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.

scholarly journals The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) with the molybdenum centre of xanthine oxidase from electron-paramagnetic-resonance spectroscopy

1984 ◽  
Vol 218 (3) ◽  
pp. 961-968 ◽  
Author(s):  
T R Hawkes ◽  
G N George ◽  
R C Bray
Keyword(s):  
Nitrogen Atom ◽  
Xanthine Oxidase ◽  
Strong Coupling ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Kinetic Studies ◽  
Heterocyclic Ring ◽  
Resonance Spectroscopy ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Studies were carried out on the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,5-diol) with xanthine oxidase, in extension of the work of Williams & Bray [Biochem. J. (1981) 195, 753-760]. By suitable regulation of the reaction conditions, up to 10% of the functional enzyme could be converted into the complex in the Mo(V) oxidation state. The e.p.r. spectrum of the complex was investigated in detail with the help of computer simulation and substitution with stable isotopes. Close structural analogy of the signal-giving species to that of the Very Rapid intermediate in enzyme turnover is shown by g-values (2.0279, 1.9593 and 1.9442) and by coupling to 33S in the cyanide-labile site of the enzyme [A(33S) 0.30, 3.10 and 0.70mT]. However, whereas in the Very Rapid signal there is strong coupling to 17O [Gutteridge & Bray, Biochem. J. (1980) 189, 615-623], instead, in the Alloxanthine signal there is strong coupling to a single nitrogen atom [A(14N) 0.35, 0.35, 0.32 mT]. This is presumed to originate from the 2-position of the heterocyclic ring system. From this work and from earlier kinetic studies it is concluded that alloxanthine, after being bound reversibly at the active centre, reacts slowly with it, in a specific manner, distinct from that in the normal catalytic reaction with substrates. This reaction involves elimination of an oxygen ligand of molybdenum and co-ordination, in this site, of alloxanthine via the N-2 nitrogen atom, to give a complex that is structurally but not chemically closely analogous to that of the Very Rapid species.

scholarly journals Rapid type 2 molybdenum(V) electron-paramagnetic resonance signals from xanthine oxidase and the structure of the active centre of the enzyme

1980 ◽  
Vol 185 (3) ◽  
pp. 767-770 ◽  
Author(s):  
J P Malthouse ◽  
S Gutteridge ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
Computer Simulations ◽  
Active Centre ◽  
Geometrical Isomers ◽  
Paramagnetic Resonance ◽  
The Relationship ◽  
Electron Paramagnetic

Rapid type 2 molybdenum(V) e.p.r. signals from reduced functional xanthine oxidase have been further investigated. These signals, which show strong coupling of two protons to molybdenum, have been obtained under a variety of new conditions: specifically either at pH 8.2 in the presence of borate ions, or at pH 10.1-10.7 with or without various other additions. Parameters of the signals were obtained with the help of computer simulations. In at least some of these signals, the coupled protons must be located on the enzyme rather than on bound species. The relationship between type 1 and type 2 Rapid signals is discussed. They may represent geometrical isomers, or alternatively, hydroxyl uptake as a ligand of molybdenum may be involved in formation of type 2 species.

scholarly journals Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase

1976 ◽  
Vol 157 (2) ◽  
pp. 469-478 ◽  
Author(s):  
R Cammack ◽  
M J Barber ◽  
R C Bray
Keyword(s):  
Xanthine Oxidase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Bovine Milk ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Reduction Potentials ◽  
Point Reduction ◽  
Oxidation Reduction ◽  
Mediator System ◽  
Electron Paramagnetic

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, −343 +/- 15mV; Fe/S II, −303 +/- 15mV; FAD/FADH-; −351 +/- 20mV; FADH/FADH2, −236 +/-mV; Mo(VI)/Mo(V) (Rapid), −355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), −355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), −440 +/- 25mV; Mo(V) (Slow)/Mo(IV), −480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.

scholarly journals Studies by electron-paramagnetic-resonance spectroscopy on the mechanism of action of xanthine dehydrogenase from Veillonella alcalescens

1976 ◽  
Vol 153 (2) ◽  
pp. 287-295 ◽  
Author(s):  
H Dalton ◽  
D J Lowe ◽  
R T Pawlik ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Aldehyde Oxidase ◽  
Detailed Comparison ◽  
Xanthine Dehydrogenase ◽  
Resonance Spectroscopy ◽  
Enzyme Complex ◽  
Partial Reduction ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

E.p.r- (electron-paramagnetic-resonance) spectroscopy was used to compare chemical environment and reactivity of molybdenum, flavin and iron-sulphur centres in the enzyme xanthine dehydrogenase from Veillonella alcalescens (Micrococcus lactilyticus) with those of the corresponding centres in milk xanthine oxidase. The dehydrogenase is frequently contaminated with small but variable amounts of a species resistant to oxidation and giving a new molybdenum (V) e.p.r. signal, “Resting I”. There is also a “desulpho” form of the enzyme giving a Slow Mo(V) signal, indistinguishable from that of the milk enzyme. Molybdenum of the active enzyme behaves in a manner analogous to that of the milk enzyme, giving a Rapid Mo(V) signal on partial reduction with substrates or dithionite. Detailed comparison shows that molybdenum in each enzyme must have the same ligand atoms arranged in the same manner. As with the milk enzyme, complex-formation between reduced dehydrogenase and purine substrate molecules, presumably interacting at the normal substrate-binding site, modifies the Rapid signal, confirming that such substrates interact near molybdenum. The dehydrogenase-flavin semiquinone signal is identical with that of the oxidase but, in contrast, there is only one iron-sulphur signal. The latter gives an e.p.r. spectrum similar to that of aldehyde oxidase.

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.

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