Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase

Barber, Michael J.; Bray, Robert C.; Lowe, David J.; Coughlan, Michael P.

doi:10.1042/bj1530297

Cited by 52 publications

(12 citation statements)

References 24 publications

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“…5) and the EPR parameters found are presented in Tables 2 and 3. The signals obtained are closely related to those observed and described for bovine milk XO and for avian liver XD [39][40][41][42][43][44][45]. Both active and desulfo enzymes are reduced simultaneously by dithionite and, accordingly, the Mo(V) Fig.…”

Section: Steady-state Kinetics Of Osupporting

confidence: 81%

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

et al. 2007

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To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O 2•-source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O 2•-molecule and half a H 2 O 2 molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min -1 , respectively). EPR spectra of NADHreduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 lM -1 min -1 ) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 lM -1 min -1 ). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O 2•-source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O 2•-than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.Keywords Xanthine oxidoreductase Á Xanthine oxidase Á Xanthine dehydrogenase Á Rat liver Á Reactive oxygen species Á NADH Abbreviations AFR Activity-to-flavin ratio AO Aldehyde oxidase EPR Electron paramagnetic resonance FAD Flavin adenine dinucleotide ROS Reactive oxygen species Sim Simulated XD Xanthine dehydrogenase XO Xanthine oxidase XOR Xanthine oxidoreductase

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Section: Steady-state Kinetics Of Osupporting

confidence: 81%

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

et al. 2007

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“…Integrations were compared with 2 mM Cu2'-EDTA as standard, with corrections as described in earlier work (e.g. Barber et al, 1976). …”

Section: 75mentioning

confidence: 99%

Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy

Bennett

Benson

McEwan

et al. 1994

European Journal of Biochemistry

137

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The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with g,, 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with g,, values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via 0 or N, for a cysteine co-ordinating via S. Since the two signals with g,, 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.Enzymes that depend on the pterin molybdenum cofactor (Rajagopalan, 1991) are widely distributed, have a variety of important roles in different organisms and have been extensively studied in many laboratories (for recent reviews, see Spiro, 1985;Bray, 1988;Solomonson and Barber, 1990;Wootton et al., 1991 ;Enemark and Young, 1993). Detailed structural information from X-ray crystallography is not yet available on any of these enzymes (however, see Romao et al., 1993a,b). All but one of the enzymes contain, in addition to molybdenum, redox centres such as flavin, haem or ironsulphur. These complicate spectroscopic investigations and, in particular, their strong absorption in the ultraviolethisible region effectively masks the rather weak absorption of the molybdenum chromophore. Nevertheless, other spectroscopic methods have provided important insights into the local structure around the molybdenum atom. EPR spectroscopy has been very important in this context and has been supplemented particularly by extended X-ray absorption fine structure (EXAFS) and to a lesser extent by magnetic circular Abbreviations. MCD, magnetic circular dichroism ; EXAFS, extended X-ray absorpt...

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“…Spin densities of paramagnetic samples were estimated from integrations of EPR absorption spectra by comparison with a 2 m M CuZ+-EDTA standard with corrections for relative transition probabilities and tube diameters being applied as elsewhere (e.g. Barber et al, 1976). Where employed, difference spectroscopy techniques were based on methods developed by Bray and co-workers (see Bray, 1988) and were performed using Bruker ER200-SH software.…”

Section: G Chemicals Ltd) the Apparent P*h (Pd) Of Samples In 'Hmentioning

confidence: 99%

Mo(V) Electron Paramagnetic Resonance Signals from the Periplasmic Nitrate Reductase of Thiosphaera Pantotropha

Bennett

Berks

Ferguson

et al. 1994

European Journal of Biochemistry

100

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A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphueru pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrifcans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of 7: pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.Molybdenum-containing enzymes are found in all forms of life from bacteria to higher plants and man (Coughlan, 1980). With the exception of the molybdenum-containing nitrogenases which contain a novel molybdenum-iron-sulphur cluster (Burgess, 1990;Kim and Rees, 1992), the molybdenum-containing enzymes all contain one of a series of pterin molybdenum cofactors (Rajagopalan and Johnson, 1992). Enzymes of this latter type, the oxomolybdenum enzymes, participate in a wide variety of biologically important processes including nitrate assimilation in plants and microorganisms, many modes of bacterial respiration and, in higher animals, purine catabolism and sulphite oxidation. Largely on the basis of their spectroscopic properties, Wootton et al. (1991) classified the oxomolybdenum enzymes into the families of molybdenum hydroxylases, sulphite oxidases, plant and fungal assimilatory nitrate reductases and bacterial respiratory reductases. The latter class includes the respiratory nitrate reductases and dimethylsulphoxide reductases, though recent work (Bennett et al., 1994a,b) has highlighted significant differences within this group.Three types of molybdenum-containing nitrate reductases are found in bacteria ; the membrane-bound and periplasmic respiratory enzymes and the assimilatory enzyme. The membrane-bound respiratory enzymes are used by cells to utilise nitrate as the terminal electron acceptor in the absence of Correspondence to B. Bennett, School of Chemical Sciences, University of East Anglia, University Plain, Nonvich, England NR4 7TJAbbreviations. ENDOR, electron-nuclear double resonance ; EX-AFS, extended X-ray absorption fine structure; MCD, magnetic circular dichroism; pD,, , apparent pD (p*H).oxygen. The periplasmic respiratory enzymes c...

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Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase

Cited by 52 publications

References 24 publications

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy

Mo(V) Electron Paramagnetic Resonance Signals from the Periplasmic Nitrate Reductase of Thiosphaera Pantotropha

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