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ABSTRACT: A study of the molecular, electronic, and vibrational characteristics of the molybdenum-containing enzyme complex xanthine oxidase with violapterin has been carried out using density functional theory calculations and resonance Raman spectroscopy. The electronic structure calculations were carried out on a model consisting of the enzyme molybdopterin cofactor [in the four-valent, reduced state; Mo(IV)O(SH)] covalently linked to violapterin (1H,3H,8H-pteridine-2,4,7-trione in the neutral form) via an oxygen bridge, Mo-O-C7. Full geometry optimizations were performed for all models using the SDD basis set and the three-parameter exchange functional of Becke combined with the Lee, Yang, and Parr correlational functional. Harmonic vibrational frequencies were determined for a variety of isotopes in an attempt to correlate experimentally observed shifts upon 18O-labeling of the Mo-OR bridge to bound product as well as shifts seen upon substitution of solvent-exchangeable protons in samples prepared in D2O. The theoretical vibrational frequencies compared favorably with experimentally observed vibrational modes in the resonance Raman spectra of the reduced xanthine oxidase-violapterin complex prepared in H2O and D2O and with 18O-labeled product. Correlating the isotopic shifts from the calculations with those from the resonance Raman experiments resulted in complete normal mode assignments for all modes observed in the 350-1750 cm(-1) range. The present work demonstrates that a model in which the violapterin is coordinated to the molybdenum of the active site in a simple end-on manner via the hydroxyl group introduced by an enzyme accurately predicts the observed resonance Raman spectrum of the complex. Given the numerous modes involving the bridging oxygen, a side-on binding mode can be eliminated.
The Journal of Physical Chemistry B 03/2005; 109(7):3023-31. · 3.70 Impact Factor
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ABSTRACT: Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.
Journal of Biological Chemistry 10/2004; 279(39):40437-44. · 4.77 Impact Factor
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ABSTRACT: Recent studies of the reaction mechanism of the molybdenum-containing enzyme xanthine oxidase are presented. The pH-dependence of both the steady-state and rapid reaction kinetics of the enzyme exhibits is bell-shaped, with pK(a)s for the acid and alkaline limbs of 6.6 and 7.4, respectively. These are assigned to ionizations of an active site base and substrate, respectively, with the implication that enzyme acts on the neutral rather than monoanionic form of the purine substrate. A computational study provides evidence that in the course of the reaction tautomerization of substrate occurs, with a proton moving from N-3 to N-9 in the course of the reaction - enzyme facilitation of this tautomerization may contribute as much as 24 kcal/mol in transition state stabilization for the reaction. Electron spin echo (ESEEM) and electron-nuclear double resonance (ENDOR) studies of the so-called "very rapid" Mo(V) intermediate of the reaction, the latter work using a newly synthesized form of the substrate 2-hydroxy-6-methylpurine that has been selectively isotopically labeled at C-8, indicates that product is bound to the molybdenum of the active site in a simple, end-on fashion, consistent with a reaction mechanism involving nucleophilic attack of a (deprotonated) Mo-OH on the C-8 position of substrate. A kinetic study using a series of purines has failed to identify a correlation between the one-electron reduction potential for substrate and catalytic effectiveness, indicating that a reaction mechanism initiated by one-electron, outer-sphere electron transfer is unlikely. Finally, a consideration of the active site structure in the context of the above work suggests specific amino acid residues to target for site-directed mutagenesis studies. Preliminary experiments with two such mutants are entirely consistent with the proposed catalytic roles of two active site glutamate residues.
Journal of Inorganic Biochemistry 06/2004; 98(5):841-8. · 3.35 Impact Factor
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ABSTRACT: Current research on xanthine oxidase has favored a mechanism involving base-catalyzed proton abstraction from a Mo-OH group, allowing nucleophilic attack on the substrate and hydride transfer from the substrate to Mo=S group in the active site. During the course of this reaction mechanism, the molybdenum redox cycles from MoVI to MoIV, with reoxidation of the MoIV speices to form the EPR active MoV intermediate. However, it has also been suggested that the reaction occurs in two subsequent one-electron steps. We have determined kinetic parameters kred and kred/Kd for a variety of plausible substrates as well as the one-electron reduction potentials for these substrates. Our data indicate no correlation between these kinetic parameters and their one-electron reduction potentials, as would be expected if the enzyme were using two subsequent one-electron reduction steps. Our results provide additional support to current evidence for the favored two-electron reduction mechanism.
Journal of the American Chemical Society 01/2003; 124(49):14554-5. · 9.91 Impact Factor
