35 GHz ENDOR characterization of the "very rapid" signal of xanthine oxidase reacted with 2-hydroxy-6-methylpurine ((13)C8): Evidence against direct Mo-C8 interaction

Xanthine oxidase is a molybdenum-containing enzyme that catalyzes the hydro xylation of xanthine and a wide variety of other aromatic heterocycles. In the course of the reaction with xanthine and substrates such as 2-hydroxy-6 -methylpurine (HMP), the enzyme gives rise to a Mo(V) EPR signal, denoted " very rapid", that arises from an authentic catalytic intermediate. The two alternative catalytic mechanisms proposed for this enzyme differ critically in whether the distance between Mo and C8 of the purine nucleus in this in termediate is short enough to admit a direct bonding interaction. To examin e this distance, we have performed C-13 ENDOR measurements, of the "very ra pid" EPR signal generated by xanthine oxidase during reaction with (13)C8-H MP. The resulting (13)C8 hyperfine tensor, A = [10.2(1), 7.0(1), 6.5(1)] MH z, is discussed in the framework of a detailed consideration of factors inv olved in extracting metrical parameters from an anisotropic hyperfine inter action composed of contributions from multiple sources, in particular, the effect of the local contributions from spin density on (13)C8. The analysis presented here gives a Mo . . .C distance whose value is expected to be ca . 2.7-2.9 Angstrom in the "very rapid" intermediates formed with both xanth ine and HMP, consistent with plausible bond lengths for a Mo-O-C8 fragment where C8 is a trigonal-planar aromatic carbon. The difference from earlier conclusions is explained. The data thus do not support the existence of a d irect Mo-C bond in the signal-giving species. This conclusion supports a me chanism that does not involve such an interaction and which begins with bas e-assisted nucleophilic attack of the Mo-VI-OH group on the C-8 of substrat e, with concomitant hydride transfer to the Mo=S group to give Mo-IV-SH; th e EPR-active "very rapid" species then forms by one-electron oxidation and deprotonation to yield the EPR-detectable (MoOS)-O-V(OR) species. We furthe r discuss the complexities and limitations of the semiempirical method used to arrive at these conclusions.