Inhibition Of Cytochrome Oxidase Research Articles

A Functional Approach towards Understanding the Role of the Mitochondrial Respiratory Chain in an Endomycorrhizal Symbiosis

Arbuscular mycorrhizal fungi (AMF) are crucial components of fertile soils, able to provide several ecosystem services for crop production. Current economic, social and legislative contexts should drive the so-called “second green revolution” by better exploiting these beneficial microorganisms. Many challenges still need to be overcome to better understand the mycorrhizal symbiosis, among which (i) the biotrophic nature of AMF, constraining their production, while (ii) phosphate acts as a limiting factor for the optimal mycorrhizal inoculum application and effectiveness. Organism fitness and adaptation to the changing environment can be driven by the modulation of mitochondrial respiratory chain, strongly connected to the phosphorus processing. Nevertheless, the role of the respiratory function in mycorrhiza remains largely unexplored. We hypothesized that the two mitochondrial respiratory chain components, alternative oxidase (AOX) and cytochrome oxidase (COX), are involved in specific mycorrhizal behavior. For this, a complex approach was developed. At the pre-symbiotic phase (axenic conditions), we studied phenotypic responses of Rhizoglomus irregulare spores with two AOX and COX inhibitors [respectively, salicylhydroxamic acid (SHAM) and potassium cyanide (KCN)] and two growth regulators (abscisic acid – ABA and gibberellic acid – Ga3). At the symbiotic phase, we analyzed phenotypic and transcriptomic (genes involved in respiration, transport, and fermentation) responses in Solanum tuberosum/Rhizoglomus irregulare biosystem (glasshouse conditions): we monitored the effects driven by ABA, and explored the modulations induced by SHAM and KCN under five phosphorus concentrations. KCN and SHAM inhibited in vitro spore germination while ABA and Ga3 induced differential spore germination and hyphal patterns. ABA promoted mycorrhizal colonization, strong arbuscule intensity and positive mycorrhizal growth dependency (MGD). In ABA treated plants, R. irregulare induced down-regulation of StAOX gene isoforms and up-regulation of genes involved in plant COX pathway. In all phosphorus (P) concentrations, blocking AOX or COX induced opposite mycorrhizal patterns in planta: KCN induced higher Arum-type arbuscule density, positive MGD but lower root colonization compared to SHAM, which favored Paris-type formation and negative MGD. Following our results and current state-of-the-art knowledge, we discuss metabolic functions linked to respiration that may occur within mycorrhizal behavior. We highlight potential connections between AOX pathways and fermentation, and we propose new research and mycorrhizal application perspectives.

Eight fatalities due to drinking methanol-tainted alcohol in Pakistan: A case report

Methanol has a widespread commercial use as a solvent in paints, varnishes, anti-freeze solutions and denaturant for ethanol. Exposure may occur due to accidental, suicidal ingestion or as a result of consuming adulterated liquor. Fatalities were reported in Pakistan in an incident after consuming methanol-tainted liquor. Postmortem specimens of eight deceased males, ages ranged from 16 to 40yrs, were submitted for toxicological analysis. Presence of blurred vision, severe metabolic acidosis with decreased serum bicarbonate level, increased serum osmolality and mean anion gap before death strongly suggested methanol toxicity. Headspace gas chromatograph coupled to flame ionization detector was used to quantify volatiles in blood and stomach contents of victims. Lethal levels of methanol in addition to ethanol were detected. The most probable mechanism of methanol-related deaths was sudden cessation of respiration due to inhibition of cytochrome oxidase that led to histotoxic hypoxia.

Successful Treatment of Aluminum Phosphide Poisoning with Continuous Veno-venous Hemofiltration: A Case Report

Address for Correspondence: Ayca Acikalin, Department of Emergency Medicine, Cukurova University School of Medicine, Adana, Turkey E-mail: aycaacikalin@yahoo.com ©Copyright 2016 by Emergency Physicians Association of Turkey Available online at www.jemcr.org Introduction Aluminum phosphide (AP) is a pesticide that has been used since the 1940s (1). It is a metal phosphide compound (Na3P, AlP) and is used as a rodenticide and insecticide. AP in an evaporable tablet form is used in wheat storage to reduce the number of mice attacks. This form causes fatalities, in suicidal exposure in particular. In countries such as Turkey, Iran, and India, public awareness of deadly exposure results in oral intake during suicide attempts (1). Gastric hydrochloric acid (HCl) causes the release of phosphine in the stomach after swallowing metal phosphide compounds. Phosphine induces cellular lipid peroxidation by means of the inhibition of mitochondrial cytochrome oxidase and oxygen utilization. This influence usually presents with hypotension, acidosis, and multiple organ failure (MOF) resistant to treatment. Mortality is extremely high and there is no identified antidote. Gastric irrigation with potassium and the replacement of intravenous magnesium have been suggested for treatment (1).

Oleanolic Acid Inhibits High Salt-Induced Exaggeration of Warburg-like Metabolism in Breast Cancer Cells.

Cancer cells have a proliferative advantage by utilizing intermediates of aerobic glycolysis (Warburg effect) for their macromolecule synthesis. Although the exact causes of this Warburg effect are unclear, high osmotic stress in solid tumor microenvironment is considered one of the important factors. Oleanolic acid (OA) is known to exert anti-inflammatory and anti-cancer effect. In our current studies, using breast cancer cell lines, we determined the protective role of OA in high salt-mediated osmotic stress-induced cancer growth. Hypertonic (0.16M NaCl) culture conditions enhanced the cancer cell growth (26%, p<0.05) and aerobic glycolysis as marked by increased glucose consumption (34%, p<0.05) and lactate production (25%, p<0.05) over untreated cells. This effect was associated with increased expression and activity of key rate-limiting enzymes of aerobic glycolysis, namely hexokinase, pyruvate kinase type M2, and lactate dehydrogenase A. Interestingly, this high salt-mediated enhanced expression of aerobic glycolytic enzymes was efficiently reversed by OA along with the decreased cancer cell proliferation. In cancer cells, enhanced aerobic glycolysis is associated with the decreased mitochondrial activity and mitochondrial-associated caspase activity. As expected, high salt further inhibited the mitochondrial related cytochrome oxidase and caspase-3 activity. However, OA efficiently reversed the high salt-mediated inhibition of cytochrome oxidase, caspase activity, and pro-apoptotic Bax expression, thus suggesting that OA induced mitochondrial activity and enhanced apoptosis. Taken together, our data indicate that OA efficiently reverses the enhanced Warburg-like metabolism induced by high salt-mediated osmotic stress along with potential application of OA in anti-cancer therapy.

Positive feedback during sulfide oxidation fine-tunes cellular affinity for oxygen

Sulfide (H2S in the gas form) is the third gaseous transmitter found in mammals. However, in contrast to nitric oxide (NO) or carbon monoxide (CO), sulfide is oxidized by a sulfide quinone reductase and generates electrons that enter the mitochondrial respiratory chain arriving ultimately at cytochrome oxidase, where they combine with oxygen to generate water. In addition, sulfide is also a strong inhibitor of cytochrome oxidase, similar to NO, CO and cyanide. The balance between the electron donor and the inhibitory role of sulfide is likely controlled by sulfide and oxygen availability. The present study aimed to evaluate if and how sulfide release and oxidation impacts on the cellular affinity for oxygen. Results: i) when sulfide delivery approaches the maximal sulfide oxidation rate cells become exquisitely dependent on oxygen; ii) a positive feedback makes the balance between sulfide-releasing and -oxidizing rates the relevant parameter rather than the absolute values of these rates, and; iii) this altered dependence on oxygen is detected with sulfide concentrations that remain in the low micromolar range. Conclusions: i) within the context of continuous release of sulfide stemming from cellular metabolism, alterations in the activity of the sulfide oxidation pathway fine-tunes the cell's affinity for oxygen, and; ii) a decrease in the expression of the sulfide oxidation pathway greatly enhances the cell's dependence on oxygen concentration.

Ceramide channel: Structural basis for selective membrane targeting

A ceramide commonly found in mammalian cells, C16-ceramide (N-palmitoyl-d-erythro-sphingosine), is capable of forming large, protein-permeable channels in the mitochondrial outer membrane (MOM). However, C16-ceramide is unable to permeabilize the plasma membrane of erythrocytes. This specificity is unexpected considering that ceramide forms channels in simple phosphoglycerolipid membranes. Synthetic analogs of C16-ceramide with targeted changes at each of the functional regions of the molecule including methylation, altered hydrocarbon chain length, and changes in the stereochemistry, were tested to probe the role of ceramide’s molecular features on its ability to form channels in these two different membrane types. The ability to permeabilize the MOM was relatively insensitive to modifications of the various functional groups of ceramide whereas the same modifications resulted in plasma membrane permeabilization (a gain of function rather than a loss of function). Some analogs (ceramine, NBD-labeled ceramide, C18,1 ceramide) gained another function, the ability to inhibit cytochrome oxidase. The gain of deleterious functions indicates that constraints on the structure of ceramide that is formed by the cell's synthetic machinery includes the avoidance of deleterious interactions. We propose that the specific structure of ceramide limits the size of its interactome (both proteins and lipids) thus reducing the likelihood of unwanted side effects.

COX assembly factor ccdc56 regulates mitochondrial morphology by affecting mitochondrial recruitment of Drp1

Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.

Bedaquiline for the treatment of multidrug-resistant tuberculosis: great promise or disappointment?

Acquired drug resistance by Mycobacterium tuberculosis (MTB) may result in treatment failure and death. Bedaquiline was recently approved for the treatment of multidrug-resistant tuberculosis (MDR-TB). This report examines the available data on this novel drug for the treatment of MDR-TB. PubMed searches, last updated 18 February 2015, using the terms bedaquiline, TMC 207 and R207910 identified pertinent English citations. Citation reference lists were reviewed to identify other relevant reports. Pertinent MDR-TB treatment reports on the US Food and Drug Administration, Centers for Disease Control and Prevention (CDC), World Health Organization (WHO) and Cochrane websites were also evaluated. Bedaquiline is an adenosine triphosphate (ATP) synthase inhibitor specific for MTB and some nontuberculous mycobacteria. The early bactericidal activity (EBA) of bedaquiline is delayed until ATP stores are depleted but subsequently it is similar to the EBA of isoniazid and rifampin. Bedaquiline demonstrated excellent minimum inhibitory concentrations (MICs) against both drug-sensitive and MDR-TB. Adding it to the WHO-recommended MDR-TB regimen reduced the time for sputum culture conversion in pulmonary MDR-TB. Rifampin, other cytochrome oxidase 3A4 inducers or inhibitors alter its metabolism. Adverse effects are common with MDR-TB treatment regimens with or without bedaquiline. Nausea is more common with bedaquiline and it increases the QTcF interval. It is not recommended for children, pregnant or lactating women. More patients died in the bedaquiline-treatment arms despite better microbiological outcomes in two recent trials. The WHO and CDC published interim guidelines that recommend restricting its use to patients with MDR-TB or more complex drug resistance who cannot otherwise be treated with a minimum of three effective drugs. It should never be added to a regimen as a single drug nor should it be added to a failing regimen to prevent the emergence of bedaquiline-resistant strains.

Analysis of some enzymes activities of hydrogen sulfide metabolism in plants.

Hydrogen sulfide (H2S) which is considered as a novel gasotransmitter after reactive oxygen species and nitric oxide in plants has dual character, that is, toxicity that inhibits cytochrome oxidase at high concentration and as signal molecule which is involved in plant growth, development, and the acquisition of tolerance to adverse environments such as extreme temperature, drought, salt, and heavy metal stress at low concentration. Therefore, H2S homeostasis is very important in plant cells. The level of H2S in plant cells is regulated by its synthetic and degradative enzymes, L-/D-cysteine desulfhydrase (L-/D-DES), sulfite reductase (SiR), and cyanoalanine synthase (CAS), which are responsible for H2S synthesis, while cysteine synthase (CS) takes charge of the degradation of H2S, but its reverse reaction also can produce H2S. Here, after crude enzyme is extracted from plant tissues, the activities of L-/D-DES, SiR, CAS, and CS are measured by spectrophotometry, the aim is to further understand homeostasis of H2S in plant cells and its potential mechanisms.

Cardiolipin plays a role in KCN-induced necrosis

Cardiolipin (CL) is a unique anionic, dimeric phospholipid found almost exclusively in the inner mitochondrial membrane and is essential for the function of numerous enzymes that are involved in mitochondrial energy metabolism. While the role of cardiolipin in apoptosis is well established, its involvement in necrosis is enigmatic. In the present study, KCN-induced necrosis in U937 cells was used as an experimental model to assess the role of CL in necrosis. KCN addition to U937 cells induced reactive oxygen species (ROS) formation, while the antioxidants inhibited necrosis, indicating that ROS play a role in KCN-induced cell death. Further, CL oxidation was confirmed by the monomer green fluorescence of 10-N-nonyl acridine orange (NAO) and by TLC. Utilizing the red fluorescence of the dimeric NAO, redistribution of CL in mitochondrial membrane during necrosis was revealed. We also showed that the catalytic activity of purified adenosine triphosphate (ATP) synthase complex, known to be modulated by cardiolipin, decreased following KCN treatment. All these events occurred at an early phase of the necrotic process prior to rupture of the cell membrane. Furthermore, CL-deficient HeLa cells were found to be resistant to KCN-induced necrosis as compared with the wild type cells. We suggest that KCN, an effective reversible inhibitor of cytochrome oxidase and thereby of the respiratory chain leads to ROS increase, which in turn oxidizes CL (amongst other membrane phospholipids) and leads to mitochondrial membrane lipid reorganization and loss of CL symmetry. Finally, the resistance of CL-deficient cells to necrosis further supports the notion that CL, which undergoes oxidation during necrotic cell death, is an integral part of the milieu of events taking place in mitochondria leading to membrane disorganization and mitochondrial dysfunction.

Cation Binding Site of cytochrome c oxidase: Progress report

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca2+-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the “slow” form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Successful treatment of cardiogenic shock with an intraaortic balloon pump following aluminium phosphide poisoning

Aluminium phosphide (AlP) is a highly toxic pesticide that inhibits cytochrome oxidase c and causes oxidative stress. Death results from refractory cardiogenic shock due to myocardial dysfunction. There is very little information regarding extracorporeal life support in severe AlP poisoning. Although several therapies are available, none are curative. We report on the use of an intra-aortic balloon pump (IABP) in a 24-year-old woman brought to our hospital after an intentional ingestion of a tablet of AlP (3 g), which caused refractory AlP-induced cardiogenic shock and acute respiratory distress syndrome (ARDS). The patient underwent gastric lavage with potassium permanganate, received sodium bicarbonate intravenously, and was admitted to the intensive care unit. Echocardiography at 36 h post ingestion showed a left ventricular ejection fraction (LVEF) of <20 %. An IABP was inserted and the patient’s vital signs stabilised. After eight days, the IABP was removed and on day 20, the patient’s LVEF increased to 50 %. IABP was successfully used and may improve future prognoses for severely poisoned AlP patients with refractory cardiogenic shock. We encourage clinical toxicologists to examine this new treatment.

Direct Regulation of Cytochrome c Oxidase by Calcium Ions

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50–60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s−1 or less). No inhibitory effect of Ca2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s−1 at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca2+ and is reversed by EGTA. Na+ ions that compete with Ca2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca2+. The Ca2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca2+ binding with cytochrome oxidase. Ca2+- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.

Oxygen consumption measurement is a strong tool not only to elucidate the metabolism of blastocyst, but also to select suitable blastocyst for transfer

Combination of morphology and oxygen (O2) consumption measurement has been reported to be effective for embryo selection. The present study was conducted to develop a novel method to assess the viability of blastocyst (BL) using mitochondrial oxidative phosphorylation (OXPHOS) indicated by O2 consumption with FCCP:Carbonyl cyanide-p-trifluoromethoxyphenyl hydrazine (uncoupler) and cyanide (inhibitor of mitochondrial cytochrome oxidase).

Acute toxicity of some synthetic cyanogens in rats: Time-dependent cyanide generation and cytochrome oxidase inhibition in soft tissues after sub-lethal oral intoxication

Cyanogens include complex nitrile-containing compounds that can generate free cyanide of toxicological significance. Acute toxicity, time-dependent cyanide generation and cytochrome oxidase (CYTOX) inhibition in soft tissues, and urinary thiocyanate levels were measured after acute cyanogen intoxication in rats. Order of cyanogens in terms of LD₅₀ was: malononitrile (MCN)>propionitrile (PCN)≈sodium nitroprusside (SNP)>acrylonitrile (ACN)>succinonitrile (SCN)>acetonitrile (ATCN) for oral, and SNP>MCN>ACN>PCN>SCN>ATCN for intraperitoneal and subcutaneous routes. MCN was most toxic by oral (LD₅₀=66.4 mg/kg) and SNP by intraperitoneal (LD₅₀=16.7 mg/kg) and subcutaneous (LD₅₀=11.9 mg/kg) routes. Minimum survival time (25 min) was recorded after 4.0 LD₅₀ ATCN. Order of cyanogens (0.75 LD₅₀; oral) on the basis of maximum blood cyanide and time of peak cyanide generation were: ATCN>SNP>SCN>PCN>MCN>ACN, and MCN (30 min)<SNP (1h)<PCN≈ACN (8h)<SCN (24h)<ATCN (72 h), respectively. In most cases, time profile of cyanide generation correlated with corresponding CYTOX inhibition and urinary thiocyanate levels. With the understanding of time-dependent toxicity of different cyanogens, suitable therapeutic windows can be designed for their management.

In the eye of the storm: mitochondrial damage during heart and brain ischaemia

We review research investigating mitochondrial damage during heart and brain ischaemia, focusing on the mechanisms and consequences of ischaemia-induced and/or reperfusion-induced: (a) inhibition of mitochondrial respiratory complex I; (b) release of cytochrome c from mitochondria; (c) changes to mitochondrial phospholipids; and (d) nitric oxide inhibition of mitochondria. Heart ischaemia causes inhibition of cytochrome oxidase and complex I, release of cytochrome c, and induction of permeability transition and hydrolysis and oxidation of mitochondrial phospholipids, but some of the mechanisms are unclear. Brain ischaemia causes inhibition of complexes I and IV, but other effects are less clear.

Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells

The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O2) on mitochondrial function in neonatal rat type-1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P50 of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca2+-free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (ψm, measured using rhodamine 123) with a P50 of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca2+-free Tyrode and Tyrode containing the Ca2+ channel antagonist Ni2+, respectively. In the presence of oligomycin and low carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) ψm is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised ψm/inhibited electron transport with a P50 of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl-p-phenylenediamine. Under these conditions ψm is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised ψm) with a P50 of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P50= 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type-1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained.

Effects of exogenous hydrogen sulphide on calcium signalling, background (TASK) K channel activity and mitochondrial function in chemoreceptor cells

It has been proposed that endogenous H2S mediates oxygen sensing in chemoreceptors; this study investigates the mechanisms by which H2S excites carotid body type 1 cells. H2S caused a rapid reversible increase in intracellular calcium with EC50 ≈ 6 μM. This [Ca2+]i response was abolished in Ca-free Tyrode. In perforated patch current clamp recordings, H2S depolarised type 1 cells from −59 to −35 mV; this was accompanied by a robust increase in [Ca2+]i. Voltage clamping at the resting membrane potential abolished the H2S-induced rise in [Ca2+]i. H2S inhibited background K+ current in whole cell perforated patch and reduced background K+ channel activity in cell-attached patch recordings. It is concluded that H2S excites type 1 cells through the inhibition of background (TASK) potassium channels leading to membrane depolarisation and voltage-gated Ca2+ entry. These effects mimic those of hypoxia. H2S also inhibited mitochondrial function over a similar concentration range as assessed by NADH autofluorescence and measurement of intracellular magnesium (an index of decline in MgATP). Cyanide inhibited background K channels to a similar extent to H2S and prevented H2S exerting any further influence over channel activity. These data indicate that the effects of H2S on background K channels are a consequence of inhibition of oxidative phosphorylation. Whilst this does not preclude a role for endogenous H2S in oxygen sensing via the inhibition of cytochrome oxidase, the levels of H2S required raise questions as to the viability of such a mechanism.

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus) microplus Embryos

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5′-triphosphate synthesis in hard tick embryos.

Protein Phosphorylation and Prevention of Cytochrome Oxidase Inhibition by ATP: Coupled Mechanisms of Energy Metabolism Regulation

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.