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 Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases

1977 ◽  
Vol 163 (2) ◽  
pp. 279-289 ◽  
Author(s):  
M J Barber ◽  
R C Bray ◽  
R Cammack ◽  
M P Coughlan
Keyword(s):  
Uric Acid ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Xanthine Dehydrogenase ◽  
Resonance Spectroscopy ◽  
Redox Potentials ◽  
Paramagnetic Resonance ◽  
Reduction Potentials ◽  
Oxidation Reduction ◽  
Temperature Electron ◽  
Full Reduction

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.

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 Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid

1978 ◽  
Vol 171 (3) ◽  
pp. 653-658 ◽  
Author(s):  
R C Bray ◽  
M J Barber ◽  
D J Lowe
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Uric Acid ◽  
Xanthine Oxidase ◽  
Catalytic Reaction ◽  
Active Site ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Substrate Molecule ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

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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