User:Adam Mirando/Sandbox 1: Difference between revisions

<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced [http://en.wikipedia.org/wiki/FAD flavin] to a final [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NAD⁺] acceptor, creating NADH <ref name="thermo" />. 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. The reduction of O₂ produces substantial amounts of H₂O₂ and superoxide as byproducts <ref name="gluarg" /><ref name="conver">PMID:15878860</ref>.  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>.

Xanthine oxidoreductase has two functional forms: xanthine dehydrogenase and xanthine oxidase. This conversion is controlled by the oxidation state of Cys535, Cys992, Cys1316, and Cys1324. When these residues are reduced, the enzyme functions as a dehydrogenase, using NAD⁺ as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopyridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie [http://en.wikipedia.org/wiki/Dithiothreitol dithiothreitol]) will restore the enzyme to the the dehydrogenase form <ref name="conver" />. Studies involving the C535A/C992R/C1316S triple mutant, however, were unable to convert to the oxidase form. Consequently, crystal structures of this mutant revealed a monomeric structure, in contrast to the normally homodimeric wild type enzyme <ref name="conver" />. The mechanism of this conversion is thought to be the formation of [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bridges] between Cys535 and Cys992 and Cys1316 and Cys1324<ref name="gluarg" /><ref name="conver" />.  Crystallographic data have show that residues 535 and 992 are capable of forming disulfide bonds<ref name="gluarg" />. Due to a distance of 15.7 Ǻ between the α-carbons of Cys535 and 992, the formation of a bond would require a substantial conformational change <ref name="structure" />. Further structural analyses reveal a peptide cluster composed of Arg426, Arg334, Trp335, and Phe549 that are tightly packed in the XDH but dispersed following disulfide bond formation. This modification is then transmitted to a loop consisting of residues 422-432 in the FAD domain restricting NAD⁺ from binding while opening a channel accessible for O₂ in the now dispersed peptide cluster<ref name="gluarg" />. As a consequence of the loop shifting, Asp429 interactions with the flavin are eliminated while new interactions between the flavin and Arg426 are introduced. These new electrostatic interactions modify the flavin potential in favor of oxidase functiondomain<ref name="structure" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/4'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/3'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, XDH only, Cys535 could not be solved in either structure), peptide cluster (teal), Arg426 (blue), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). Similar effects are also noticed in the case of irreversible conversion to XO by trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the 422-432 loop and an exchange of Asp429 for Arg426 <ref name="structure" />. The oxidation Cys1316 and 1324 also eliminates the NAD⁺ binding properties of XOR. The two residues are 20.5 Ǻ on the C-terminal tail of the enzyme. The insertion of this tail into the FAD domain appears to be essential for the binding of NAD⁺. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" /> (<scene name='User:Adam_Mirando/Sandbox_1/C-terminal_tail_xdh/1'>XDH structure</scene>: shown are Cys1317 (fuchsia), Cys1325 (green), and the C-terminal tail (yellow); C-terminal tail not solved in XO structure).

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. This mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1261 <ref>PMID:15265866</ref>, an event computed to occur readily in the presence of the substrate <ref name="theoretical">PMID:17564439</ref>. The electrons from the deprotonated oxygen are then free to attack the electrophilic C8 atom of the bound <scene name='User:Adam_Mirando/Sandbox_1/Xanthine_in_active_site/1'>xanthine</scene>. The formation of glutamic acid stabilizes this structure through hydrogen bond interactions with the N1 atom <ref>PMID:15148401</ref>. Crystalographic data has also suggested possible stabilizing interactions between Arg880 of the active site and enolate tautomerization at C6 <ref name="SubOri">PMID:19109252</ref>. 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 <ref name="gluarg">PMID:18513323</ref>. 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 <ref>PMID:15134930</ref>. 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 <ref name="theoretical" />.