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 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 The NosX and NirX Proteins of Paracoccus denitrificans Are Functional Homologues: Their Role in Maturation of Nitrous Oxide Reductase

2000 ◽  
Vol 182 (18) ◽  
pp. 5211-5217 ◽  
Author(s):  
Neil F. W. Saunders ◽  
Jorrit J. Hornberg ◽  
Willem N. M. Reijnders ◽  
Hans V. Westerhoff ◽  
Simon de Vries ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Nitrous Oxide ◽  
Double Mutant ◽  
Paracoccus Denitrificans ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Nitrous Oxide Reductase ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic ◽  
Functional Homologues

ABSTRACT The nos (nitrous oxide reductase) operon ofParacoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the CuA center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon.

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 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.

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