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 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 Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli

1985 ◽  
Vol 227 (3) ◽  
pp. 925-931 ◽  
Author(s):  
G N George ◽  
R C Bray ◽  
F F Morpeth ◽  
D H Boxer
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Strong Coupling ◽  
Low Ph ◽  
High Ph ◽  
Single Proton

The interconversion of nitrate reductase from Escherichia coli between low-pH and high-pH Mo(V) e.p.r. signal-giving species was re-investigated [cf. Vincent & Bray (1978) Biochem. J. 171, 639-647]. The process cannot be described by a single pK value, since the apparent pK for interconversion is raised by the presence of various anions. The low-pH form of the enzyme exists as a series of complexes with different anion ligands of molybdenum. Each complex has specific and slightly different e.p.r. parameters, but all show strong coupling of Mo(V) to a single proton, exchangeable with the solvent, having A(1H)av. 1.0 to 1.3 mT. Complexes with Cl-, F- [A(19F)av. 0.7 mT], NO3- and NO2- give particularly well-defined spectra. The high-pH form of the enzyme is now shown to bear a coupled proton. Like that in the low-pH species, this proton is exchangeable with the solvent, but the coupling is much weaker, with A(1H)av. 0.3 mT. Thus, contrary to earlier assumptions, the proton detectable by e.p.r. is probably not identical with the proton whose dissociation controls interconversion between the two species; the latter proton could be located in the protein rather than on a ligand of molybdenum. Treatment of the enzyme with trypsin [Morpeth & Boxer (1985) Biochemistry 24, 40-46] did not affect its Mo(V) e.p.r. signals.

scholarly journals Campylobacter, Salmonella, and Escherichia coli on broiler carcasses subjected to a high pH scald and low pH postpick chlorine dip

2011 ◽  
Vol 90 (4) ◽  
pp. 896-900 ◽  
Author(s):  
M.E. Berrang ◽  
W.R. Windham ◽  
R.J. Meinersmann
Keyword(s):  
Escherichia Coli ◽  
Low Ph ◽  
High Ph

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.

Electronic and Raman spectroscopic studies of copper adrenalin complexes and copper induced compounds

1976 ◽  
Vol 54 (24) ◽  
pp. 3815-3823 ◽  
Author(s):  
Mohammed S. Rahaman ◽  
Stephen M. Korenkiewicz
Keyword(s):  
Free Radical ◽  
Hydrochloric Acid ◽  
Raman Spectra ◽  
Spectroscopic Studies ◽  
Low Ph ◽  
Basic Solution ◽  
High Ph ◽  
Raman Spectroscopic ◽  
Ph Values

Electronic and Raman spectra of adrenalin–copper(II) complexes and copper catalyzed compounds have been studied. Adrenalin reacts with copper(II) ion at pH 9.2 and higher to produce a very short lived violet free radical, a brown adrenochrome, a yellow conjugated salt, indolyl-indoquinone, and melanin. Results indicate that copper does not form complexes with adrenalin in basic solution. Between pH 6.5 and 8.5 adrenalin transforms into adrenochrome in presence of copper. The adrenochrome in 1.5 N hydrochloric acid produces the conjugate salt that is produced in the solution of high pH. At low pH values, between pH 4.0 to 5.5, adrenalin forms a brown complex with copper(II). Copper is entirely chelated to the phenolic groups of the amines. The complex in 1.5 N hydrochloric acid produces a black polymeric pigment.

AN IMMUNOLOGICAL ENQUIRY INTO THE IDENTITY OF ASSIMILATORY AND DISSIMILATORY NITRATE REDUCTASE FROM ESCHERICHIA COLI

1963 ◽  
Vol 9 (6) ◽  
pp. 781-790 ◽  
Author(s):  
E. D. Murray ◽  
B. D. Sanwal
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Electron Donor ◽  
Enzyme Induction ◽  
Specific Antibody ◽  
Particulate Material ◽  
Enzyme Form ◽  
E Coli ◽  
Antigenic Activity

The properties of the respiratory (dissimilatory) form of nitrate reductase and the assimilatory form of the same enzyme from E. coli cells were investigated. It was shown that each enzyme form was attached to cellular "particulate" material. Although each enzyme is reported in the literature to differ in its electron donor requirements, no differential assay could be established.Immunochemical investigations showed that the specific antibody against the highly purified respiratory form of enzyme could cross react with the assimilatory enzyme. Induction studies indicated that the increase in activity of the assimilatory form of nitrate reductase paralleled the increase in antigenic activity. These similarities between the respiratory and assimilatory form of nitrate reductase would indicate that few, if any, differences exist between the two forms.

Negative chemotaxis in Cytophaga johnsonae

1996 ◽  
Vol 42 (5) ◽  
pp. 515-518 ◽  
Author(s):  
Zheng-Xian Liu ◽  
Irwin Fridovich
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Salmonella Typhimurium ◽  
Nalidixic Acid ◽  
Sodium Phosphate ◽  
Low Ph ◽  
Chemotactic Response ◽  
High Ph ◽  
Gliding Bacteria ◽  
Gliding Bacterium

Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria. The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl−, and N-chlorotaurine, as well as by low pH. Its response to H2O2 was eliminated by catalase. Nalidixic acid at 200 μM, which inhibits the growth but not the motility of C. johnsonae, did not interfere with its negative chemotactic response to H2O2, whereas sodium phosphate at 10 mM, which inhibits motility, did so. Cytophaga johnsonae was not repelled by taurine, n-octanol, phenol, L-valine, or high pH. Chemotaxis can be conveniently studied in gliding bacteria such as C. johnsonae.Key words: gliding bacteria, Cytophaga johnsonae, negative chemotaxis, hydrogen peroxide, N-chlorotaurine.

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.

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