scholarly journals The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals

1979 ◽  
Vol 177 (1) ◽  
pp. 357-360 ◽  
Author(s):  
R C Bray ◽  
S Gutteridge ◽  
D A Stotter ◽  
S J Tanner
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
Low Ph ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Alternative Pathways ◽  
Ph Values ◽  
Covalent Intermediate ◽  
The Relationship ◽  
Electron Paramagnetic

On the basis of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897] and of other data in the literature, a mechanism for the reaction of xanthine oxidase with reducing substrates is proposed. In the Michaelis complex, xanthine is bound to molybdenum via the N-9 nitrogen atom. Coupled transfer of two electrons to molybdenum and the C-8 proton to the enzyme yields (Enzyme)-Mo-SH. Concerted with this process, reaction of the xanthine residue with a nucleophile in the active centre yields a covalent intermediate that breaks down to give the product by alternative pathways at high and at low pH values.

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 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 Hydrogels from a Protein in a Molten Globule-like State

2020 ◽  
Author(s):  
Seyed Hamidreza Arabi ◽  
Behdad Aghelnejad ◽  
Jonas Volmer ◽  
Dariush Hinderberger
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Bovine Serum Albumin ◽  
Stearic Acid ◽  
Molten Globule ◽  
High Concentration ◽  
Paramagnetic Resonance ◽  
Ph Values ◽  
Amphiphilic Molecules ◽  
Viscous Solutions ◽  
Electron Paramagnetic

We demonstrate that a molten globule-like (MG) state of a protein, usually described as a compact yet non-folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, hydrogels made from MG-BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR-active stearic acid derivatives (16-DSA), the MG-state hydrogel is the least long-lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules.

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 Coupling of [33S]sulphur to molybdenum(V) in different reduced forms of xanthine oxidase

1981 ◽  
Vol 199 (3) ◽  
pp. 629-637 ◽  
Author(s):  
J P G Malthouse ◽  
G N George ◽  
D J Lowe ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
The Other ◽  
Reduced Form ◽  
Sulphur Atom ◽  
Paramagnetic Resonance ◽  
Different Types ◽  
Resonance Coupling ◽  
Electron Paramagnetic ◽  
Reduced Forms

Different reduced forms of xanthine oxidase, labelled specifically in the cyanide-labile site with 33S, were prepared and examined by electron paramagnetic resonance. Coupling of this isotope to molybdenum(V) was quantified with the help of computer simulations and found to differ markedly from one reduced form to another. The xanthine Very Rapid signal shows strong, highly anisotropic, coupling with A(33S)av. 1.27 mT. For this signal, axes of the g- and A(33S)-tensors are rotated relative to one another. One axis of the A-tensor is in the plane of gxx ang gyy, but rotated by 40 degrees relative to the gxx axis, whereas the direction of weakest coupling to sulphur deviates by 10 degrees from the gzz axis. In contrast with this signal, only rather weaker coupling was observed in different types of Rapid signal [A(33S)av. 0.3--0.4 mT], and in the Inhibited signal coupling was weaker still [A(33S)av. 0.1--0.2 mT]. Clearly, there must be substantial differences in the structures of the molybdenum centre in the different signal-giving species, with the sulphur atom perhaps in an equatorial type of ligand position in the Very Rapid species but in a more axial one in the other species. Structures are discussed in relation to the mechanism of action of the enzyme and the nature of the proton-accepting group that participates in turnover.

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