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

Abstract

Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5=OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.

Catalytic Mechanisms for Cofactor-Free Oxidase-Catalyzed Reactions: Reaction Pathways of Uricase-Catalyzed Oxidation and Hydration of Uric Acid

“…Recent X-ray and neutron crystal structures of uricase binding with different inhibitors and UA have provided fundamental insights into the possible enzyme-substrate binding mode in the catalytic site of the enzyme. 11h–11k As revealed in the crystal structure of thermophilic Bacillus sp. TB-90 12 bound with inhibitor 8-azaxanthine, extensive electrostatic and hydrogen-bonding interactions with the inhibitor are contributed by highly conserved residues Arg201, Gln250, Asn276, and Gln304 from one subunit, and residues Thr73 and Asp74 from another subunit.…”

“…The binding site of O 2 has also been determined to be at a site ~3 Å above the inhibitor and formed a hydrogen-bonding network with residues Asn276, Gln304, and Thr73 from the neighboring subunit. 11i,11k The X-ray structure of Aspergillus flavus uricase 11h in complex with UA and chloride ion (PDB entry of 3L9G with resolution of 1.75 Å) showed that the binding mode of UA with the uricase was very similar as the binding mode of 8-azaxanthine with Bacillus sp. TB-90 uricase.…”

“…10 It has been suggested that the catalytic reaction starts from the binding of O 2 molecule, and proceeded by the release of an intermediate hydroperoxide (H 2 O 2 ). 10d,11 However, these experimental and computational studies failed to identify the molecular species of actual substrate. Recent X-ray and neutron crystal structures of uricase binding with different inhibitors and UA have provided fundamental insights into the possible enzyme-substrate binding mode in the catalytic site of the enzyme.…”

Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

“…Online Raman spectroscopy was performed on beamline ID29 as described previously (Bui et al, 2014) using a setup specifically designed for the collection of X-ray and Raman data in an interleaved manner (von Stetten et al, unpublished work). In brief, Raman spectra were recorded using an inVia Raman instrument (Renishaw PLC, Wotton-under-Edge, England) equipped with a near-infrared (785 nm) 300 mW diode laser source.…”

Refining the reaction mechanism of O2towards its co-substrate in cofactor-free dioxygenases

“…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-dioxygenase active towards (1 H )-3-hydroxy-4-oxoquinolines (EC 1.13.11.47), but recent computational results have been interpreted as contradicting this hypothesis, as the computed reaction energy for the electron transfer from substrate to O 2 (8–11 kcal  mol −1 ) would imply an “endothermic process […] unlikely to happen spontaneously in the protein or in solvent” (Hernández-Ortega et al, 2015).…”