{"id":723,"date":"2017-04-26T11:36:59","date_gmt":"2017-04-26T15:36:59","guid":{"rendered":"http:\/\/sites.williams.edu\/bigchem\/?page_id=723"},"modified":"2017-05-01T10:19:42","modified_gmt":"2017-05-01T14:19:42","slug":"ascorbate-oxidase","status":"publish","type":"page","link":"https:\/\/sites.williams.edu\/bigchem\/ascorbate-oxidase\/","title":{"rendered":"Ascorbate Oxidase"},"content":{"rendered":"<h1 style=\"text-align: center\"><b>Ascorbate Oxidase (AO)<\/b><\/h1>\n<hr \/>\n<h1><strong>Locations &amp; Activity<\/strong><\/h1>\n<p><span style=\"font-weight: 400\">AO found only in higher plants, present in the cell wall and cytoplasm. It catalyzes the oxidation of L-ascorbate or vitamin C at one site, shuttling a total of four electrons and four protons to reduce oxygen to water at another site (overall reaction shown below).<\/span><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\" wp-image-811 aligncenter\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Rxn-Scheme-AOODJ--300x96.png\" alt=\"\" width=\"442\" height=\"142\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Rxn-Scheme-AOODJ--300x96.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Rxn-Scheme-AOODJ-.png 452w\" sizes=\"auto, (max-width: 442px) 100vw, 442px\" \/><\/p>\n<h1><strong>Protein Structure<\/strong><\/h1>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"size-medium wp-image-808 alignleft\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Enzyme-Structure-AOODJ-300x178.png\" alt=\"\" width=\"300\" height=\"178\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Enzyme-Structure-AOODJ-300x178.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Enzyme-Structure-AOODJ-768x456.png 768w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Enzyme-Structure-AOODJ-1024x607.png 1024w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Enzyme-Structure-AOODJ.png 1600w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/p>\n<p>140 kD homodimer, 9 total Cu(II)<\/p>\n<p style=\"padding-left: 30px\">\u00a0 \u00a0 \u00a0 3 domains in each subunit: 4 Cu each<\/p>\n<ul>\n<li style=\"font-weight: 400\"><span style=\"font-weight: 400\">Mononuclear and Trinuclear Cu Centers<\/span><\/li>\n<\/ul>\n<ul>\n<li style=\"font-weight: 400\"><span style=\"font-weight: 400\">1 Cu(II) in the subunit interface<\/span>\n<ul>\n<li style=\"font-weight: 400\"><span style=\"font-weight: 400\">Function unknown<\/span><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h1><strong>Copper Centers<\/strong><\/h1>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-810 aligncenter\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Cu-Centers-AOODJ-300x88.png\" alt=\"\" width=\"433\" height=\"127\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Cu-Centers-AOODJ-300x88.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Cu-Centers-AOODJ.png 712w\" sizes=\"auto, (max-width: 433px) 100vw, 433px\" \/><\/p>\n<p>&nbsp;<\/p>\n<h1><strong>Redox Mechanism<\/strong><\/h1>\n<p><img loading=\"lazy\" decoding=\"async\" class=\" wp-image-809 alignleft\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Active-Site-AOODJ-300x168.png\" alt=\"\" width=\"366\" height=\"205\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Active-Site-AOODJ-300x168.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Active-Site-AOODJ-768x430.png 768w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Active-Site-AOODJ-1024x573.png 1024w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Active-Site-AOODJ.png 1330w\" sizes=\"auto, (max-width: 366px) 100vw, 366px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p><span style=\"font-weight: 400\">AO uses its different type copper centers to shuttle electrons. AO uses a T1 Cu site in conjunction with a single T2 and a pair T3 site 12-13 angstroms away, arranged in a trinuclear copper cluster (TNC), to reduce O<\/span><span style=\"font-weight: 400\">2<\/span><span style=\"font-weight: 400\"> to 2H<\/span><span style=\"font-weight: 400\">2<\/span><span style=\"font-weight: 400\">O. From the figure, above, we see there is a distinct geographic separation of redox activity: ascorbic acid (substrate) gets oxidized at the T1 center whereas molecular oxygen is reduced at the trinuclear (TNC) site. The TNC is linked to the T1 protein by Cys and His residues \u00a0(C1021, H1020, H1022) Ascorbate binds and is oxidized at the T1 center and oxygen is reduced at the TNC.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Ascorbic acid needs to be aligned properly in order to fit into the binding pocket at the T1 bound by histidine and tryptophan residues (pictured above). \u00a0Through homolytic cleavage of the OH bond, ascorbate gets oxidized (Cu reduced to Cu(I)) to an unstable radical monodehydroascorbate (MDHA) that ultimately becomes DHA. <\/span><\/p>\n<p>&nbsp;<\/p>\n<p><span style=\"font-weight: 400\">The electron are transferred from the T1 \u00a0copper center to the TNC via His and Cys residues. Electrons transfer between Cu atoms of the TNC, reducing them. Reduction of the T3 copper atoms destroys the OH moiety bridge between them and increases separation between the two centers. Electrons are stored in these copper centers until they reach a bound oxygen and reduce it. The exact nature of oxygen binding is undefined. Some research with laccases suggests that it binds to all the copper atoms in the TNC, but a 1993 study with zucchini suggested the existence of a peroxide\/oxygen substitute that binds to only one T3 copper that ultimately leaves as a water molecule. This repeats until 4e are transferred and oxygen is reduced to 2 molecules of water. All Cu reductions are Cu(II) to Cu(I). this only accounts for 3e<\/span><span style=\"font-weight: 400\">&#8211;<\/span><span style=\"font-weight: 400\"> when 4e<\/span><span style=\"font-weight: 400\">&#8211;<\/span><span style=\"font-weight: 400\"> are required.<\/span><\/p>\n<p><strong>Possible Pathway for Oxygen reduction to water<\/strong>:<\/p>\n<ol>\n<li style=\"font-weight: 400\">Transfer of a radical proton to the T2 copper via the hydroxyl group<\/li>\n<li style=\"font-weight: 400\">Reduction of a T3 center, whereby protons are held by the conserved E464 residue.<\/li>\n<li style=\"font-weight: 400\">Reduction of the second T2 center, with a concurrent break of the hydroxyl bridge between the two T3 centers<\/li>\n<li style=\"font-weight: 400\">Fully reduced trinuclear center engages oxygen and produces water\n<ol>\n<li style=\"font-weight: 400\">Details remain unknown<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<p><strong><em>Main Idea: Research suggests that this happens in a two-step, 2e-<\/em><em> reduction.<\/em><\/strong><\/p>\n<h1 style=\"text-align: left\"><strong>Model Systems<\/strong><\/h1>\n<p><span style=\"font-weight: 400\">Why do we want to study small models of these enzymes?<\/span><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\" wp-image-812 alignleft\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Syntetic-Model-AOODJ-300x238.png\" alt=\"\" width=\"283\" height=\"225\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Syntetic-Model-AOODJ-300x238.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/Syntetic-Model-AOODJ.png 346w\" sizes=\"auto, (max-width: 283px) 100vw, 283px\" \/><\/p>\n<p><span style=\"font-weight: 400\">Small model molecules allow us to better understand biological functions, such as electron transfer, hydrolitic rxns, and structural importance in oxygen reduction.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Construction of a Yttrium-Copper complex provides insight to oxygen reduction to water.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Yttrium bound to heptadentate [O<\/span><span style=\"font-weight: 400\">3<\/span><span style=\"font-weight: 400\">N<\/span><span style=\"font-weight: 400\">4<\/span><span style=\"font-weight: 400\">]- trisphenoxide-trisimine amine. Platform for trinuclear site; plays no role in redox rxns.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Copper centers 3-4 Angstroms apart in tricationic form, within range of multicopper oxidase centers.<img loading=\"lazy\" decoding=\"async\" class=\"size-medium wp-image-813 alignleft\" src=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/SyntheticModels2-AOODJ-300x248.png\" alt=\"\" width=\"300\" height=\"248\" srcset=\"https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/SyntheticModels2-AOODJ-300x248.png 300w, https:\/\/sites.williams.edu\/bigchem\/files\/2017\/05\/SyntheticModels2-AOODJ.png 403w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/span><\/p>\n<p><span style=\"font-weight: 400\">Complex 5 extracted at low temp (-40 and -78 <\/span><span style=\"font-weight: 400\">o<\/span><span style=\"font-weight: 400\">C) and characterized.<\/span><\/p>\n<p><span style=\"font-weight: 400\">\u00a0 \u00a0 \u00a0 \u00a0 \u00a0Cu<\/span><span style=\"font-weight: 400\">3<\/span><span style=\"font-weight: 400\">O<\/span><span style=\"font-weight: 400\">2<\/span><span style=\"font-weight: 400\"> stoichiometry.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Successful oxygen reduction in the presence of thiol reducing agent, 4-tert-butylthiophenol, indicating successful transfer of 4 e<\/span><span style=\"font-weight: 400\">&#8211;<\/span><span style=\"font-weight: 400\"> and 4H<\/span><span style=\"font-weight: 400\">+<\/span><span style=\"font-weight: 400\"> for reduction of O<\/span><span style=\"font-weight: 400\">2<\/span><span style=\"font-weight: 400\"> to H<\/span><span style=\"font-weight: 400\">2<\/span><span style=\"font-weight: 400\">O. Mimics biological system!<\/span><\/p>\n<p><span style=\"font-weight: 400\">Lionetti <\/span><i><span style=\"font-weight: 400\">et al<\/span><\/i><span style=\"font-weight: 400\"> studied mononuclear sites and other lanthanide based complexes, but found no reactivity with oxygen, so trinuclear site structure is very important to overall reactivity.<\/span><\/p>\n<h1><span style=\"font-weight: 400\">References<\/span><\/h1>\n<p><i><span style=\"font-weight: 400\">Chem. Sci.<\/span><\/i><span style=\"font-weight: 400\">, 2013,4, 785-790<\/span><\/p>\n<p><span style=\"font-weight: 400\">Berg, P.; Singer, M. <\/span><i><span style=\"font-weight: 400\">Dealing with Genes: The Language of Heredity<\/span><\/i><span style=\"font-weight: 400\">; 1st ed.; University Science Books: Mill Valley, Calif., 1992; p. 87.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Gallie, D. R., L-Ascorbic Acid: A Multifunctional Molecule Supporting Plant Growth and Development. Scientifica 2013, 2013 (4118), 1-24.<\/span><\/p>\n<p><span style=\"font-weight: 400\">Kjaergaard, C. H.; Jones, S. M.; Gounel, S.; Mano, N.; Solomon, E. I., Two-Electron Reduction versus One-Electron Oxidation of the Type 3 Pair in the Multicopper Oxidases. Journal of the American Chemical Society 2015, 137 (27), 8783-8794.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Ascorbate Oxidase (AO) Locations &amp; Activity AO found only in higher plants, present in the cell wall and cytoplasm. It catalyzes the oxidation of L-ascorbate or vitamin C at one site, shuttling a total of four electrons and four protons &hellip; <a href=\"https:\/\/sites.williams.edu\/bigchem\/ascorbate-oxidase\/\">Continue reading <span class=\"meta-nav\">&rarr;<\/span><\/a><\/p>\n","protected":false},"author":1557,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-723","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/pages\/723","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/users\/1557"}],"replies":[{"embeddable":true,"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/comments?post=723"}],"version-history":[{"count":5,"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/pages\/723\/revisions"}],"predecessor-version":[{"id":815,"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/pages\/723\/revisions\/815"}],"wp:attachment":[{"href":"https:\/\/sites.williams.edu\/bigchem\/wp-json\/wp\/v2\/media?parent=723"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}