Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme

Turner, Nigel; Bray, Robert C.; Diakun, G.P.

doi:10.1042/bj2600563

Cited by 56 publications

(69 citation statements)

References 31 publications

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“…11, with the curve-fitting results summarized in Table V. The XDH alloxanthine complex is very similar to that of bovine XO described by Bray and co-workers (26). The metal is coordinated by one oxo-ligand at 1.71 Å, three thiolates at 2.37 Å, and one oxygen or nitrogen at 2.01 Å (Table V).…”

Section: Investigation Of the Fe/s Clusters Of R Capsulatus Xdh Usinsupporting

confidence: 66%

“…should provide insight into the diverse modes of substrate binding by the enzyme. EXAFS data of the R. capsulatus XDH alloxanthine-bound complex are very similar to that of bovine XO (26), showing a mono-oxo molybdenum site with the nitrogen atom of alloxanthine directly coordinated to molybdenum, and an Mo-S ligand in addition to the two thiolates from the MPT-dithiolene group. The recent crystallography of the XDHalloxanthine complex suggested a similar coordination sphere so that the EXAFS and the crystallography data are in excellent agreement.…”

Section: Discussionmentioning

confidence: 73%

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Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Leimkühler

Hodson

George

et al. 2003

Journal of Biological Chemistry

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Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (␣␤) 2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten L III -edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active (␣␤) 2 heterotetrameric form and an inactive (␣␤) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.Xanthine dehydrogenase/oxidase (XDH 1 /XO) is a complex metallo-flavoprotein catalyzing the oxidative hydroxylation of purines, pyrimidines, pterines, and aldehyde substrates using NAD ϩ or molecular oxygen as electron acceptor. Mammalian XDH (EC 1.1.1.204) is a homodimer (290 kDa), with each subunit containing a single molybdenum cofactor (Moco) as well as two iron-sulfur clusters and an FAD molecule. Inherited XDH deficiency, referred to as xanthinuria I, is an autosomal recessive disease that is characterized by hyperuricemia, hyperuricosuria, and xanthinuria and is based on base pair exchanges in the structural gene for XDH (1). The affected individuals may develop urinary tract calculi, acute renal failure, or myositis, because of tissue deposition of xanthine, although some subjects with homozygous xanthinuria remain asymptomatic (1). Several mutations in the human XDH gene in patients with classical xanthinuria have been reported (2-4).In their native form, mammalian xanthine oxidizing enzymes exist as dehydrogenases that transfer electrons to NAD but can undergo a dehydrogenase to oxidase conversion by proteolytic cleavage or by oxidation, with the concomitant loss of the ability to use NAD ϩ as electron acceptor, and nearly 10-fold increase in the oxidase activity. The crystal structures of bovine milk XDH in both the dehydrogenase and oxidase forms have been solved at 2.1 and 2.5 Å resolution, respectively (5). It was shown that during the conversion of XDH to XO, a specific structural rearrangement blocks access of NAD ϩ to its binding site near the FAD moiety and changes ...

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Section: Investigation Of the Fe/s Clusters Of R Capsulatus Xdh Usinsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 73%

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Leimkühler

Hodson

George

et al. 2003

Journal of Biological Chemistry

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“…The most likely structure for this species, based on EPR and EXAFS data, is shown in Scheme 2a (Wootton et al, 1991;Turner et al, 1989;Bray, 1988). In view of the remarkable similarity (Table 1) of the parameters of this signal to those of the Low-g type-1 signal from Me,SO reductase, we infer that the structure illustrated applies also to this enzyme, when in this signal-giving state.…”

Section: General Structural Inferences From the Epr Parametersmentioning

confidence: 72%

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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“…Xanthine oxidase and xanthine dehydrogenase are two different forms of the same enzyme, and they catalyze the conversion of xanthine to uric acid, a step in the catabolic metabolism of purine bases. The two forms differ in the chemical nature of the oxidizing substrate, O 2 for xanthine oxidase and NAD + for xanthine dehydrogenase, and details of the purification procedure determine which form is isolated (46,47). All enzymes in this family have broad and partially overlapping substrate specificities.…”

Section: Xanthine Oxidase Familymentioning

confidence: 99%

“…All enzymes in this family have broad and partially overlapping substrate specificities. EXAFS spectroscopy has shown that the Mo is coordinated by two sulfur ligands originating from the pterin cofactor, the additional cyanide-labile sulfido group, an oxo group, and another oxygen or nitrogen ligand (28,46). Removal of the cyanide-labile sulfido group results in formation of the inactive desulfo-enzyme, with an oxygen ligand replacing the sulfido group on the Mo.…”

Section: Xanthine Oxidase Familymentioning

confidence: 99%

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

Kisker

Schindelin

Rees

1997

Annu. Rev. Biochem.

492

370

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Molybdenum-containing enzymes catalyze basic metabolic reactions in the nitrogen, sulfur, and carbon cycles. With the exception of the nitrogenase cofactor, molybdenum is incorporated into proteins as the molybdenum cofactor that contains a mononuclear molybdenum atom coordinated to the sulfur atoms of a pterin derivative named molybdopterin. Certain microorganisms can also utilize tungsten in a similar fashion. Molybdenum-cofactor-containing enzymes catalyze the transfer of an oxygen atom, ultimately derived from or incorporated into water, to or from a substrate in a two-electron redox reaction. On the basis of sequence alignments and spectroscopic properties, four families of molybdenum-cofactorcontaining enzymes have been identified. The available crystallographic structures for members of these families are discussed within the framework of the active site structure and catalytic mechanisms of molybdenum-cofactor-containing enzymes. Although the function of the molybdopterin ligand has not yet been conclusively established, interactions of this ligand with the coordinated metal are sensitive to the oxidation state, indicating that the molybdopterin may be directly involved in the enzymatic mechanism. CONTENTS

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Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme

Cited by 56 publications

References 31 publications

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

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

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

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