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 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 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 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.

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 Acid- and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis

1999 ◽  
Vol 181 (7) ◽  
pp. 2209-2216 ◽  
Author(s):  
Darcy Blankenhorn ◽  
Judith Phillips ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Low Ph ◽  
Anaerobic Growth ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
High Ph ◽  
Significant Difference ◽  
Aerobic And Anaerobic Growth ◽  
Aerobic And Anaerobic

ABSTRACT Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified inEscherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). Thegad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Expression patterns of two carbonic anhydrase genes, Na+/K+-ATPase and V-type H+-ATPase, in the freshwater crayfish, Cherax quadricarinatus, exposed to low pH and high pH

2017 ◽  
Vol 65 (1) ◽  
pp. 50 ◽  
Author(s):  
Muhammad Yousuf Ali ◽  
Ana Pavasovic ◽  
Peter B. Mather ◽  
Peter J. Prentis
Keyword(s):  
Carbonic Anhydrase ◽  
Expression Patterns ◽  
Maximum Level ◽  
Low Ph ◽  
Freshwater Crayfish ◽  
Α Subunit ◽  
Cherax Quadricarinatus ◽  
High Ph ◽  
Internal Ph ◽  
Ph Conditions

Carbonic anhydrase (CA), Na+/K+-ATPase (NKA) and Vacuolar-type H+-ATPase (HAT) play vital roles in osmoregulation and pH balance in decapod crustaceans. As variable pH levels have a significant impact on the physiology of crustaceans, it is crucial to understand the mechanisms by which an animal maintains its internal pH. We examined expression patterns of cytoplasmic (CAc) and membrane-associated form (CAg) of CA, NKA α subunit and HAT subunit a in gills of freshwater crayfish, Cherax quadricarinatus, at three pH levels – 6.2, 7.2 (control) and 8.2 – over 24 h. Expression levels of CAc were significantly increased at low pH and decreased at high pH conditions 24 h after transfer. Expression increased at low pH after 12 h, and reached its maximum level by 24 h. CAg showed a significant increase in expression at 6 h after transfer at low pH. Expression of NKA significantly increased at 6 h after transfer to pH 6.2 and remained elevated for up to 24 h. Expression for HAT and NKA showed similar patterns, where expression significantly increased 6 h after transfer to low pH and remained significantly elevated throughout the experiment. Overall, CAc, CAg, NKA and HAT gene expression is induced at low pH conditions in freshwater crayfish.

Sign in / Sign up

Export Citation Format

Share Document