Neutron diffraction studies of reactive intermediates in UOX catalysis

QUICK INFORMATION
Type
Seminar
Start Date
11-03-2020 14:30
End Date
11-03-2020 15:30
Location
Room 337, Central Building
Speaker's name
Lindsay McGREGOR
Speaker's institute
Institut Laue-Langevin
Contact name
Eleanor Ryan
Host name
Montserrat SOLER LOPEZ
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There are still many unanswered questions in O2 biology. Most enzymes that utilize O2 in their catalytic process contain a metal or organic co-factor to generate organic radicals to react with O2, or to activate the molecule directly. However, there also exist O2-dependent enzymes that do not possess any co-factor and therefore have very limited chemical tools at their disposal.

Urate oxidase (UOX) is the archetypal co-factor-free oxidase enzyme. It catalyses the O2- mediated degradation of uric acid (UA) to 5-hydroxyisourate (5-HIU). Recent research has previously shown unambiguously that UOX-mediated catalysis proceeds via a 5- peroxyisourate (5-PIU) intermediate, highlighting possible mechanistic similarities with the large family of flavin-dependent monooxygenases. Despite these advances, exact details of the protonation states of the relevant chemical groups involved in catalysis remain unknown.

Neutron crystallography is an invaluable structural technique, providing complementary information to X-ray crystallography. Exploiting the ability of hydrogen, and more importantly deuterium atoms, to scatter neutrons, elusive protonation states and hydrogen bonding networks can be identified. I will present two neutron structures of key intermediates in the UOX catalytic pathway, namely the anaerobic-mimic UOX:AZA complex and the peroxide-bound UOX:PMUA complex. The 8-azaxanthine (AZA) inhibitor prevents O2 from binding, allowing us an insight into the protonation states of the active site in an anaerobic-like environment. The second complex involves structural analogue 9-methyl uric acid (MUA). This ligand traps the peroxide complex in the 5-peroxyisourate intermediate (5-PMUA). This allows the examination of protonation states in this intermediate, previously undetermined. Neutron structures for these two complexes will allow us to determine the exact chemical identity of the UOX:ligand-bound active site before and after the presence of O2 identify the nature of the peroxide and aid our understanding of what role protons play in this process.

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