Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme–Substrate–Dioxygen Configuration in a Cofactor‐free Oxidase

Abstract

This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) (grant number BB/I020411/1) to R.A.S. We acknowledge the use of the NIH Biowulf cluster and the EPSRC UK National Service for Computational Chemistry Software (NSCCS) in carrying out this work. We thank B. Castro and M. El Hajji (Sanofi-Aventis, Montpellier, France) for supplying purified UOX for some experiments. We are grateful to D. Heyes and N. S. Scrutton of the Manchester Institute of Biotechnology for the use of the anaerobic chamber. We thank A. Pandini (King's College London) and J. Foadi (Diamond Light Source) for advice on R scripting. We thank the ESRF and DLS for providing access to their beamlines. Beamline scientists (particularly P. Romano, DLS) are gratefully acknowledged for their support. The Cryobench is a platform of the Grenoble Instruct centre (ISBG UMS 3518 CNRS-CEA-UJF-EMBL) with support from ESRF, FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Partnership for Structural Biology (PSB).

Radiation Damage in Macromolecular Crystallography

“…More recently, enzyme catalysis in copper nitrite reductase has been elucidated by serial synchrotron crystallography (SSX) at 100 K [75] (see below). Furthermore, the catalytic cycle of the non-chromophore-containing urate oxidase, kick-started by X-ray absorption, has been studied by combining in crystallo Raman spectroscopy, QM/MM simulations and X-ray crystallography [76].…”

Cations in motion: QM/MM studies of the dynamic and electrostatic roles of H+ and Mg2+ ions in enzyme reactions

“…Describing both the quantum chemistry required for accurate description of reactions as well as the complex structural/dynamical aspects of the protein/nucleic acid environment, hybrid quantum/classical QM/MM methods have a unique, unprecedented capability to examine reaction mechanisms at sub-atomic details. An extensive amount of work has been done toward understanding various enzymatic systems such as lipoxygenases [1,2], cofactor-free oxidases [3,4], Kemp eliminase [5,6], serine hydroxymethyltransferase [7] and many others [8][9][10][11][12][13]. Here we focus on phosphate catalytic reactions, which contribute probably the most important catalytic reactions in living organisms [14][15][16][17][18][19].…”

Peer Review #1 of "Refining the reaction mechanism of O2 towards its co-substrate in cofactor-free dioxygenases (v0.2)"

“…Extensive computational details of the reaction mechanisms of coproporphyrinogen oxidase (Silva & Ramos, 2008) and vitamin K.-dependent glutamate carboxylase (Silva & Ramos, 2007) confirmed that substrate deprotonation is indeed required for their catalytic action. Evidence for substrate deprotonation is also available for urate oxidase (Bui et al, 2014), although in this instance a more complex mechanism involving transient protein-based free radicals was proposed to be operative, based on EPR measurements of anaerobic preparations of substrate-bound enzyme (Gabison et al, 2011). Based on the reaction profile towards a superoxide-scavenging spin probe, radical-pair reactivity towards O 2 has also been suggested to occur (Thierbach et al, 2014) in a bacterial ring-cleaving 2,4-PeerJ reviewing PDF | (2016:01:8687:3:0:NEW 10 Nov 2016)…”