NADH Respiration Research Articles

Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacterium Eikenella corrodens grown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N’-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2 without the bc1 complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitive cbb' cytochrome c oxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system of E. corrodens ATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Discovery of extracellular respiration of NADH and its application in novel NADH regeneration

Discovery of extracellular respiration of NADH and its application in novel NADH regeneration

Mutant Huntingtin Expression Induces Mitochondrial Calcium Handling Defects in Clonal Striatal Cells

Huntington disease (HD) is caused by a pathological elongation of CAG repeats in the huntingtin protein gene and is characterized by atrophy and neuronal loss primarily in the striatum. Mitochondrial dysfunction and impaired Ca2+ homeostasis in HD have been suggested previously. Here, we elucidate the effects of Ca2+ on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) huntingtin-expressing cells of striatal origin. When treated with increasing Ca2+ concentrations, mitochondria from mutant huntingtin-expressing cells showed enhanced sensitivity to Ca2+, as they were more sensitive to Ca2+-induced decreases in state 3 respiration and DeltaPsim, than mitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2+ uptake capacity in comparison with wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The DeltaPsim defect was attenuated in the presence of ADP and the decreases in Ca2+ uptake capacity were abolished in the presence of Permeability Transition Pore (PTP) inhibitors. These findings clearly indicate that mutant huntingtin-expressing cells have mitochondrial Ca2+ handling defects that result in respiratory deficits and that the increased sensitivity to Ca2+ induced mitochondrial permeabilization maybe a contributing mechanism to the mitochondrial dysfunction in HD.

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H 2O 2-producing side-reaction of respiratory complex I with O 2

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with succinate or ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation kinetics for O 2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O 2-affinity system ( K m app = 1.5 μM) was abolished by low cyanide concentration; it corresponded to cytochrome aa 3. The low-O 2-affinity system ( K m app = 120 μM) was resistant to cyanide. Similar O 2 saturation kinetics, using succinate or ascorbate–TMPD as electron donor, showed only the high-O 2-affinity cyanide-sensitive component. Horse cytochrome c increased 2–3 times the rate of electron flow across the cyanide-sensitive pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant amounts of H 2O 2 (at least 10% of the total O 2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H 2O 2. Production of H 2O 2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O 2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O 2, producing H 2O 2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.

Human quadriceps muscle mitochondria: a functional characterization.

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in micromol O min(-1) g protein(-1) (25 degrees C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 micromol O min(-1) g protein(-1)). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 micromol ATP min(-1) g protein(-1)). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.

Effects of Photodynamic Action on Respiration in Nonphosphorylating Mitochondria

We have studied the effects of singlet oxygen produced by photodynamic action on respiration in nonphosphorylating mitochondria (state 4). Isolated rat liver mitochondria were incubated with 3 μM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 W/m2. After short durations of irradiation, state 4 respiration with β-hydroxybutyrate as substrate increases while respiration with succinate is negligibly affected. When mitochondria have been uncoupled with carbonylcyanide-p-trifluoromethoxyphenyl hydrazone before irradiation, no change occurs in β-hydroxybutyrate-driven respiration, while succinate-driven respiration strongly decreases. Stimulation of state 4 NADH respiration cannot be explained by slippage of the NADH ubiquinone oxidoreductase because the stoichiometry of the redox pump was found insensitive to photodynamic action. In the light of the metabolite theory for linear enzymatic chains applied to state 4 respiration (Brandet al., Biochem. J.255, 535–539, 1988), these results suggest that stimulation of NADH respiration is simply due to an increase of membrane leaks which occurs after irradiation. In the case of succinate-driven respiration, a strong inhibition of succinate dehydrogenase activity has been demonstrated after irradiation. It can be suggested that this inhibition introduces a negative control coefficient over state 4 respiration, counterbalancing the effects due to leakage.

Requirement for the Proton‐Pumping NADH Dehydrogenase I of Escherichia Coli in Respiration of NADH to Fumarate and Its Bioenergetic Implications

In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration. The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around -309, -277, and downstream of -231 bp, respectively, relative to the transcriptional-start site. The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL. For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II. The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I. The mutant used acetyl-CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol. Therefore, NADH dehydrogenase I is essential for NADH-->fumarate respiration, and is able to use menaquinone as an electron acceptor. NADH-->dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I. The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed.

Immobilized thermophilic bacteria as a source of respiratory chain for the recycling of NAD +

Immobilization of thermophilic bacterium strain PS3 has been performed by crosslinking with 0.4% glutaraldehyde in the presence of 4.6% bovine serum albumin at − 20°C. After immobilization of bacteria the plasma membrane became permeable to NADH. The yield of NADH respiration of the immobilized strain was ∼10%. The apparent K m for NADH with the immobilized thermophilic strain was 6 × 10 −4 m . After immobilization of this strain no variations of activities were observed between pH 4.5 and 9.5. Recycling of NAD + is at least 10 times better with the thermophilic strain compared to Escherichia coli. In a preliminary experiment at 45°C the half life obtained with Escherichia coli was 1 h and with PS3 was 12 h. This temperature increased the rate of respiration by a factor of ∼4 (compared to 20°C) and may avoid most of bacterial contaminations (most bacteria are not able to grow above 42°C).

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP(+) using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP(+) was recycled more than 10-fold.We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation. 1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm. 2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration. 3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2). o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity.Protection against heat inactivation was obtained for the two activities with NAD(+), NADP(+), NADH, and NADPH.All these results suggested the possibility of recycling of NADP(+) under similar conditions to those previously described for NAD(+) (Burstein et al., 1981). It becomes thus possible to use various NAD(+) and NADP(+)-dependent dehydrogenases in enzyme technology.

Effect of syringomycin and syringotoxin produced by Pseudomonas syringae pv. syringae on structure and function of mitochondria isolated from holcus spot resistant and susceptible maize lines

Syngomycin (SR) and syringotoxin (ST), two toxigenic peptides produced by ecotypic strains of Pseuomonas syringae pv. syringae, caused several changes in oxidative metabolism of mithochodria isolated from etiolated shoots of ACCO maize lines WF9 and B37 which differed in susceptibility to holus spot disease. Evidence was obtained that both toxins, in a concentration range of 6 to 24 μg mg−1 mitochondrial protein, depending on the toxin and on the substrate being oxidized, caused uncoupling of oxidative phosphorylation, stimulation of succinate, malate/glutamate and NADH respiration, stimulation of ATPase activity, inhibition of ATP formation, increased swelling, and a decrease in mitochoondrial membrane potential. Generally the effect induced by ST was greater than that of SR. ST at 6 μg mg−1 protein caused complete uncoupling with all three substrates tested, while SR had the same effect at 12 μg mg−1 protein with NADH and succinate and at 24 μg mg−1 protein with malate/glutamate. This difference between SR and ST was probably due to a difference in specificity for their sites of action. Electron micrographs of B37 mithochodria indicated that SR and ST caused swelling with fragmentation of the inner membrane.The effects of the two toxins on WF9 and B37 mithochondria were the same, although, based on number of lesions and population dynamics of the pathogen in leaf tissues, pathogenicity tests indicated that leaves of WF9 were more susceeptible to P. syringae pv. syringae than those of B37. However, when leaf tissues were treated with SR or ST, B37 was more sensitive to the toxins than WF9. Both toxins caused typical holcus spot symptoms. Based on assays of bacterial populations in leaves, susceptibility of WF9 in contrast to the resistance of B37 to holcus spot was apparently due to the effects of host substrate on the extent and rate of growth of pathogenic bacteria in relation to production of SR.

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208x and 66x less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.

Relationship of cytochrome content to the sensitivity of bacteria to NaCl on freezing and thawing.

Eight species of bacteria representing rod, coccus, gram-positive, and gram-negative forms were tested for their sensitivity to sodium chloride during freezing and thawing. Six of the eight species tested were salt-sensitive, though to different degrees, while Lactobacillus casei and Streptococcus faecalis were resistant. Escherichia coli grown anaerobically exhibited only 38% of the salt sensitivity of aerobically grown cells. Analysis of cytochrome pigments in the organisms revealed that the six sensitive organisms all contained these pigments but in varying amounts, while the two resistant ones were devoid of them. Anaerobically grown E. coli contained 50% of the cytochromes of aerobically grown cells. A relationship between cytochrome content of the organisms and salt sensitivity during freezing and thawing was demonstrated with a correlation coefficient of 0.76 (P less than 0.05); the higher the cytochrome content, the more salt-sensitive the organism. This indicated that 58% of the salt sensitivity was due to the cytochrome content. Using a model organism E. coli, the effect of salt during freezing and thawing on the respiratory activity was examined. Freezing and thawing in water or saline decreased the respiration by whole cells of substrates expected to be NAD-linked while NADH-stimulated respiration was increased. In cell-free extracts derived from unfrozen cells or those frozen and thawed in water or saline, the respiration of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was constant. The respiration of NADH, succinate, and lactate in cell-free extracts derived from cells frozen and thawed in saline was reduced compared with those extracts derived from unfrozen cells or cells frozen and thawed in water. Studies with E. coli showed that the decreased respiratory activity caused by disruptions in the electron-transport chain could not account for the salt sensitivity on freezing and thawing. More likely, salt sensitivity is related to the presence of bonds between cytochromes and other membrane components which are disrupted by sodium chloride on freezing and thawing. This would then result in loss of membrane integrity and function.

Biochemistry of the dog hookworm: III. Oxidative phosphorylation

Mitochondria isolated from the dog hookworm Ancylostoma caninum were capable of esterifying inorganic phosphate when succinate was oxidized. P O ratios ranging from 0.1 to 0.7 were observed. 2,4-Dinitrophenol at a concentration of 10 −4 M uncoupled the phosphorylation. The mitochondria lacked respiratory control. NADH-amytal-sensitive respiration was observed in hookworm mitochondria. The NADH respiration was not coupled to phosphorylation. 3-OH-butanoic acid was not oxidized by A. caninum mitochondria. Isolation of hookworm mitochondria in 10 −4 M EDTA and 5 × 10 −3 M phosphate buffer stabilized the phosphorylating properties of the mitochondrial suspensions.

The effect of respiratory inhibitors on NADH, succinate and malate oxidation in corn mitochondria.

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.