Mitochondrial metabolic states and membrane potential modulate mtNOS activity

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.     

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O₂, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.     

Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I

The present study shows that rat liver and brain mitochondrial nitric oxide synthase (mtNOS) are functionally associated with mitochondrial respiratory chain complex I. When complex I is activated, mtNOS exerts high activity and generates nitric oxide, whereas inactivation of complex I leads mtNOS to abandon its NOS activity. Functional association of mtNOS with complex I is potentially important in regulating mtNOS activity and mitochondrial functions.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H⁺ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

Complex I syndrome in myocardial stunning and the effect of adenosine

Isolated rabbit hearts were exposed to ischemia (I; 15 min) and reperfusion (R; 5-30 min) in a model of stunned myocardium. I/R decreased left-ventricle O₂ consumption (46%) and malate-glutamate-supported mitochondrial state 3 respiration (32%). Activity of complex I was 28% lower after I/R. The pattern observed for the decline in complex I activity was also observed for the reduction in mitochondrial nitric oxide synthase (mtNOS) biochemical (28%) and functional (50%) activities, in accordance with the reported physical and functional interactions between complex I and mtNOS. Malate-glutamate-supported state 4 H₂O₂ production was increased by 78% after I/R. Rabbit heart Mn-SOD concentration in the mitochondrial matrix (7.4 ± 0.7 μM) was not modified by I/R. Mitochondrial phospholipid oxidation products were increased by 42%, whereas protein oxidation was only slightly increased. I/R produced a marked (70%) enhancement in tyrosine nitration of the mitochondrial proteins. Adenosine attenuated postischemic ventricular dysfunction and protected the heart from the declines in O₂ consumption and in complex I and mtNOS activities and from the enhancement of mitochondrial phospholipid oxidation. Rabbit myocardial stunning is associated with a condition of dysfunctional mitochondria named “complex I syndrome.” The beneficial effect of adenosine could be attributed to a better regulation of intracellular cardiomyocyte Ca²⁺ concentration.     

Sugar binding in lactose permease: anomeric state of a disaccharide influences binding structure

Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with β(α)-d-galactopyranosyl-(1,1)-β-d-galactopyranoside (ββ-(Galp)₂) and αβ-(Galp)₂ result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and αβ-(Galp)₂ has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp)₂ than ββ-(Galp)₂, i.e. −10.7(±0.7) and −3.1(±1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with ββ-(Galp)₂ binding resulting in larger distortion than αβ-(Galp)₂.     

12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium

12(S)-Hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.     

Transport and metabolism of l-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added l-lactate can enter cerebellar granule cells, we investigated whether and how l-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions.

(1)

l-lactate enters mitochondria, perhaps via an l-lactate/H⁺ symporter, and is oxidized in a manner stimulated by ADP. The existence of an l-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the K_m and V_max values changed en route to apoptosis.

(2)

In both normal and apoptotic cell homogenates, externally added l-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored l-lactate uptake by mitochondria and occurred with a hyperbolic dependence on l-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of l-lactate. The rate of the process depended on l-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular l-lactate/pyruvate shuttle, whose activity was limited by the putative l-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier.

(3)

l-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative l-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

     

Relationship between exercise intervention and NO pathway in patients with heart failure with preserved ejection fraction

Objective: Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.

Material and methods: Sixty-two patients (65?±?6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.

Results: There were baseline associations for adjusted values of maximum workload with ADMA (r=??0.322, p?=?0.028) and L-Arg/ADMA ratio (r?=?0.331, p?=?0.015), and for the 6-min walk test (6MWT) with ADMA (r=??0.314, p?=?0.024) and L-Arg/ADMA ratio (r?=?0.346, p?=?0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak O₂) at follow-up: 3.4?±?2.8 vs. 1.1?±?2.9?mL/min/kg (p?=?0.005).

Conclusions: Exercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak O₂.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells

l-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta-oxidation. It is known that l-carnitine exists as a zwitterion and that member of the OCTN family play an important role in its transport. The aims of this study were to characterize l-carnitine transport in the intestine by using Caco-2 cells and to elucidate the effects of levofloxacin (LVFX) and grepafloxacin (GPFX), which are zwitterionic drugs, on l-carnitine uptake. Kinetic analysis showed that the half-saturation Na⁺ concentration, Hill coefficient and K_m value of l-carnitine uptake in Caco-2 cells were 10.3 ± 4.5 mM, 1.09 and 8.0 ± 1.0 μM, respectively, suggesting that OCTN2 mainly transports l-carnitine. LVFX and GPFX have two pK_a values and the existence ratio of their zwitterionic forms is higher under a neutral condition than under an acidic condition. Experiments on the inhibitory effect of LVFX and GPFX on l-carnitine uptake showed that LVFX and GPFX inhibited l-carnitine uptake more strongly at pH 7.4 than at pH 5.5. It was concluded that the zwitterionic form of drugs plays an important role in inhibition of OCTN2 function.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.     

Jerusalem artichoke mitochondria can export reducing equivalents in the form of malate as a result of D-lactate uptake and metabolism

We found that as a result of d-lactate uptake and metabolism by Jerusalem artichoke mitochondria, reducing equivalents were exported from the mitochondrial matrix to the exterior in the form of malate. The rate of malate efflux, as measured photometrically using NADP+ and malic enzyme, depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics (K(m) = 5 mM; V(max) = 9 nmol/min mg of mitochondrial protein) and was inhibited by non-penetrant compounds. We conclude that reducing equivalent export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from other mitochondrial carriers, as shown by the different inhibitor sensitivity.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Dielectric properties of two diastereoisomers of the arabinose and their equimolar mixture

Dielectric relaxation measurements were performed on two enantiomers, d- and l-arabinose and their equimolar mixture, and compared to dielectric data obtained for d-ribose. d-Arabinose differs from d-ribose by having the opposite configuration at C2. This study reveals that both d- and l- of arabinose exhibit α-relaxation peaks with the same shape for the same α-relaxation time τ_α, and the same steepness index for the T_g-scale T-dependence of τ_α. However, the two isomers have slightly different glass transition temperatures T_g’s, and their secondary γ-relaxation times also differ slightly from the previously observed γ-relaxation in d-ribose at the same temperature. However, when samples of both investigated monosaccharides are annealed at higher temperatures, their glass transition temperatures become nearly identical. This is an effect of the mutarotation process, which leads to the formation of pairs of the enantiomers and accordingly they should have the same physical properties. The width of the α-relaxation of d- and l-arabinose is broader than that of d-ribose, as reflected by the smaller stretch exponent in the Kohlrausch-Williams-Watts function used to fit the data of the former (β_KWW = 0.46 ± 0.01) than the latter (β_KWW = 0.55 ± 0.01). The width of the α-relaxation of racemic mixture of the d- and l-arabinose is slightly broader than that of the pure isomers. While the dielectric loss data of d-ribose in the glassy state at ambient and elevated pressures show an inflexion indicating the presence of the JG β-relaxation, the data of d- and l-arabinose show no such feature for identification of the supposedly universal JG β-relaxation. Nevertheless, on comparing the loss spectra of d-arabinose with that of d-ribose, the presence of the JG β-relaxation in d-arabinose has been rationalized.