scholarly journals Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase

1983 ◽  
Vol 213 (1) ◽  
pp. 137-142 ◽  
Author(s):  
S Gutteridge ◽  
R C Bray ◽  
B A Notton ◽  
R J Fido ◽  
E J Hewitt
Keyword(s):  
Nitrate Reductase ◽  
Prolonged Exposure ◽  
Spinacia Oleracea ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Reaction Times ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Sulphite Oxidase ◽  
Electron Paramagnetic ◽  
Reduced Forms

The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as ‘signal A’ and ‘signal B’, analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.

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 Reduction and inactivation of superoxide dismutase by hydrogen peroxide

1974 ◽  
Vol 139 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Robert C. Bray ◽  
Stephen A. Cockle ◽  
E. Martin Fielden ◽  
Peter B. Roberts ◽  
Giuseppe Rotilio ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Prolonged Exposure ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Limited Range ◽  
Enzymic Activity ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Visible Spectra ◽  
Electron Reduction ◽  
Electron Paramagnetic

Reactions of H2O2 with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu2+ of the enzyme is reduced by H2O2, although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H2O2 is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O2−. On somewhat more prolonged exposure to H2O2, the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H2O2 concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m−1·s−1 at 20–25°C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.

scholarly journals Electron-paramagnetic-resonance spectroscopy studies on the dissimilatory nitrate reductase from Pseudomonas aeruginosad

1984 ◽  
Vol 224 (2) ◽  
pp. 601-608 ◽  
Author(s):  
C Godfrey ◽  
C Greenwood ◽  
A J Thomson ◽  
R C Bray ◽  
G N George
Keyword(s):  
Nitrate Reductase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Complex Signal ◽  
Resonance Spectroscopy ◽  
High Catalytic Activity ◽  
Paramagnetic Resonance ◽  
Activity Form ◽  
Spectroscopy Studies ◽  
Nitrate And Nitrite ◽  
Electron Paramagnetic

Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a ‘high-catalytic-activity’ form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.

scholarly journals Evidence from electron-paramagnetic-resonance spectroscopy for a complex of sulphite ions with the molybdenum centre of sulphite oxidase

1982 ◽  
Vol 201 (1) ◽  
pp. 241-243 ◽  
Author(s):  
R C Bray ◽  
M T Lamy ◽  
S Gutteridge ◽  
T Wilkinson
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Hyperfine Coupling ◽  
Resonance Spectrum ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Low Ph ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Ph Values ◽  
Sulphite Oxidase ◽  
Electron Paramagnetic

Reduction of sulphite oxidase by sulphite at low pH values in Mes (4-morpholine-ethanesulphonic acid) buffer gives rise to a new molybdenum(V) electron-paramagnetic-resonance spectrum different from that obtained by photoreduction of the enzyme in the same medium. The spectrum is attributed to a sulphite complex of the enzyme, showing g-values of about 2.000, 1.972 and 1.963. The complex is analogous to that with the inhibitor phosphate in that it gives rise to no observable hyperfine coupling of Mo(V) to exchangeable protons.

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.

scholarly journals Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of aldehyde oxidase

1982 ◽  
Vol 203 (1) ◽  
pp. 263-267 ◽  
Author(s):  
R C Bray ◽  
G N George ◽  
S Gutteridge ◽  
L Norlander ◽  
J G Stell ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Aldehyde Oxidase ◽  
Resonance Spectroscopy ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic ◽  
Reduced Forms

Molybdenum(V) e.p.r. spectra from reduced forms of aldehyde oxidase were obtained and compared with those from xanthine oxidase. Inhibited and Desulpho Inhibited signals from aldehyde oxidase were fully characterized, and parameters were obtained with the help of computer simulations. These differ slightly but significantly from the corresponding parameters for the xanthine oxidase signals. Rapid type 1 and type 2 and Slow signals were obtained from aldehyde oxidase, but were not fully characterized. From the general similarities of the signals from the two enzymes, it is concluded that the ligands of molybdenum must be identical and that the overall co-ordination geometries must be closely similar in the enzymes. The striking differences in substrate specificity must relate primarily to structural differences in a part of the active centre concerned with substrate binding and not involving the catalytically important molybdenum site.

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