The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.     

Role of the Transmembrane Potential in the Membrane Proton Leak

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨ_m) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨ_m (similar to ΔΨ_m in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨ_m. The application of ΔΨ_m up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨ_m and the increase of FA membrane concentration bring about the significant exponential G_m increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA⁻ remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨ_m more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨ_m may be key in mitochondrial respiration and metabolism.     

Intrinsic and extrinsic uncoupling of oxidative phosphorylation

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H⁺/e⁻ stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential ΔΨ, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential ΔΨ and maintains high ΔΨ values (150-200 mV). The second occurs only in mitochondria, is suggested to keep ΔΨ at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of ΔΨ and the production of reactive oxygen species (ROS) in mitochondria at high ΔΨ values (150-200 mV) are discussed.     

Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

High efficiency versus maximal performance — The cause of oxidative stress in eukaryotes: A hypothesis

Degenerative diseases are in part based on elevated production of ROS (reactive oxygen species) in mitochondria, mainly during stress and excessive work under stress (strenuous exercise). The production of ROS increases with increasing mitochondrial membrane potential (ΔΨ_m). A mechanism is described which is suggested to keep ΔΨ_m at low values under normal conditions thus preventing ROS formation, but is switched off under stress and excessive work to maximize the rate of ATP synthesis, accompanied by decreased efficiency. Low ΔΨ_m and low ROS production are suggested to occur by inhibition of respiration at high [ATP]/[ADP] ratios. The nucleotides interact with phosphorylated cytochrome c oxidase (COX), representing the step with the highest flux-control coefficient of mitochondrial respiration. At stress and excessive work neural signals are suggested to dephosphorylate the enzyme and abolish the control of COX activity (respiration) by the [ATP]/[ADP] ratio with consequent increase of ΔΨ_m and ROS production. The control of COX by the [ATP]/[ADP] ratio, in addition, is proposed to increase the efficiency of ATP production via a third proton pumping pathway, identified in eukaryotic but not in prokaryotic COX. We conclude that ‘oxidative stress’ occurs when the control of COX activity by the [ATP]/[ADP] ratio is switched off via neural signals.     

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH-ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP and ATP turns on, and subsequent incubation with Ca²⁺ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS⁴⁴¹ at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser⁴⁴¹ turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca²⁺]] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg⁻¹ at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the K_m for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP andATP turns on, and subsequent incubation with Ca2+ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS441 at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser441 turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca2+] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that:

(1)

phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles.

(2)

Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation.

(3)

The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O₂ concentrations and mitochondrial O₂ consumption by high-resolution respirometry over a range of O₂ concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC₅₀ on [O₂] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O₂ and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O₂-dependent oxidation. It also explains the non-linear dependence of IC₅₀ on O₂ concentration, and the hyperbolic increase of c₅₀ as a function of NO concentration.     

Metabolic regulation of pyruvate kinase isolated from autotrophically and heterotrophically grown Paracoccus denitrificans

Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophycally grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent M _m for ADP and PEP in the autotrophic enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent K _m for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.Non-Common Abbreviations PEP phosphoenolpyruvate - R-5-P ribose-5-phosphate - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate     

ATP-triggered ADP release from the asymmetric chaperonin GroEL/GroES/ADP7 is not the rate-limiting step of the GroEL/GroES reaction cycle

The GroEL/GroES protein folding chamber is formed and dissociated by ATP binding and hydrolysis. ATP hydrolysis in the GroES-bound (cis) ring gates entry of ATP into the opposite unoccupied trans ring, which allosterically ejects cis ligands. While earlier studies suggested that hydrolysis of cis ATP is the rate-limiting step of the cycle (t_½ ∼ 10 s), a recent study suggested that ADP release from the cis ring may be rate-limiting (t_½ ∼ 15-20 s). Here we have measured ADP release using a coupled enzyme assay and observed a t_½ for release of ?4-5 s, indicating that this is not the rate-limiting step of the reaction cycle.     

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca²⁺ oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca²⁺] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca²⁺ oscillations in the cytosol that are transduced into mitochondrial Ca²⁺ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]_mito and [ATP]_cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca²⁺]_cyto and [ATP]_cyto during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.     

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect

In the present study, we describe the existence of mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoK_ATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoK_ATP in insect mitochondria. MitoK_ATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.     

Control of lysyl oxidase activity through site-specific deuteration of lysine

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d₂-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V_max/K_m as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D₂Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.