Acidianus ambivalens type-II NADH dehydrogenase: genetic characterisation and identification of the flavin moiety as FMN

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely (31)P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8alpha-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.     

The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O(2) min(-1) mg(-1), which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 degrees C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 micromol NADH oxidized min(-1) mg(-1) at 60 degrees C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.     

Crystallisation and preliminary structure determination of a NADH: quinone oxidoreductase from the extremophile Acidianus ambivalens

NADH:quinone oxidoreductases (NDHs), constitute one of the electron entry points into membrane-bound respiratory chains, oxidising NADH and reducing quinones. Type-II NDHs (NDH-2) are functionally unable to translocate protons and are typically constituted by a single approximately 50 kDa subunit lacking iron-sulfur clusters and containing one flavin as the sole redox centre. No three dimensional crystal structure is yet available for NDHs. We describe the crystallisation and preliminary structure determination of a NDH-2 that contains a covalently bound FAD, isolated from the membrane fraction of Acidianus ambivalens, a hyperthermoacidophilic archaeon capable of growing at 80 degrees C and pH 2.0. NDH-2 was solubilised with the detergent n-dodecyl-beta-d-maltoside and crystallised using ammonium phosphate as precipitant. The structure was solved by MIRAS using Pt and I derivatives.     

Acidianus ambivalens Complex II typifies a novel family of succinate dehydrogenases

Complex II from the thermoacidophilic archaeon Acidianus ambivalens, an archetype of an emerging class of succinate dehydrogenases (SDH), was extracted from intact membranes and purified to homogeneity. The complex contains one molecule of covalently bound FAD and 10 Fe atoms. EPR studies showed that the complex contains the canonical centres S1 ([2Fe-2S]2+/1+) and S2 ([4Fe-4S]+2/+1) but lacks centre S3 ([3Fe-4S]+1/0); these observations agree with the fact that the iron-sulfur subunit contains an extra cysteine that may allow the binding of a new centre, most probably a tetranuclear one. Succinate-driven oxygen consumption is observed in intact membranes indicating that in vivo, complex II operates as a succinate:quinone oxidoreductase, despite missing the typical anchor domain subunits. The pure complex was found to contain bound caldariella quinone, the enzyme physiological partner. An alternative membrane anchoring for this new type of SDHs, based on the amphipathic nature of the putative helices found in SdhC, is suggested.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

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.     

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.     

Pullulanase,an enzyme of starch catabolism,is associated with the outer membrane of Klebsiella

Late-log phase cells of Klebsiella sp. 5246 could be converted into spheroplasts with a yield of better than 90% by ethylenediamine tetraacetate/lysozyme treatment in osmotically stabilizing media. Membrane fragments obtained after ultrasonication of spheroplasts were separated by centrifugation to sedimentation equilibrium on a sucrose density gradient. A light membrane fraction with a buoyant density of 1.17±0.02g/cm³ was sought and found to contain the enzymes NADH oxidase, succinate dehydrogenase and D-lactate dehydrogenase. A heavy membrane fraction having a buoyant density of 1.23 ±0.01g/cm³ was characterized by phospholipase A₁ activity and lipopolysaccharide content. By analogy to other gram-negative bacteria, the light and the heavy fraction were assigned, respectively, to the cytoplasmic and the outer membrane of Klebsiella sp. 5246.The organism produced pullulanase in a cellbound form during the exponential phase of growth on soluble starch. Pullulanase was localized exclusively on the outer membrane. Pullulanase is the second protein of the outer membrane with defined enzyme function to become known among gram-negative bacteria, the other one being phospholipase A₁.What had been inferred from physiological studies of growth characteristics on various carbon sources can now be proven directly: Pullulanase implicated in the utilization of branched -glucans in Klebsiella is capable of acting on macromolecular substrates in the environment of the cell by virtue of its association with the outer membrane.Non-Standard Abbreviations EDTA ethylenediamine tetraacetate - SDS sodium dodecyl sulphate - OD optical density List of Enzymes EC 3.2.1. 23 -galactosidase or -D-galactoside galactohydrolase - EC 1.1.1.28 D-lactate dehydrogenase or D-lactate: NAD⁺ oxidoreductase - EC 3.2.1.17 lysozyme or mucopeptide N-acetylmuramoylhydrolase - EC 2.4.1.1 maltodextrin phosphorylase or 1,4--D-glucan: orthophosphate -glucosyltransferase - EC 1.6.99.3 NADH oxidase or NADH: (acceptor) oxidoreductase - EC 3.1.1.32 phospholipase A₁ or phosphatide 1-acylhydrolase - EC 3.2.1.41 pullulanase or pullulan 6-glucanohydrolase - EC 1.3.99.1 succinate dehydrogenase or succinate: (acceptor) oxidoreductase     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

The rotenone sensitive NADH: menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50°C and pH 8.1, and the enzyme presents a K _M of 9 M for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 ± 3.5 iron atoms and 3.7 menaquinone per FMN. At least five iron—sulfur centers are observed by EPR spectroscopy: two [2Fe–2S]^2+/1+ and three [4Fe–4S]^2+/1+ centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

The action of the Notch locus in Drosophila melanogaster

Summary It is shown that the Notch⁸ deficiency in Drosophila melanogaster affects a number of enzyme activities localized in the mitochondria, such as NADH oxidase (activity of the complete respiratory chain), NADH dehydrogenase (the first step in the respiratory chain before transfer to ubiquinone), Succinate dehydrogenase and -Glycerophosphate dehydrogenase. The experiments reported here do not exclude the possibility of involvement of other genes in the deficiency. The effect of duplications of the Notch locus on NADH oxidase and NADH dehydrogenase suggest that this locus determines the enzyme activities.The dosage effects of the Notch locus on activity suggest that this locus contains the structural genes for these enzymes.     

Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K _m) of 5.87 μM, a turnover rate (k _cat) of 0.24/sec, and a catalytic efficiency (k _cat/K _m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k _cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k _cat/K _m, 107.75/mM/sec).     

A portable multi-flash kinetic fluorimeter for measurement of donor and acceptor reactions of Photosystem 2 in leaves of intact plants under field conditions

A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the double-flash kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439–448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 s) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% F_max) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 s after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.Abbreviations DCMU 3-(3,4-Dichlorophenol)-1,1-dimethylurea - PS 2 photosystem 2 - PS 1 photosystem 1 - P₆₈₀ primary electron donor of the PS 2 reaction center - Q_A primary acceptor quinone of PS 2 - Q_B secondary acceptor quinone of PS 2 - CCCP carbonyl cyanide-m-chlorophenylhydrazone - Y_z donor to P₆₈₀ ⁺ - F₀ level of fluorescence with all PS 2 centers open - F_max maximum level of fluorescence with all PS 2 centers closed - P₆₈₀Q_A Open reaction centers with P₆₈₀ reduced and Q_A oxidized (low fluorescence) - P₆₈₀Q_A ^- Closed reaction centers, in which P₆₈₀ is reduced (high fluorescence) - P₆₈₀ ⁺Q_A ^- Closed reaction centers, in which P₆₈₀ is oxidized (low fluorescence)     

NADH dehydrogenase and NADH oxidation inBacillus megaterium

Only one type (membrane-bound form) of NADH dehydrogenase could be detected in the log-phase cells ofBacillus megaterium. By sonification this enzyme could be effectively solubilized, while NADH oxidase remained bound to the membrane. A molecular weight of about 40 Kd was estimated for the dehydrogenase by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) with an activity stain. Mercuric chloride and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were inhibitors for both the NADH dehydrogenase and oxidase inB. megaterium. The inhibition studies of NADH oxidation suggested that NADH dehydrogenase provided the primary electron source for NADH oxidase in this organismin vitro. NADH dehydrogenase was highly specific for NADH, and K_m was estimated to be 28.2 M. The enzyme was subjected to end-product inhibition of a competitive type.     

A light-enhanced metabolism of sulfite in cells of Cucumis sativus L. cotyledons

The effects of light and several photosynthetic inhibitors on the rate of sulfite metabolism in cells obtained from Cucumis sativus L. cotyledons was studied. The cells were treated with 200 M Na₂SO₃ and the disappearance of sulfite was monitored using either dithiobisnitrobenzoic acid or fuchsin. The rate of sulfite disappearance in light was double the dark rate. Disalicylidene propanediamine at 1 mM increased this light-enhanced metabolism approx. 50%; neither 1 M 3,4-dichlorophenyl-N,N-dimethylurea nor 0.1 mM cyanazine, which completely inhibited CO₂-dependent oxygen evolution, affected the rate of sulfite metabolism. Addition of 200 M Na₂SO₃ to the cells partially inhibited ¹⁴CO₂ fixation. The rate of sulfite consumption by the cells did not affect this inhibition. We conclude that light-dependent sulfite metabolism is cucumber cells may utilize reduced ferredoxin generated as a result of photosynthetic electron transport. An injurious interaction between CO₂ fixation and sulfite appears to occur independently of the sulfite-metabolism process.Abbreviations DCMU 3,4-dichlorophenyl-N,N-dimethylurea - DSPD disalicylidene propanediamine - DTNB 5,5-dithiobis-(2-nitrobenzoic acid)     

A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon

Abstract Membranes of the extremely thermoacidophilic archaeon Desulfurolobus ambivalens grown under aerobic conditions contain a quinol oxidase of the cytochrome aa ₃-type as the most prominent hemoprotein. The partially purified enzyme consists of three polypeptide subunits with apparent molecular masses of 40, 27 and 20 kDa and contains two heme A molecules and one copper atom. CO difference spectra suggest one heme to be a heme a ₃-centre. The EPR spectra indicate the presence of a low-spin and a high-spin heme species. Redox titrations of the solubilized enzyme show the presence of two reduction processes, with apparent potentials of + 235 and + 330 mV. The enzyme cannot oxidize reduced cytochrome c , but rather serves as an oxidase of caldariella quinone. Due to their very simple composition, D . ambivalens cell appear as a promising candidate to study Structure-function relationships of cytochrome aa ₃ in the integral membrane state.     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase     

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II)

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans , we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.