scholarly journals Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12

1978 ◽  
Vol 171 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Stephen P. Vincent ◽  
Robert C. Bray
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Low Ph ◽  
Paramagnetic Resonance ◽  
Escherichia Coli K12 ◽  
Exchangeable Proton ◽  
Ph Range ◽  
Nitrate And Nitrite

Nitrate reductase was purified from anaerobically grown Escherichia coli K12 by a method based on the Triton X-100 extraction procedure of Clegg[(1976) Biochem. J.153, 533–541], but hydrophobic interaction chromatography was used in the final stage. E.p.r. spectra obtained from the enzyme under a variety of conditions are well resolved and were interpreted with the help of the computer-simulation procedures of Lowe [(1978) Biochem. J.171, 649–651]. Parameters for five molybdenum(V) species from the enzyme are given. The low-pH species (gav. 1.9827) is in pH-dependent equilibrium with the high-pH species (gav. 1.9762), the pK for interconversion of the species being 8.26. Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified molybdenum(V) e.p.r. spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton. The fifth molybdenum(V) species, sometimes detected in small amounts, appears not to be due to functional nitrate reductase. After full reduction of the enzyme with dithionite, addition of nitrate caused reoxidation of molybdenum to the quinquivalent state, in a time less than the enzyme turnover. Activity of the enzyme in the pH range 6–10 is controlled by a pK of 8.2. It is suggested that the low-pH signal-giving species is the form of the enzyme involved in the catalytic cycle. Iron–sulphur and other e.p.r. signals from the enzyme are briefly described and the enzymic reaction mechanism is discussed.

scholarly journals X-ray-absorption and electron-paramagnetic-resonance spectroscopic studies of the environment of molybdenum in high-pH and low-pH forms of Escherichia coli nitrate reductase

1989 ◽  
Vol 259 (3) ◽  
pp. 693-700 ◽  
Author(s):  
G N George ◽  
N A Turner ◽  
R C Bray ◽  
F F Morpeth ◽  
D H Boxer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Spectroscopic Studies ◽  
Variable Number ◽  
Low Ph ◽  
Paramagnetic Resonance ◽  
High Ph ◽  
Enzyme Form ◽  
Dependent Change ◽  
Small Change

Previous e.p.r. work [George, Bray, Morpeth & Boxer (1985) Biochem. J. 227, 925-931] has provided evidence for a pH- and anion-dependent transition in the structure of the Mo(V) centre of Escherichia coli nitrate reductase, with the low-pH form bearing both an anion and probably a hydroxy-group ligand. Initial e.x.a.f.s. measurements [Cramer, Solomonson, Adams & Mortenson (1984) J. Am. Chem. Soc. 106, 1467-1471] demonstrated the presence of sulphur (or chloride) ligands in the Mo(IV) and Mo(VI) oxidation states, as well as a variable number of terminal oxo (Mo = O) groups. To synthesize the e.p.r. and e.x.a.f.s. results better, we have conducted new e.p.r. experiments and complementary e.x.a.f.s. measurements under redox and buffer conditions designed to give homogeneous molybdenum species. In contrast with results on other molybdoenzymes, attempts to substitute the enzyme with 17O by dissolving in isotopically enriched water revealed only very weak hyperfine coupling to 17O. The significance of this finding is discussed. Experiments with different buffers indicated that buffer ions (e.g. Hepes) could replace the Cl- ligand in the low-pH Mo(V) enzyme form, with only a small change in e.p.r. parameters. E.x.a.f.s. studies of the oxidized and the fully reduced enzyme were consistent with the e.p.r. work in indicating a pH- and anion-dependent change in structure. However, in certain cases non-stoichiometric numbers of Mo = O interactions were determined, complicating the interpretation of the e.x.a.f.s. Uniquely for a molybdenum cofactor enzyme, a substantial proportion of the molecules in a number of enzyme samples appeared to contain no oxo groups. No evidence was found in our samples for the distant ‘heavy’ ligand atom reported in the previous e.x.a.f.s. study. The nature of the high-pH-low-pH transition is briefly discussed.

scholarly journals Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12

1976 ◽  
Vol 155 (1) ◽  
pp. 201-203 ◽  
Author(s):  
R C Bray ◽  
S P Vincent ◽  
D J Lowe ◽  
R A Clegg ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Electron Paramagnetic Resonance ◽  
Nitrate Reductase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Escherichia Coli K12 ◽  
Electron Paramagnetic ◽  
Respiratory Nitrate Reductase

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.

Purification and characterization of catalase HPII from Escherichia coli K12

1986 ◽  
Vol 64 (7) ◽  
pp. 638-646 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Unusual Structure ◽  
Escherichia Coli K12 ◽  
Molecular Weights ◽  
Ph Range ◽  
Heme Cofactor ◽  
Native Molecular Weight

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90 000 and 92 000. Only a single 92 000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532 000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4–11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.

scholarly journals Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12

1987 ◽  
Vol 168 (2) ◽  
pp. 451-459 ◽  
Author(s):  
Chantal IOBBI ◽  
Claire-Lise SANTINI ◽  
Violaine BONNEFOY ◽  
Gerard GIORDANO
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Escherichia Coli K12

Genetic mapping of the chl C gene of the nitrate reductase a system in Escherichia coli K12

1969 ◽  
Vol 35 (5) ◽  
pp. 659-662 ◽  
Author(s):  
Juan Puig ◽  
Edgard Azoulay ◽  
Francis Pichinoty ◽  
Jacqueline Gendre
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Genetic Mapping ◽  
Escherichia Coli K12

scholarly journals Purification and further characterization of the second nitrate reductase of Escherichia coli K12

1990 ◽  
Vol 188 (3) ◽  
pp. 679-687 ◽  
Author(s):  
Chantal IOBBI-NIVOL ◽  
Claire-Lise SANTINI ◽  
Francis BLASCO ◽  
Gerard GIORDANO
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Escherichia Coli K12

scholarly journals Investigation by electron paramagnetic resonance spectroscopy of the molybdenum centre of respiratory nitrate reductase from Paracoccus denitrificans

1988 ◽  
Vol 252 (3) ◽  
pp. 925-926 ◽  
Author(s):  
N Turner ◽  
A L Ballard ◽  
R C Bray ◽  
S Ferguson
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Paracoccus Denitrificans ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Species Comparison ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic ◽  
Respiratory Nitrate Reductase ◽  
Active Centres

The molybdenum centre of respiratory nitrate reductase from Paracoccus denitrificans has been investigated by e.p.r. spectroscopy of Mo(V). In common with the centres of the analogous enzymes from Escherichia coli and Pseudomonas aeruginosa, it undergoes a pH- and anion-dependent transition between two different e.p.r. signal-giving species. Comparison of the relevant e.p.r. parameters extracted with the help of computer simulations reveals ligation of the metal in the active centres of the three enzymes to be identical.

scholarly journals pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine

1978 ◽  
Vol 175 (3) ◽  
pp. 879-885 ◽  
Author(s):  
A D Tsopanakis ◽  
S J Tanner ◽  
R C Bray
Keyword(s):  
Nitrate Reductase ◽  
Xanthine Oxidase ◽  
Low Ph ◽  
High Ph ◽  
Ph Values ◽  
Exchangeable Proton ◽  
Subzero Temperatures ◽  
Ph Jump ◽  
Unique Nature ◽  
Prototropic Equilibrium

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at–82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to–60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.

scholarly journals Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

1998 ◽  
Vol 180 (16) ◽  
pp. 4192-4198 ◽  
Author(s):  
Andrew J. Darwin ◽  
Eva C. Ziegelhoffer ◽  
Patricia J. Kiley ◽  
Valley Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Binding Site ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Operon Expression ◽  
Nitrate And Nitrite ◽  
K 12

ABSTRACT The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, thenapF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napFcontrol region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

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