35 GHz ENDOR Characterization of the “Very Rapid” Signal of Xanthine Oxidase Reacted with 2-Hydroxy-6-methylpurine (¹³C8):  Evidence against Direct Mo−C8 Interaction

Abstract

Xanthine oxidase is a molybdenum-containing enzyme that catalyzes the hydroxylation 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 intermediate is short enough to admit a direct bonding interaction. To examine this distance, we have performed 13C ENDOR measurements of the "very rapid" EPR signal generated by xanthine oxidase during reaction with 13C8-HMP. The resulting (13)C8 hyperfine tensor, A = [10.2(1), 7.0(1), 6.5(1)] MHz, is discussed in the framework of a detailed consideration of factors involved in extracting metrical parameters from an anisotropic hyperfine interaction 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 A in the "very rapid" intermediates formed with both xanthine 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 direct Mo-C bond in the signal-giving species. This conclusion supports a mechanism that does not involve such an interaction and which begins with base-assisted nucleophilic attack of the Mo(VI)-OH group on the C-8 of substrate, with concomitant hydride transfer to the Mo=S group to give Mo(IV)-SH; the EPR-active "very rapid" species then forms by one-electron oxidation and deprotonation to yield the EPR-detectable Mo(V)OS(OR) species. We further discuss the complexities and limitations of the semiempirical method used to arrive at these conclusions.

Substrate Orientation in Xanthine Oxidase

“…The orientation of substrate with respect to the molybdenum center seen here in Fig. 4B, with product coordinated to the metal via the catalytically introduced hydroxyl group in a simple end-on fashion, is consistent with that proposed previously on the basis of ESEEM and ENDOR studies of this EPR-active intermediate (although the Mo-C distance seen here is again somewhat longer than predicted) (30,31). This orientation provides important support of our previously proposed reaction mechanism of xanthine oxidoreductase (33), involving base-assisted nucleophilic attack by the planar molybdenum oxygen on the C8 position of substrate, with concomitant hydride transfer to the planar sulfur.…”

“…The orientation of bound product to the molybdenum in this species, coordinated to the molybdenum in a simple end-on fashion, is in fact that expected on the basis of ESEEM and ENDOR studies of this species, although the Mo-C distance of 3.4 Å seen here is somewhat longer than the 2.9 distance expected (30,31). The orientation observed in the present crys- tal structure has important mechanistic implications, because it is inconsistent with proposed mechanisms that lead to a side-on orientation of product in the molybdenum coordination sphere (32).…”

Geometries and Electronic Structures of Cyanide Adducts of the Non-Heme Iron Active Site of Superoxide Reductases: Vibrational and ENDOR Studies

“…As discussed by Snetsinger et al for the Fe-CN linkage in transferrin (46), the anisotropic term is the sum of a local contribution proportional to the p-orbital spin density induced in the sp n orbital involved in the bond to Fe, plus a non-local contribution that is proportional to the spin density in the d π orbital on Fe and inversely proportional to the cube of r(Fe-C) (49). The two contributions both have the dipolar form with the unique direction along the Fe-C bond; the dipolar parameter for the non-local interaction has a positive sign, 2t nl > 0, whereas that for the polarization-induced local contribution is negative, 2t l < 0, like A iso .…”

ENDOR of Metalloenzymes

“…30 The above mechanism offers no role for the unique cluster iron, although it appears that the [4Fe-4S] cluster of every enzyme in the Fe-S/AdoMet family has one. To examine the coordination sphere of the unique Fe, we performed 35 GHz pulsed ENDOR spectroscopic studies of [1 + /AdoMet] labeled with 17 O/ 13 C in the carboxyl group of the methionine fragment, and with 15 N in the amino group. ENDOR signals observed with all three labels showed that both the carboxylato and amino groups of methionine are coordinated to the unique iron of the [4Fe-4S] cluster in a classical fivemembered-ring N/O chelate.…”