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'''Xanthine oxidoreductase''' (XOR) is an extensively studied metalloflavoprotein that is found in a variety of different organisms, ranging from bacteria to eukaryotes. XORs are dimeric enzymes typically around 300 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of two cysteines residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation. XOR is involved in purine catabolism, catalyzing the oxidation of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons. The transport of these electrons is facilitated by the molybdenum of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/2'>molybdopterin cofactor</scene>, two
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'''Xanthine oxidoreductase''' (XOR) is an extensively studied metalloflavoprotein that is found in a variety of different organisms, ranging from bacteria to eukaryotes. XORs are dimeric enzymes typically around 300 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of two cysteines residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation. XOR is involved in purine catabolism, catalyzing the oxidation of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons. The transport of these electrons is facilitated by the molybdenum of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/3'>molybdopterin cofactor</scene>, two
<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound
<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound
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<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/3'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced flavin to a final NAD+ acceptor, creating NADH. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. Consequently, the reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts. The prodution of these oxidative species has been implicated in the innate immune response <ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.
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<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced flavin to a final NAD+ acceptor, creating NADH. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. Consequently, the reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts. The prodution of these oxidative species has been implicated in the innate immune response <ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.

Revision as of 19:26, 26 April 2010

Bovine Milk Xanthine Dehydrogenase Y-700 Bound Form

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Contents

Overview

Xanthine oxidoreductase (XOR) is an extensively studied metalloflavoprotein that is found in a variety of different organisms, ranging from bacteria to eukaryotes. XORs are dimeric enzymes typically around 300 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of two cysteines residues or irreversible trypsin truncation. XOR is involved in purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to urate through the extraction of two electrons. The transport of these electrons is facilitated by the molybdenum of the , two , and a bound coenzyme. In XDH the electrons are then passed preferentially from the reduced flavin to a final NAD+ acceptor, creating NADH. Apart from NADH, XDH may also use O2 as a final electron acceptor. In contrast, conversion to the XO form precludes NAD+ from binding, permitting only the use of O2. Consequently, the reduction of O2 produces substantial amounts of H2O2 and superoxide as byproducts. The prodution of these oxidative species has been implicated in the innate immune response [1] and cardiovascular disease, such as atherosclerosis [2], ischemia-reperfusion injury, and chronic heart failure [3] [4].


Structure

Bovine xanthine dehydrogenase has the overall dimensions 155 Ǻ x 90 Ǻ x 70 Ǻ in its dimeric form and 100 Ǻ x 90 Ǻ x 70 Ǻ for the individual protomers. The overall structure of the enzyme can be categorized into three key domains. The (green, residues 1- 165) harbors the two Fe-S clusters. The second, (blue, residues 226-531) contains the FAD domain and the NAD/O2 binding site. The (purple, residues 590-1332) contains the Mo-pterin co-factor and is positioned close to the interface between the other two domains. This structure allows for interactions between co-factors of the same protomer. However, closest distance of co-factors between the two subunits is greater than 50 Ǻ, suggesting that the two subunits do not cross communicate [5].


Mechanism

Xanthine Oxidation

Several mechanisms have been suggested for the oxidation of xanthine to urate by xanthine oxidoreductase. However, a substantial amount of data appears to favor a mechanism in which a deprotonated molybdenum hydroxyl attacks the C8 atom of xanthine.

Image:Xanthine Oxidation Mechanism Mod.png
Xanthine oxidation mechanism. Choi et al. J. Inorg. Bioch. 2004 (98) 841-848.

This mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1261 [6], an event computed to occur readily in the presence of the substrate [7]. The electrons from the deprotonated oxygen are then free to attack the electrophilic C8 atom of the bound . The formation of glutamic acid stabilizes this structure through hydrogen bond interactions with the N1 atom [8]. Crystalographic data has also suggested possible stabilizing interactions between Arg880 of the active site and enolate tautomerization at C6 [9]. Bond formation between the substrate and the molybdenum center orients a Mo = S moiety equatorially to the substrate, positioning it favorably for a concomitant hydride transfer from xanthine N7 [10]. Extraction of this hydride produces Mo-SH and reduces the Mo center from Mo VI to Mo IV. This intermediate breaks down through electron transfer from the molybdenum center through the iron-sulfur clusters, known as Fe-S I and Fe-S II to the bound FAD, forming FADH2. In this mechanism the Fe-S clusters function as electron sinks, maintaining an oxidized Mo-cofactor and a reduced FADH2. The Mo atom serves as a transducer between the two electrons passed from the substrate to the single electron of system of the Fe-S clusters. The transfer of electrons can be monitored through the formation of the paramagnetic transient Mo V [11]. Subsequent reduction of NAD+ to NADH in the case of xanthine dehydrogenases and O2 to H2O2 regenerates the oxidized FAD. Other mechanisms involving protonated molybdenum hydroxyls have been proposed with similar calculated activation energies (40 kcal/mol). However, the products in these cases have been computationally determined to be less stable that the reactant complex [12].


Hypoxanthine Oxidation

Image:Hypoxanthine mechanism.png
Hypoxanthine oxidation mechanism. Yamaguchi et al. J. Biochem. 2007 (141) 513-524
The oxidation of hypoxanthine is theorized to occur much like the oxidation of xanthine. Once again the mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1262. The deprotonated molybdenum oxygen is then free to engage in a nucleophilic attack on C2 of hypoxanthine paired with the concomitant hydride transfer from hypoxanthine to the molybdenum bound sulfur. The reactive species are stabilized by hydrogen bond interactions between the protonated Glu1262 and N2 of hypoxanthine as well as between Glu803 and N5 and the carbonyl of C4 of hypoxanthine. The subsequent electron transfer and product release occur similarly to the xanthine oxidation reaction [13].


Redox Potential

The redox potential of bovine XOR differs between the XO and XDH forms and may partly explain the difference in specificity for their final electron acceptors. Microcoulometry measurements of the mid-point potentials for the cofactors of XO at pH 7.7, 25 °C were as follows: MoVI/MoV, -375 mV; Mov/MoIV, -405 mV; Fe-S Iox/red, -320 mV; Fe-S IIox/red, -230 mV; FAD/FADH2, -280 mV [14]. The Em of xanthine/urate at pH 7.65 was -410 mV, indicating that the oxidation of xanthine by the enzyme is favored and that the electron affinity of the enzyme cofactors approximately follows FAD ≥ Fe/S > Mo [15]. In the case of XDH, the iron sulfur potentials appear similar to those of XO, - 310 and -234 mV for Fe-S I and Fe-S II respectively. However, the midpoint two electron reduction potential of FAD/FADH2 is -340 mV. Considering that the E¬m of NAD is -335 mV, the flavin midpoint potential of XDH appears suitable for the reduction of NAD to NADH while the corresponding potential FAD in XO (-280 mV) is too large [16].

References

  1. ↑ Vorbach C, Harrison R, Capecchi MR. Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol. 2003 Sep;24(9):512-7. PMID:12967676
  2. ↑ McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol. 2003 Dec;285(6):H2290-7. Epub 2003 Sep 4. PMID:12958034 doi:http://dx.doi.org/10.1152/ajpheart.00515.2003
  3. ↑ Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol. 2004 Mar 16;555(Pt 3):589-606. Epub 2003 Dec 23. PMID:14694147 doi:10.1113/jphysiol.2003.055913
  4. ↑ Farquharson CA, Butler R, Hill A, Belch JJ, Struthers AD. Allopurinol improves endothelial dysfunction in chronic heart failure. Circulation. 2002 Jul 9;106(2):221-6. PMID:12105162
  5. ↑ Enroth C, Eger BT, Okamoto K, Nishino T, Nishino T, Pai EF. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion. Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10723-8. PMID:11005854
  6. ↑ Leimkuhler S, Stockert AL, Igarashi K, Nishino T, Hille R. The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase. J Biol Chem. 2004 Sep 24;279(39):40437-44. Epub 2004 Jul 20. PMID:15265866 doi:10.1074/jbc.M405778200
  7. ↑ Amano T, Ochi N, Sato H, Sakaki S. Oxidation reaction by xanthine oxidase: theoretical study of reaction mechanism. J Am Chem Soc. 2007 Jul 4;129(26):8131-8. Epub 2007 Jun 12. PMID:17564439 doi:10.1021/ja068584d
  8. ↑ Okamoto K, Matsumoto K, Hille R, Eger BT, Pai EF, Nishino T. The crystal structure of xanthine oxidoreductase during catalysis: implications for reaction mechanism and enzyme inhibition. Proc Natl Acad Sci U S A. 2004 May 25;101(21):7931-6. Epub 2004 May 17. PMID:15148401 doi:10.1073/pnas.0400973101
  9. ↑ Pauff JM, Cao H, Hille R. Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase: CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE. J Biol Chem. 2009 Mar 27;284(13):8760-7. Epub 2008 Dec 24. PMID:19109252 doi:10.1074/jbc.M804517200
  10. ↑ Nishino T, Okamoto K, Eger BT, Pai EF, Nishino T. Mammalian xanthine oxidoreductase - mechanism of transition from xanthine dehydrogenase to xanthine oxidase. FEBS J. 2008 Jul;275(13):3278-89. Epub 2008 May 30. PMID:18513323 doi:10.1111/j.1742-4658.2008.06489.x
  11. ↑ Choi EY, Stockert AL, Leimkuhler S, Hille R. Studies on the mechanism of action of xanthine oxidase. J Inorg Biochem. 2004 May;98(5):841-8. PMID:15134930 doi:10.1016/j.jinorgbio.2003.11.010
  12. ↑ Amano T, Ochi N, Sato H, Sakaki S. Oxidation reaction by xanthine oxidase: theoretical study of reaction mechanism. J Am Chem Soc. 2007 Jul 4;129(26):8131-8. Epub 2007 Jun 12. PMID:17564439 doi:10.1021/ja068584d
  13. ↑ Yamaguchi Y, Matsumura T, Ichida K, Okamoto K, Nishino T. Human xanthine oxidase changes its substrate specificity to aldehyde oxidase type upon mutation of amino acid residues in the active site: roles of active site residues in binding and activation of purine substrate. J Biochem. 2007 Apr;141(4):513-24. Epub 2007 Feb 14. PMID:17301077 doi:10.1093/jb/mvm053
  14. ↑ Spence JT, Barber MJ, Siegel LM. Determination of the stoichiometry of electron uptake and the midpoint reduction potentials of milk xanthine oxidase at 25 degrees C by microcoulometry. Biochemistry. 1982 Mar 30;21(7):1656-61. PMID:6282314
  15. ↑ Sanders SA, Massey V. The thermodynamics of xanthine oxidoreductase catalysis. Antioxid Redox Signal. 1999 Fall;1(3):371-9. PMID:11229448
  16. ↑ Sanders SA, Massey V. The thermodynamics of xanthine oxidoreductase catalysis. Antioxid Redox Signal. 1999 Fall;1(3):371-9. PMID:11229448

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