10.1021\/acs.biochem.0c00179<\/a><\/p>\nAuthors:<\/h4>\n
Jiafeng Geng, Andrew C. Weitz, Kednerlin Dornevil, Michael P. Hendrich, Aimin Liu<\/p>\n
Abstract:<\/h4>\n
The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans<\/i> TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by M\u00f6ssbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O2<\/sub>, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-M\u00f6ssbauer results confirmed the identity of the Michaelis complex as an O2<\/sub>-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O2<\/sub> adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse \u03b1-secondary substrate kinetic isotope effect was observed with a k<\/i>H<\/sub>\/k<\/i>D<\/sub> of 0.87 \u00b1 0.03 when (indole-d<\/i>5<\/sub>)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.<\/p>\n","protected":false},"excerpt":{"rendered":"DOI: 10.1021\/acs.biochem.0c00179 Authors: Jiafeng Geng, Andrew C. Weitz, Kednerlin Dornevil, Michael P. Hendrich, Aimin Liu Abstract: The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[],"class_list":["post-641","post","type-post","status-publish","format-standard","hentry","category-freeze-quench"],"_links":{"self":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/641","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=641"}],"version-history":[{"count":0,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/641\/revisions"}],"wp:attachment":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=641"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=641"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=641"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}