Xanthine oxidase–product complexes probe the importance of substrate/product orientation along the reaction coordinate

Abstract

A combination of reaction coordinate computations, resonance Raman spectroscopy, spectroscopic computations, and hydrogen bonding investigations have been used to understand the importance of substrate orientation along the xanthine oxidase reaction coordinate. Specifically, 4-thiolumazine and 2,4-dithiolumazine have been used as reducing substrates for xanthine oxidase to form stable enzyme-product charge transfer complexes suitable for spectroscopic study. Laser excitation into the near-infrared molybdenum-to-product charge transfer band produces rR enhancement patters in the high frequency in-plane stretching region that directly probe the nature of this MLCT transition and provide insight into the effects of electron redistribution along the reaction coordinate between the transition state and the stable enzyme-product intermediate, including the role of the covalent Mo-O-C linkage in facilitating this process. The results clearly show that specific Mo-substrate orientations allow for enhanced electronic coupling and strong hydrogen bonding interactions with amino acid residues in the substrate binding pocket.

Mechanism of Antioxidant Activity of Betanin, Betanidin and Respective C15-Epimers via Shape Theory, Molecular Dynamics, Density Functional Theory and Infrared Spectroscopy

“…The literature widely reported the antioxidant capacity of betalains [ 16 , 39 , 51 , 52 ]. Additionally, other studies [ 12 , 13 , 66 ] showed that in a reducing half-reaction, the substrate is hydroxylated oxidatively at the molybdenum (Mo) center, whose deprotonation can be facilitated by a conserved glutamate residue (Glu1261 in the bovine enzyme). Xanthine is formed from the hydroxylation of hypoxanthine at C-2 (see Figure S5 ) and is then converted to uric acid by hydroxylation at C-8 (see Figure S5 ), which involves the generation of ROS.…”

“…In general, the interactions of the this compound with the protein pocket showed favorable interactions, allowing the inhibition of the protein. This situation is critical at the cellular level since oxygen-based radicals are associated with a series of events, including the activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH), cytochrome catalyzed linoleate peroxidation, xanthine oxidase (XO), which is a form of xanthine oxidoreductase that generates reactive oxygen species, such as radicals superoxide and hydrogen peroxide [ 13 , 66 ].…”

Spectroscopic Studies of Mononuclear Molybdenum Enzyme Centers

“…This spectral change was again very weak, with an estimated extinction change of 2.4 mM −1 cm −1 for xanthine oxidase, and 1.2 mM −1 cm −1 for xanthine dehydrogenase. Lumazine [ 8 , 9 , 10 ], 4-thiolumazine [ 11 , 12 , 13 ], and 2,4-dithiolumazine [ 11 , 12 , 13 ] have also been used as substrates for bovine xanthine oxidase and bacterial xanthine dehydrogenase. Their two-electron reduction to the corresponding violopterin products leads to the formation of stable Mo(IV)-product complexes where the product is bound to Mo(IV) as the enolate tautomer through an Mo-O-C product bridge.…”

“…Their two-electron reduction to the corresponding violopterin products leads to the formation of stable Mo(IV)-product complexes where the product is bound to Mo(IV) as the enolate tautomer through an Mo-O-C product bridge. For Mo(IV)-thioviolapterin and Mo(IV)-dithioviolapterin, the single intense long wavelength absorption feature observed in the Mo(IV)-violapterin complex is shifted to the near-infrared (NIR) region of the spectrum (758–778 nm) [ 11 , 12 , 13 ]. Neither the band energy or the overall band shape of the Mo(IV) → product charge transfer feature changed between the wild-type bacterial dehydrogenase and its Q197A and Q102G variants.…”

Metal–Dithiolene Bonding Contributions to Pyranopterin Molybdenum Enzyme Reactivity

“…Experimental investigation of the electronic structures of the Mo centers of enzymes is difficult because of the intense absorptions from other chromophores (e.g., the b-type heme in sulfite oxidase and iron sulfur centers and FAD in xanthine oxidase) [37][38][39][40][41]. However, the effects of dithiolene coordination on electronic structure have been investigated for model oxo-Mo(V) compounds ( Figure 6) by electronic absorption, XAS, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopies [12,[14][15][16][17]32,33,[42][43][44][45][46][47][48][49][50][51].…”