Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of ubiquinone-1 to E. coli ML 308-225 membrane vesicles dramatically increases coupling between NADH oxidation and active transport such that initial rates and steady-state levels of lactose and amino-acid accumulation are comparable to those observed during D-lactate oxidation. Similar but less dramatic effects are observed with the quinone and succinate or L-lactate. In the presence of NADH and ubiquinone-1, the vesicles also generate a membrane potential (interior negative) that is similar in magnitude to that observed in the presence of D-lactate. Stimulation of NADH-dependent transport by ubiquinone-1 cannot be accounted for by increased rates of oxidation of NADH, and the effect of the quinone on NADH-dependent lactose transport is not observed in vesicles depleted of NADH dehydrogenase activity. Thus, it is apparent that ubiquinone-1 shunts electrons from NADH dehydrogenase [NADH:(acceptor)oxidoreductase; EC 1.6.99.3] to the portion of the respiratory chain containing the energy-coupling site. The findings demonstrate unequivocally that inefficient coupling of NADH oxidation to active transport cannot be due to the presence of inverted vesicles. In addition, they provide further support for specific localization of the energy-coupling site.
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PMID:Ubiquinone-mediated coupling of NADH dehydrogenase to active transport in membrane vesicles from Escherichia coli. 0 Jun 72

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.
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PMID:Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite. 3 82

1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl. 2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (Ki) is 0.075 plus or minus 0.012 mM. NADH dehydrogenase is not inhibited significantly. 3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with Ki equals 0.22 plus or minus 0.03 mM and for phototrophic membranes with Ki equals 0.49 plus or minus 0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl. 4. Cytochrome oxidase is inhibited only slightly. 5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95 percent, succinate-dependent reactions can be inhibited more than 95 percent only in chemotrophic membranes. In phototrophic membranes the maximum inhibition of succinate-dependent reactions is about 70 percent. 6. The type of inhibition in both cases 2 and 3 is non-competitive. 7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b. 8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.
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PMID:Separation of respiratory reactions in Rhodospirillum rubrum: inhibition studies with 2-hydroxydiphenyl. 16 37

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).
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PMID:Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart. 16 70

Various respiratory electron transport activities of Rhodopseudomonas capsulata were studied in membrane fragments prepared from photosynthetically grown cells of a parental strain and two terminal oxidase-defective mutant strains. The NADH and succinate oxidase activities of the mutant having a functional N,N,N1,N1-tetramethyl-p-phenylenediamine oxidase, M6, were consideraly more sensitive to inhibition by either antimycin A or cyanide than the corresponding activities of the mutant lacking a functional N,N,N1,N1-tetramethyl-p-phenylenediamine oxidase, M7. The parental strain, Z-1, but not the mutants, showed biphasic inhibitory responses of NADH and succinate oxidase activities with either antimycin A or cyanide. In certain reactions no differences in inhibitor susceptibility were found among the strains tested, implying that the pathways involved were unaffected in the mutants. In this category were the actions of rotenone on NADH oxidase, antimycin A on cytochrome c reductase and, in M6 and Z-1, cyanide on N,N,N'N'-tetramethyl-p-phenylenediamine oxidase. These results suggest that the respiratory chain of the parental strain branches at the ubiquinone-cytochrome b region into two pathways, each branch goes to a distinct terminal oxidase, and either may be blocked independently by genetic mutation.
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PMID:The branched respiratory system of photosynthetically grown Rhodopseudomonas capsulata. 17 46

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.
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PMID:The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes. 17 76

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.
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PMID:Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain. 17 56

(1) Studies of the steady-state kinetics of the NADH dehydrogenase activity of Complex I (NADH: Q oxidoreductase) revealed that the reaction mechanism with the one-electron acceptor ferricyanide or the two-electron acceptor 2,6-dichloro-indophenol is ping pong bi bi, with double substrate inhibition. NADH inhibits the reaction of the reduced form of the flavoprotein with the acceptors, and the acceptors prevent NADH from reacting with the oxidized form. This implies that both NADH and acceptors react with the same site on NADH dehydrogenase. (2) The velocity at infinite NADH and acceptor concentrations (corrected for the double substrate inhibition) is much larger with ferricyanide than with the indophenol. It is concluded that the latter binds to the reduced enzyme. Thus, with ferricyanide the rate constant measured refers to the dissociation of bound NAD+ from the reduced enzyme (k2) and with the indophenol to the rate constant of oxidation of reduced enzyme by bound acceptor (k4). The latter value is not an estimate for the situation in vivo, where ubiquinone is the acceptor. (3) The rate constant of the dissociation of bound NAD+ from the reduced enzyme (k2) increases with pH. It is suggested that an ionizing group on the enzyme is involved in the dissociation. (4) After extraction of ubiquinone from Complex I with pentane curve relating activity at infinite ferricyanide concentration to NADH concentration changes from hyperbolic to sigmoidal. The hyperbolic curve is restored by reincorporating ubiquinone. It is concluded that ubiquinone is an effector for NADH dehydrogenase.
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PMID:Steady-state kinetics of high molecular weight (type-I) NADH dehydrogenase. 18 Oct 89

(1) The steady-state kinetics of the NADH dehydrogenase activity of Type-II (low molecular weight) NADH dehydrogenase with the acceptors ferricyanide, cytochrome c and 2,6-dichloroindophenol are consistent with the simultaneous operation of an ordered and a ping-pong mechanism. Thus, depending on the acceptor concentration, the reduced enzyme is preferentially oxidized before or after NAD+ disociates from it. (2) The acceptors are able to oxidize the reduced enzyme and its NAD+ complex equally well. In contrast to the kinetics of the Type-I (high molecular weight) enzyme, double substrate inhibition is not found, implying that the site of oxidation of the reduced enzyme by acceptors and the NADH-binding site are remote. (3) With the indophenol, in the concentration range measured, the ordered mechanism is mainly operative. At infinite NADH and acceptor concentrations the rate constant of the reduction of enzyme by bound NADH is measured. (4) With ferricyanide and cytochrome c, in the concentration range measured, erroneous conclusions may be drawn from extrapolations owing to the fact that extrapolated lines in double-reciprocal plots of turnover number against acceptor concentration, at different NADH concentrations, intersect in the third quadrant. A method is described that allows the extrapolation of these data to zero acceptor concentrations. (5) The relation between activity and NADH concentration is sigmoidal (h = 2.0) with ferricyanide or cytochrome c as acceptor, but hyperbolic with 2,6-dichloroindophenol. The latter is also an inhibitor, competitive with respect to NADH. It is concluded that this two-electron acceptor, like ubiquinone, acts as an allosteric effector. (6) Type II is isolated from Type I without gross changes in tertiary structure, as judged by the unaltered rate constants of dissociation of NADH (k-1) and NAD+ (k4) and association of NADH (k1). (7) Type II differs from Type I in two respects, (a) The accessibility of the acceptors is greater by at least two orders of magnitude (k3). (b) The redox potential of the prosthetic group FMN is 120 mV less, as judged by a drop in the value of k2 by four orders of magnitude. It is suggested that one or more of the iron-sulphur proteins present in Type-I but lacking in Type-II dehydrogenase functions as an effector, regulating the redox potential of the FMN.
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PMID:Steady-state kinetics of low molecular weight (type-II) NADH dehydrogenase. 18 Oct 90

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.
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PMID:Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. 18 49


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