title: Spectroscopic and electronic structure studies probing covalency contributions to C-H bond activation and transition-state stabilization in xanthine oxidase.
abstract: A detailed electron paramagnetic resonance (EPR) and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed and serves as a basis for developing a valence-bond description of C-H activation and transition-state (TS) 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, (95,97)Mo hyperfine (A(Mo)), and (13)C hyperfine (A(C)) tensors. Analysis of the EPR spectra has allowed for greater insight into the electronic origin of key delocalizations within the Mo-O(eq)-C fragment and how these contribute to C-H bond activation/cleavage and 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(-1)) and C-H σ → Mo═S π* (ΔE = 20.0 kcal mol(-1)) back-donation are important in activating the substrate C-H bond for cleavage. Additional contributions to C-H activation derive from O(eq) lp → C-H σ* (lp = lone pair; ΔE = 8.2 kcal mol(-1)) and S lp → C-H σ* (ΔE = 13.2 kcal mol(-1)) stabilizing interactions. The O(eq)-donor ligand that derives from water is part of the Mo-O(eq)-C fragment probed in the EPR spectra of inhibited XO, and the observation of O(eq) lp → C-H σ* back-donation indicates a key role for O(eq) in activating the substrate C-H bond for cleavage. We also show that the O(eq) donor plays an even more important role in TS stabilization. We find that O(eq) → Mo + C charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal mol(-1)) and the Mo-O(eq)-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-O(eq)-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 wave functions.
authors.affiliation: Department of Chemistry and Chemical Biology, The University of New Mexico, MSC03 2060, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Sempombe
authors.forename: Benjamin
authors.initials: B
authors.lastname: Stein
authors.forename: Martin L
authors.initials: ML
authors.lastname: Kirk
mesh_terms: D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation; D002244:Carbon / Q000737:chemistry; D002417:Cattle; D002625:Chemistry, Organic / Q000379:methods*; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism; D012996:Solutions; D013057:Spectrum Analysis; D055672:Static Electricity; D013816:Thermodynamics; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000447:Aldehydes; D011355:Prodrugs; D012996:Solutions; D002244:Carbon; D006859:Hydrogen; D008982:Molybdenum; D014969:Xanthine Oxidase; D010100:Oxygen