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)

The soluble NADH dehydrogenase of low molecular weight, isolated from complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) of the respiratory chain, has been shown to have NADPH dehydrogenase and NADPH leads to NAD transhydrogenase activities. Both activities are greatly increased in the presence of added guanidine-HCl and at pH values less than 6.5. The chromophores of the soluble enzyme (flavin and iron--sulfur centers) are reduced by NADH and NADPH to the same extent. The latter reduction is extremely slow, and is considerably stimulated in the presence of guanidine-HCl. The soluble dehydrogenase has little or no NADH leads to NADP and NADPH leads to NADP transhydrogenase activity. The former reaction is known to be energy-linked in submitochondrial particles; the latter was shown in the present studies also to be energy-linked. In view of the above and earlier results, possible mechanisms for dehydrogenation and transhydrogenation (nonenergy-linked and energy-linked) involving reduced and oxidized NAD and NADP are proposed.
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PMID:Dehydrogenase and transhydrogenase properties of the soluble NADH dehydrogenase of bovine heart mitochondria. 1 55

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.
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PMID:The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart. 2 68

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

N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.
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PMID:Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c. 16 5

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.
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PMID:Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. 16 92

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

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th
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PMID:Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis. 16 85

1. Measurements were made at 12 degrees K of the electron-paramagnetic-resonance (e.p.r.) spectra of submitochondrial particles from Candida utilis cells grown under conditions that alter the amount of the mitochondrial NADH dehydrogenase (EC 1.6.99.3). 2. Iron-limited growth decreases the extent of iron-sulphur e.p.r. signals to undetectable values that are less than 1 percent of those normally found with glycerol-limited growth. 3. Small but significant signals attributable to the NADH dehydrogenase were detected in submitochondrial particles from sulphate-limited cells. 4. Measurements made on submitochondrial particles prepared from these and other phenotypically modified cells lead us to conclude that the presence of low-temperature e.p.r.-detectable iron-sulphur centres attributable to the NADH dehydrogenase are necessary but not sufficient for the coupling of ATP synthesis to the NADH dehydrogenase reaction in the mitochondrial membrane of C. utilis. 6. The amplitude of the g=2.01 signal observed in non-reduced submitochondrial particles is approximately tenfold diminished by iron limitation but not significantly altered by sulphate limitation.
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PMID:Electron-paramagnetic-resonance spectroscopy studies of iron-sulphur centres of submitochondrial particles from iron- and sulphur-deficient. Candida utilis. 16 15

The cell membrane-associated respiratory electron transport chain of Neisseria gonorrhoeae was examined using electron paramagnetic spectroscopy (EPR) at liquid helium temperatures and optical spectroscopy at liquid nitrogen and room temperatures. EPR spectra of dithionite-reduced particles indicated the presence of centers N-1 and N-3 in the site I region of the respiratory chain, whereas reduction with succinate revealed the existence of center S-1 from the succinate cytochrome c reductase segment. Free radical(s) resembling that due to falvin semiquinone were observed with both reductants. Low temperature (77 K) optical difference spectra indicated the presence of cytochromes with alpha band maxima at 549, 557, and 562. Bands at 567, 535, and 417 nm, characteristic of the CO compound of cytochrome o, were also identified. Cytochromes a1 and a3 were not detected; however, a broad but weak absorbance with an alpha band maximun at 600 nm and a Soret shoulder at 440 nm was observed. Hence the respiratory chain of N. gonorrhoeae appears to contain several nonheme iron centers, cytochrome c, two b cytochromes, with cytochrome o which probably serves as the terminal oxidase.
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PMID:Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae. 16 81

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


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