scholarly journals A new non-functional form of milk xanthine oxidase containing stable quinquivalent molybdenum

1976 ◽  
Vol 155 (1) ◽  
pp. 81-85 ◽  
Author(s):  
D J Lowe ◽  
M J Barber ◽  
R T Pawlik ◽  
R C Bray
Keyword(s):  
Ethylene Glycol ◽  
Xanthine Oxidase ◽  
Functional Form ◽  
Aldehyde Oxidase ◽  
Xanthine Dehydrogenase ◽  
Resonance Signal ◽  
Paramagnetic Resonance ◽  
Step Process ◽  
Nitrogen Ligand ◽  
Electron Paramagnetic

A new non-functional modified form of milk xanthine oxidase is described. This contains molybdenum in a quinquivalent state, which is resistant to both oxidation and reduction. The new species is derived from the native enzyme in a two-step process. The first step is the conversion into the desulpho form, via loss of the ‘persulphide’ sulphur, and the second involves reaction with ethylene glycol or other reagents. The species gives a characteristic Mo(V) electron-paramagnetic-resonance signal, without proton splittings, designated Resting II. This is virtually identical with signals reported previously from resting turkey liver xanthine dehydrogenase and rabbit liver aldehyde oxidase. The possibility is discussed that species Resting II, prepared with ethylene glycol, contains a -COCH2OH residue bound to a nitrogen ligand of molybdenum.

scholarly journals Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases

1978 ◽  
Vol 169 (3) ◽  
pp. 471-479 ◽  
Author(s):  
D J Lowe ◽  
R C Bray
Keyword(s):  
Xanthine Oxidase ◽  
Magnetic Coupling ◽  
Magnetic Interaction ◽  
Xanthine Dehydrogenase ◽  
Resonance Signal ◽  
Guanidinium Chloride ◽  
Paramagnetic Resonance ◽  
Interacting Species ◽  
Small Change ◽  
Electron Paramagnetic

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.

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 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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