<Emphasis Type="Italic">N,N</Emphasis>-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells,to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome <Emphasis Type="Italic">c</Emphasis> and AIF

Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.     

Redox-Linked Conformational Dynamics in Apoptosis-Inducing Factor

Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH₂-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH.     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.     

Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha

Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO₂) was supplemented to the atmosphere. In the presence of gaseous NO₂, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate. Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N₂). Nitrous oxide (N₂O) was shown to be an intermediate of denitrification. Under an N₂ atmosphere supplemented with 25 ppm NO₂ and 300 ppm CO₂, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N. eutropha increased by 5.8 × 10⁹ cells per mmol ammonia oxidized. In addition, the ATP and NADH content increased by 4.3 μmol ATP (g protein)^–1 and 6.3 μmol NADH (g protein)^–1 and was about the same in both anaerobically and aerobically grown cells. Without NO₂, the ATP content decreased by 0.7 μmol (g protein)^–1, and the NADH content decreased by 1.2 μmol (g protein)^–1. NO was shown to inhibit anaerobic ammonia oxidation. Received: 9 October 1996 / Accepted: 5 December 1996     

H. pylori‐Induced Apoptosis in Human Gastric Cancer Cells Mediated via the Release of Apoptosis‐Inducing Factor from Mitochondria

Background and Aim: Our previous study of Helicobacter pylori‐induced apoptosis showed the involvement of Bcl‐2 family proteins and cytochrome c release from mitochondria. Here, we examine the release of other factors from mitochondria, such as apoptosis‐inducing factor (AIF), and upstream events involving caspase‐8 and Bid. Methods: Human gastric adenocarcinoma (AGS) cells were incubated with a cagA‐positive H. pylori strain for 0, 3, 6, and 24 hours and either total protein or cytoplasmic, nuclear, and mitochondrial membrane fractions were collected. Results: Proteins were immunoblotted for AIF, Bid, polyadenosine ribose polymerase (PARP), caspase‐8, and β‐catenin. H. pylori activated caspase‐8, caused PARP cleavage, and attenuated mitochondrial membrane potential. A time‐dependent decrease in β‐catenin protein expression was detected in cytoplasmic and nuclear extracts, coupled with a decrease in β‐actin. An increase in the cytoplasmic pool of AIF was seen as early as 3 hours after H. pylori exposure, and a concomitant increase was seen in nuclear AIF levels up to 6 hours. A band corresponding to full‐length Bid was seen in both the cytoplasmic and the nuclear fractions of controls, but not after H. pylori exposure. Active AIF staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls. Conclusion: H. pylori might trigger apoptosis in AGS cells via interaction with death receptors in the plasma membrane, leading to the cleavage of procaspase‐8, release of cytochrome c and AIF from mitochondria, and activation of subsequent downstream apoptotic events, as reported previously for chlorophyllin. This is consistent with AIF activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.     

Apoptosis-inducing Factor (AIF) and Its Family Member Protein,AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.     

Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition

Provision of NAD⁺ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD⁺, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1^?/? and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD⁺ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.     

Toxicity of 2-mercaptobenzothiazole towards bacterial growth and respiration

Active sludge systems containing benzothiazoles may be intoxicated by 2-mercaptobenzothiazole (MBT). This toxicity towards several bacteria is now confirmed and is situated at around 100 mg MBT l^–1. Octanol-water partition coefficients indicated that MBT might interact with membrane-bound systems. This was confirmed through experiments showing that bacterial cell respiration was inhibited using lactate or succinate as substrates. Using these substrates and also NADH, it was found that their oxidation was also inhibited using isolated membrane fragments of Escherichia coli and Paracoccus denitrificans. Methylene blue reduction was also found to be inhibited. The oxidation of ascorbate was not inhibited in P. denitrificans. From these results it is suggested that MBT might interact with the respiratorychain at the level of flavoproteins or quinones and Fe-S clusters.     

Transient overexpression of TGFBR3 induces apoptosis in human nasopharyngeal carcinoma CNE-2Z cells

NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca²⁺]_i (intracellular Ca²⁺ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca²⁺]_i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development

Macrophages play a central role in the development of atherosclerosis through the accumulation of oxidized LDL (oxLDL). AIM (Spα/Api6) has previously been shown to promote macrophage survival; however, its function in atherogenesis is unknown. Here we identify AIM as a critical factor that protects macrophages from the apoptotic effects of oxidized lipids. AIM protein is induced in response to oxLDL loading and is highly expressed in foam cells within atherosclerotic lesions. Interestingly, both expression of AIM in lesions and its induction by oxidized lipids require the action of LXR/RXR heterodimers. AIM^−/− macrophages are highly susceptible to oxLDL-induced apoptosis in vitro and undergo accelerated apoptosis in atherosclerotic lesions in vivo. Moreover, early atherosclerotic lesions in AIM^−/−LDLR^−/− double knockout mice are dramatically reduced when compared to AIM^+/+LDLR^−/− controls. We conclude that AIM production facilitates macrophage survival within atherosclerotic lesions and that loss of AIM decreases early lesion development by increasing macrophage apoptosis.     

Characterization of a soluble oxidoreductase from the thermophilic bacterium Carboxydothermus ferrireducens

An NAD(P)H-dependent oxidoreductase has been purified approximately 40-fold from the soluble protein fraction of the dissimilatory iron-reducing, anaerobic, thermophilic bacterium Carboxydothermus ferrireducens. The enzyme, a flavoprotein, has broad-substrate specificity—reducing Fe³⁺, Cr⁶⁺, and AQDS with rates of 0.31, 0.33, and 3.3 U mg⁻¹ protein and calculated NADH oxidation turnover numbers of 0.25, 0.25, and 2.5 s⁻¹, respectively. Numerous quinones are reduced via a two-electron transfer from NAD(P)H to quinone, thus participating in managing oxidative stress by avoiding the formation of semiquinone radicals.     

Helicobacter pylori Induces Apoptosis of T- and B-Cell Lines and Translocates Mitochondrial Apoptosis-Inducing Factor to Nucleus

Immune cell apoptosis may play a role in human persistent Helicobacter pylori infection. We planned to study the apoptosis of T and B cells by H. pylori strains. T (Jurkat) and B (Raji) cell lines were co-cultured with cagA-positive H. pylori strains carrying different vacA genotypes (s1a/m1, s1a/m2, and s2/m2). Apoptosis was detected by microscopy, DNA fragmentation assay, and flow cytometry. Apoptosis-inducing factor (AIF) transfer from mitochondria to nucleus was studied by immunoblot analysis. Apoptosis of T and B cells was significantly higher in H. pylori-infected cells than in uninfected controls (s1a/m1 80%, s1a/m2 78%, s2m2 69% vs. control 16% for T cells, P < 0.001; s1 a/m1 78%, s1a/m2 73%, s2m2 62% vs. control 24% for B cells, P < 0.001 by flow cytometry) with no difference among the genotypes. AIF transfer from mitochondria to nucleus was demonstrated in both apoptotic cell lines. Thus, H. pylori induces apoptosis in T- and B-cell lines and translocates AIF. T and B cells deletion through apoptosis may explain the persistence of H. pylori infection; its role in pathogenesis needs further research.     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

Effects of quinones on NADH-dependent enzymatic bioluminescent systems

The effects of a number of quinones on the bioluminescence characteristics of a three-component enzymatic system containing alcohol dehydrogenase, bacterial luciferase, and NADH-FMN oxidoreductase were studied to find the most sensitive kinetic parameters of the system intended to be used in biological testing. Both direct and back reactions catalyzed by alcohol dehydrogenase were studied in the presence and in the absence of quinones. The kinetic parameters of the bioluminescent system were found to depend on the redox potentials and concentrations of quinones. The quinone-induced effects were shown to be associated with changes in the NAD⁺/NADH ratio in the chain of NADH-dependent enzymes. The three-enzyme system based on alcohol dehydrogenase is suggested as a bioluminescence test for ecological monitoring of waste water.     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

A New Type-II NADH Dehydrogenase from the Archaeon Acidianus ambivalens: Characterization and in vitro Reconstitution of the Respiratory Chain

A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein^-1/min^-1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K_m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa₃ type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.