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"Rapidly appearing" molybdenum electron-paramagnetic-resonance signals from reduced xanthine oxidase.

ABSTRACT: Further electron-paramagnetic-resonance studies relating to the role of molybdenum in the enzymic mechanisms of xanthine oxidase were carried out. The classification of the various molybdenum signals obtained on reducing the enzyme is briefly discussed. The group of ;Rapidly appearing' signals, which are obtained with all substrates within the turnover time and which show interaction with exchangeable protons, were studied in detail. Signals with salicylaldehyde, purine and xanthine in H(2)O and in 95% D(2)O were examined at 9 and 35GHz and interpreted with the help of computer simulation. Molybdenum atoms in a number of different chemical environments are involved, each substrate giving rise to two superimposed spectra with slightly different parameters; g values and proton splittings were determined. The spectrum with salicylaldehyde is believed to represent the reduced enzyme alone not in the form of a complex with substrate and its two constituents are believed to represent the two molybdenum atoms bonded slightly differently within the enzyme molecule. With purine and xanthine the spectra are thought to represent complexes of reduced enzyme with substrate molecules. With xanthine one signal-giving species shows coupling to two equivalent protons, whereas in all the other species observed two non-equivalent protons are involved. The origin of the protons is discussed in the light of the direct hydrogen-transfer mechanism implicated earlier for the enzyme. It is concluded that the proton derived from the substrate is located at least 3å from the molybdenum atom with which it interacts.

SUBMITTER: Bray RC

PROVIDER: S-EPMC1184959 | biostudies-other | 1969 Oct

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1186375 | biostudies-other

Complex-formation between reduced xanthine oxidase and purine substrates demonstrated by electron paramagnetic resonance.

Project description:The origin of the Rapid molybdenum electron-paramagnetic-resonance signals, which are obtained on reducing xanthine oxidase with purine or with xanthine, and whose parameters were measured by Bray & Vänngård (1969), was studied. It is concluded that these signals represent complexes of reduced enzyme with substrate molecules. Xanthine forms one complex at high concentrations and a different one at low concentrations. Purine forms a complex indistinguishable from the low-concentration xanthine complex. There are indications that some other substrates also form complexes, but uric acid, a reaction product, does not appear to do so. The possible significance of the complexes in the catalytic cycle of the enzyme is discussed and it is suggested that they represent substrate molecules bound at the reduced active site, waiting their turn to react there, when the enzyme has been reoxidized. Support for this role for the complexes was deduced from experiments in which frozen samples of enzyme-xanthine mixtures, prepared by the rapid-freezing method, were warmed until the signals began to change. Under these conditions an increase in amplitude of the Very Rapid signal took place. Data bearing on the origin of the Slow molybdenum signal are also discussed. This signal disappears only slowly in the presence of oxygen, and its appearance rate is unaffected by change in the concentration of dithionite. It is concluded that, like other signals from the enzyme, it is due to Mo(v) but that a slow change of ligand takes place before it is seen. The Slow species, like the Rapid, seems capable of forming complexes with purines.

| S-EPMC1184960 | biostudies-other

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

Project description: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.

| S-EPMC1161456 | biostudies-other

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

Project description: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.

| S-EPMC1184010 | biostudies-other

Pulsed electron paramagnetic resonance spectroscopy of (33)S-labeled molybdenum cofactor in catalytically active bioengineered sulfite oxidase.

Project description:Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ? 0) near the Mo(V) (d(1)) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from (33)S-labeled MPT in this catalytically active SO variant are dominated by the "interdoublet" transition arising from the strong nuclear quadrupole interaction, as also occurs for the (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

| S-EPMC3927148 | biostudies-literature

Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of aldehyde oxidase.

Project description: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.

| S-EPMC1158218 | biostudies-other

Electron-paramagnetic-resonance parameters of molybdenum(V) in sulphite oxidase from chicken liver.

Project description:A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme.

| S-EPMC1161366 | biostudies-other

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.

Project description: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.

| S-EPMC1153429 | biostudies-other

MoV electron paramagnetic resonance of sulfite oxidase revisited: the low-pH chloride signal.

Project description:Valuable information on the active sites of molybdenum enzymes has been provided by Mo(V) electron paramagnetic resonance (EPR) spectroscopy. In recent years, multiple resonance techniques have been extensively used to examine details of the active-site structure, but basic continuous-wave (CW) EPR has not been re-evaluated in several decades. Here, we present a re-examination of the CW EPR spectroscopy of the sulfite oxidase low-pH chloride species and provide evidence for direct coordination of molybdenum by chloride.

| S-EPMC2841525 | biostudies-literature

Evidence from electron-paramagnetic-resonance spectroscopy for a complex of sulphite ions with the molybdenum centre of sulphite oxidase.

Project description: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.

| S-EPMC1163632 | biostudies-other

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