13C-ENDOR Studies of the Inhibited Species of Xanthine Oxidase: The First Direct Evidence for a Molybdenum-Carbon Bond in a Biological System

Howes, Barry D.; Bennett, Brian; Bray, Robert C.; Richards, Raymond L.; Lowe, David J.

doi:10.1021/ja00104a075

Cited by 25 publications

(35 citation statements)

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“…However, it is clear from the behavior of the 13 C ENDOR features as a function of field that there is significant noncoincidence of the A-and g tensors; see Hoffman et al (1984) and True et al (1988)]. The 13 Chyperfine tensor determined in the present work (Table 1) is identical to the one reported in our preliminary investigations (Howes et al, 1994).…”

Section: C-endor From the Inhibited Mo(v) Epr Signalsupporting

confidence: 82%

“…2.0 Å), thus increasing the anisotropy expected, the agreement between the expected and the measured anisotropy is good. 13 C-ENDOR thus provides very strong evidence favoring a Mo-C bond in the Inhibited species, confirming the preliminary conclusions of Howes et al (1994).…”

Section: C-endor From the Inhibited Mo(v) Epr Signalsupporting

confidence: 78%

“…In a recent preliminary publication, Howes et al (1994) reported 13 C-ENDOR spectroscopy studies of the Inhibited species. Mo-C distance estimates from dipolar coupling calculations based on the results provided evidence for a Mo-C bond in this species, the first direct evidence for such a bond in a biological system.…”

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confidence: 99%

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Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies

et al. 1996

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The reaction mechanism of the molybdoenzyme xanthine oxidase has been further investigated by 13C and 17O ENDOR of molybdenum(V) species and by kinetic studies of exchange of oxygen isotopes. Three EPR signal-giving species were studied: (i) Very Rapid, a transient intermediate in substrate turnover, (ii) Inhibited, the product of an inhibitory side reaction with aldehyde substrates, and (iii) Alloxanthine, a species formed by reaction of reduced enzyme with the inhibitor, alloxanthine. The Very Rapid signal was developed either with [8-13C]xanthine or with 2-oxo-6-methylpurine using enzyme equilibrated with [17O]H2O. The Inhibited signal was developed with 2H13C2HO and the Alloxanthine signal by using [17O]H2O. Estimates of Mo-C distances were made, from the anisotropic components of the 13C-couplings, by corrected dipolar coupling calculations and by back-calculation from assumed possible structures. Estimated distances in the Inhibited and Very Rapid species were about 1.9 and less than 2.4 A, respectively. A Mo-C bond in the Inhibited species is very strongly suggested, presumably associated with side-on bonding to molybdenum of the carbonyl of the aldehyde substrate. For the Very Rapid species, a Mo-C bond is highly likely. Coupling from a strongly coupled 17O, not in the form of an oxo group, and no coupling from other oxygens was detected in the Very Rapid species. No coupled oxygens were detected in the Alloxanthine species. That the coupled oxygen of the Very Rapid species is the one that appears in the product uric acid molecule was confirmed by new kinetic data. It is concluded that this oxygen of the Very Rapid species does not, as frequently assumed, originate from the oxo group of the oxidized enzyme. A new turnover mechanism is proposed, not involving direct participation of the oxo ligand group, and based on that of Coucouvanis et al. [Coucouvanis, D., Toupadakis, A., Lane, J. D., Koo, S. M., Kim, C. G., Hadjikyriacou, A. (1991) J. Am. Chem. Soc. 113, 5271-5282]. It involves formal addition of the elements of the substrate (e.g., xanthine) across the Mo = S double bond, to give a Mo(VI) species. This is followed by attack of a "buried" water molecule (in the vicinity of molybdenum and perhaps a ligand of it) on the bound substrate carbon, to give an intermediate that on intramolecular one-electron oxidation gives the Very Rapid species. The latter, in keeping with the 13C, 17O, and 33S couplings, is presumed to have the 8-CO group of the uric acid product molecule bonded side-on to molybdenum, with the sulfido molybdenum ligand retained, as in the oxidized enzyme.

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Section: C-endor From the Inhibited Mo(v) Epr Signalsupporting

confidence: 82%

Section: C-endor From the Inhibited Mo(v) Epr Signalsupporting

confidence: 78%

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Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies

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“…37). A close carbon metal interaction is proposed in the "inhibited" species of xanthine oxidase by electron nuclear double resonance studies (38). The presence of product or product analogs bound to the reduced MoIV center of these enzymes is also supported by EXAFS (19,20) and resonance Raman studies (39).…”

Section: Discussionmentioning

confidence: 89%

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

Huber

Hof

Duarte

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

239

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“…Structural information from X-ray crystallography has only very recently started to become available on them (Romso et al, 1995). Extensive information has, however, for some while been available from spectroscopic studies, particularly EPR spectroscopy (Bray, 1988;Howes et al, 1994), as well as from sequencing work (Amaya et al, 1991;Wootton et al, 1991;Turner et al, 1995). Reducing substrates act at the molybdenum centre of the enzymes, reducing the metal from the Mo(V1) to the Mo(1V) state.…”

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confidence: 99%

Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

Doyle

Burke

Chovnick

et al. 1996

European Journal of Biochemistry

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Xanthine dehydrogenase, a molybdenum, iron-sulfur tlavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant flies. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]xanthine dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme, LE89Klxanthine dehydrogenase. These both had ultraviolet-visible absorption spectra similar to milk xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and IS357FIxanthine dehydrogenase mutated in the flavin domain, and [GI 01 IEIxanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD' as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]xanthine dehydrogenase, there was an increase in apparent K,, values both for NAD ' and NADH. These findings indicate involvement of this part of the sequence in the NAD+-binding site. The G l O l l E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the flies, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romiio, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, 1170-11761 from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [GI 01 IEIxanthine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activit...

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13C-ENDOR Studies of the Inhibited Species of Xanthine Oxidase: The First Direct Evidence for a Molybdenum-Carbon Bond in a Biological System

Cited by 25 publications

References 0 publications

Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies

Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

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13C-ENDOR Studies of the Inhibited Species of Xanthine Oxidase: The First Direct Evidence for a Molybdenum-Carbon Bond in a Biological System

Cited by 25 publications

References 0 publications

Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: 17O- and 13C-ENDOR and Kinetic Studies

Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: 17O- and 13C-ENDOR and Kinetic Studies

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

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Product

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Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies

Evidence Favoring Molybdenum−Carbon Bond Formation in Xanthine Oxidase Action: ¹⁷O- and ¹³C-ENDOR and Kinetic Studies