-1 mouse cortical brain sheets with an in vitro ischaemic mimic and evaluated mitochondrial respiration capacity and redox regulation following 15 or 30 min of ischaemia or ischaemia/reperfusion (I/R). We found that, relative to mice, the NMR brain largely retains mitochondrial function and redox balance post-ischaemia and I/R. Specifically: (i) ischaemia reduced complex I and II-linked respiration ∼50-70% in mice, vs. ∼20-40% in NMR brain, (ii) NMR but not mouse brain maintained relatively steady respiration control ratios and robust mitochondrial membrane integrity, (iii) electron leakage post-ischaemia was lesser in NMR than mouse brain and NMR brain retained higher coupling efficiency, and (iv) free radical generation during and following ischaemia and I/R was lower from NMR brains than mice. Taken together, our results indicate that NMR brain mitochondria are more tolerant of ischaemia and I/R than mice and retain respiratory capacity while avoiding redox derangements. Overall, these findings support the hypothesis that hypoxia-tolerant NMR brain is also ischaemia-tolerant and suggest that NMRs may be a natural model of ischaemia tolerance in which to investigate evolutionarily derived solutions to ischaemic pathology. KEY POINTS: Ischaemia is highly deleterious to the mammalian brain and this damage is largely mediated by mitochondrial dysfunction. Naked mole-rats are among the most hypoxia-tolerant mammals and their brain tolerates ischaemia ex vivo, but the impact of ischaemia on mitochondrial function is unknown. Naked mole-rat but not mouse brain mitochondria retain respiratory capacity and membrane integrity following ischaemia or ischaemia/reperfusion. Differences in free radical management and respiratory pathway control between species may mediate this tolerance. These results help us understand how natural models of hypoxia tolerance also tolerate ischaemia in the brain.

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through ß-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP- and low-ADP-stimulated respiration that accelerating flux through ß-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (∼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within ß-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.

Graduate Student Literature Review: Mitochondrial adaptations across lactation and their molecular regulation in dairy cattle.

As milk production in dairy cattle continues to increase, so do the energetic and nutrient demands on the dairy cow. Difficulties making the necessary metabolic adjustments for lactation can impair lactation performance and increase the risk of metabolic disorders. The physiological adaptations to lactation involve the mammary gland and extramammary tissues that coordinately enhance the availability of precursors for milk synthesis. Changes in whole-body metabolism and nutrient partitioning are accomplished, in part, through the bioenergetic and biosynthetic capacity of the mitochondria, providing energy and diverting important substrates, such as AA and fatty acids, to the mammary gland in support of lactation. With increased oxidative capacity and ATP production, reactive oxygen species production in mitochondria may be altered. Imbalances between oxidant production and antioxidant activity can lead to oxidative damage to cellular structures and contribute to disease. Thus, mitochondria are tasked with meeting the energy needs of the cell and minimizing oxidative stress. Mitochondrial function is regulated in concert with cellular metabolism by the nucleus. With only a small number of genes present within the mitochondrial genome, many genes regulating mitochondrial function are housed in nuclear DNA. This review describes the involvement of mitochondria in coordinating tissue-specific metabolic adaptations across lactation in dairy cattle and the current state of knowledge regarding mitochondrial-nuclear signaling pathways that regulate mitochondrial proliferation and function in response to shifting cellular energy need.

Doxorubicin causes lesions in the electron transport system of skeletal muscle mitochondria that are associated with a loss of contractile function.

Doxorubicin is an anthracycline-based chemotherapeutic that causes myotoxicity with symptoms persisting beyond treatment. Patients experience muscle pain, weakness, fatigue, and atrophy, but the underlying mechanisms are poorly understood. Studies investigating doxorubicin-induced myotoxicity have reported disrupted mitochondrial function. Mitochondria are responsible for regulating both cellular energy status and Ca2+ handling, both of which impact contractile function. Moreover, loss of mitochondrial integrity may initiate muscle atrophy. Skeletal muscle mitochondrial dysregulation may therefore contribute to an overall loss of skeletal muscle quality and performance that may be mitigated by appropriately targeted mitochondrial therapies. We therefore assessed the impact of doxorubicin on muscle performance and applied a multiplexed assay platform to diagnose alterations in mitochondrial respiratory control. Mice received a clinically relevant dose of doxorubicin delivered systemically and were euthanized 72 h later. We measured extensor digitorum longus and soleus muscle forces, fatigue, and contractile kinetics in vitro, along with Ca2+ uptake in isolated sarcoplasmic reticulum. Isolated skeletal muscle mitochondria were used for real-time respirometry or frozen for protein content and activity assays. Doxorubicin impaired muscle performance, which was indicated by reduced force production, fatigue resistance, and sarcoplasmic reticulum-Ca2+ uptake, which were associated with a substrate-independent reduction in respiration and membrane potential but no changes in the NAD(P)H/NAD(P)+ redox state. Protein content and dehydrogenase activity results corroborated these findings, indicating that doxorubicin-induced mitochondrial impairments are located upstream of ATP synthase within the electron transport system. Collectively, doxorubicin-induced lesions likely span mitochondrial complexes I-IV, providing potential targets for alleviating doxorubicin myotoxicity.

The mitochondrial alternative oxidase from Chlamydomonas reinhardtii enables survival in high light.

Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.

Understanding molecular biology of codon usage in mitochondrial complex IV genes of electron transport system: Relevance to mitochondrial diseases.

The mitochondrial cytochrome oxidase (CO) genes are involved in complex IV of the electron transport system, and dysfunction of CO genes leads to several diseases. However, no work has been reported on the codon usage pattern of these genes. We used bioinformatic methods to analyze the compositional properties and the codon usage pattern of the COI, COII, and COIII genes in fishes, birds, and mammals to understand the similarities and dissimilarities of codon usage in these genes, which gave an insight into the molecular biology of these genes. The effective number of codons (ENC) value of genes was high in different species of fishes, birds and mammals, which indicates that the codon bias of CO genes was low and the ENC values were significantly different among fishes, birds, and mammals, as revealed from the t test. The overall guanine and cytosine (GC) content in fishes, birds, and mammals was lower than 50% in all genes, indicating that the genes were AT-rich and significantly different among fishes, birds, and mammals. The TCA codon was overrepresented in fishes, birds, and mammals for the COI gene, in birds and mammals for the COII gene, but it was not overrepresented in others. Only three codons, namely CTA, CGA, and AAA, were overrepresented in all three groups for the COI, COII, and COIII genes, repectively. From the neutrality plot in fishes, birds, and mammals, it was observed that the slopes of the regression lines (regression coefficients) in the COI, COII, and COIII genes were <0.5, suggesting that natural selection played a major role, whereas mutation pressure played a minor role.

Effects of carbon nanotube on denitrification performance of Alcaligenes sp. TB: Promotion of electron generation, transportation and consumption.

Multi-walled carbon nanotubes (MWCNTs) promote biodegradation in water treatment, but the effect of MWCNT on denitrification under aerobic conditions is still unclear. This investigation focused on the denitrification performance of MWCNT and its toxic effects on Alcaligenes sp. TB which showed that 30 mg/L MWCNTs increased NO3- removal efficiency from 84% to 100% and decreased the NO2-and N2O accumulation rates by 36% and 17.5%, respectively. Nitrite reductase and nitrous oxide reductase activities were further increased by 19.5% and 7.5%, respectively. The mechanism demonstrated that electron generation (NADH yield) and electron transportation system activity increased by 14.5% and 104%, respectively. Cell membrane analysis found that MWCNT caused an increase in polyunsaturated fatty acids, which had positive effects on electron transportation and membrane fluidity at a low concentration of 96 mg/kg but caused membrane lipid peroxidation and impaired membrane integrity at a high concentration of 115 mg/L. These findings confirmed that MWCNT affects the activity of Alcaligenes sp. TB and consequently enhances denitrification performance.

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Mitochondrial phenotype during torpor: Modulation of mitochondrial electron transport system in the Chilean mouse-opossum Thylamys elegans.

Mammalian torpor is a phenotype characterized by a controlled decline of metabolic rate, generally followed by a reduction in body temperature. During arousal from torpor, both metabolic rate and body temperature rapidly returns to resting levels. Metabolic rate reduction experienced by torpid animals is triggered by active suppression of mitochondrial respiration, which is rapidly reversed during rewarming process. In this study, we analyzed the changes in the maximal activity of key enzymes related to electron transport system (complexes I, III and IV) in six tissues of torpid, arousing and euthermic Chilean mouse-opossums (Thylamys elegans). We observed higher maximal activities of complexes I and IV during torpor in brain, heart and liver, the most metabolically active organs in mammals. On the contrary, higher enzymatic activities of complexes III were observed during torpor in kidneys and lungs. Moreover, skeletal muscle was the only tissue without significant differences among stages in all complexes evaluated, suggesting no modulation of oxidative capacities of electron transport system components in this thermogenic tissue. In overall, our data suggest that complexes I and IV activity plays a major role in initiation and maintenance of metabolic suppression during torpor in Chilean mouse-opossum, whereas improvement of oxidative capacities in complex III might be critical to sustain metabolic machinery in organs that remains metabolically active during torpor.