Spectroscopic and Electronic Structure Studies Probing Covalency Contributions to C–H Bond Activation and Transition-State Stabilization in Xanthine Oxidase

Abstract

A detailed EPR and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed which serves as a basis for developing a valence bond description of C-H activation and transition state stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde Inhibited XO have been analyzed in order to provide information regarding the relationship between the g-, 95,97Mo hyperfine (AMo), and the 13C hyperfine (AC) tensors. The analysis of the EPR spectra have allowed for greater insight into the electronic origin of key delocalizations within the Mo-Oeq-C fragment, and how these contribute to C-H bond activation/cleavage and transition state (TS) stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo=S π→C-H σ* (ΔE= 24.3 kcal/mol) and C-H σ → Mo=S π* (ΔE = 20.0 kcal/mol) back donation are important in activating the substrate for C-H bond for cleavage. Additional contributions to C-H activation derive from Oeq lp→C-H σ* (lp = lone pair; ΔE = 8.2 kcal/mol), and S lp→C-H σ* (ΔE = 13.2 kcal/mol) stabilizing interactions. The Oeq donor ligand that derives from water is part of the Mo-Oeq-C fragment probed in the EPR spectra of XO Inhibited, and the observation of Oeq lp→C-H σ* back donation indicates a key role for the Oeq in activating the substrate C-H bond for cleavage. We also show that the Oeq donor plays an even more important role in transition state (TS) stabilization. We find that Oeq→(Mo + C) charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal/mol) and the Mo-Oeq-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-Oeq-C delocalization at the TS allows for the TS to be described in valence bond terms as a resonance hybrid of the reactant (R) and product (P) valence bond wavefunctions.

Orbital contributions to CO oxidation in Mo–Cu carbon monoxide dehydrogenase

“…A natural bond orbital (NBO 17, 18 ) analysis of 1 supports this partially reduced character, and further reveals 1 to be an approximate 1:1 resonance hybrid of Mo(VI) and Mo(IV) structures arising from C-Cu σ → Mo-S π* charge donation and Mo-S π → C-Cu σ* donation (Figure 4 A–D). The NBO description of the bonding in 1 is remarkably similar to what we previously observed in CODH related XO 11 (Figure 4). Specifically, the XO intermediate formed by nucleophillic attack of a metal activated water on the carbonyl carbon of aldehyde substrates possesses an analogous combination of C-H σ → Mo-S π* and Mo-S π → C-H σ* charge donations.…”

“…Interestingly, EPR and ENDOR spectroscopic studies of the related XO enzyme reveal the presence of an enzyme-substrate C-S bond in an inhibited XO enzyme form (aldehyde inhibited XO) obtained under turnover conditions with certain aldehydes. 6, 11, 12 In light of the inhibitory nature of C-S bond formation in both XO and CODH, we wondered whether the formation of highly covalent enzyme-substrate C-S bonds is a necessary condition for catalysis in CODH. We thought it possible that the elimination of stable C-S bonded structures such as 2 (Figure 1) would lead to reduced activation barriers for CO oxidation and minimal geometric changes at the active site during catalysis.…”

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

“…This is interesting in that XO/XDH catalyzed hydroxylations do not proceed via radical intermediates with H-atom transfer. We recently showed through an NBO analysis[3, 38] that this unique reactivity can be achieved through a mechanism that utilizes a combination of Mo=S π → C-H σ* and C-H σ → Mo=S π* donor-acceptor interactions. These donor-acceptor interactions represent both proton-like and hydride-like contributions to C-H bond scission that contribute to lowering the IM1 → TS activation energy.…”

“…The fact that the Mo-O-C substrate/product covalent linkage is never broken along the reaction coordinate from IM1 to IM2 indicates that this bonding interaction is important in TS stabilization and Mo reduction, as has been shown for aldehyde substrates. [38]…”

“…This is important since the Mo-O-C product linkage provides a pathway for facilitating the two-electron oxidation of XO substrates, and has previously been shown to contribute to TS stabilization in XO with aldehyde substrates. [3, 38] Finally, the intrinsic hydrogen bonding capability of lumazine substrates has been analysed. Dual hydrogen bonding interactions involving the N1 and N8 violapterin ring nitrogen atoms and the carboxylate functionality of E880 may lead to proper alignment of the substrate in the binding pocket to promote electron density redistribution between Mo(xy) and substrate during the reduction half reaction of the enzyme, enhance stabilization of IM1 leading to an increase in the activation energy required to reach the TS, and an increase in E-P stabilization, with the latter leading to our ability to spectroscopically probe the rich electronic structure of this E-P intermediate.…”