Electron spin-echo spectroscopy of the iron–sulphur clusters of xanthine oxidase from milk

Abstract

Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The environment of each type of cluster has been examined by electron spin-echo envelope modulation (ESEEM) spectroscopy, where modulations were observed for the iron–sulphur clusters in the reduced state. The spectral contributions of the two clusters were distinguished (a) by poising the samples at redox potentials such that either Fe-S(II) was predominantly reduced, or both iron–sulphur clusters were fully reduced and (b) by measurements at their characteristic g factors. Spectra of both [2Fe-2S] clusters showed weak ¹⁴N hyperfine interactions, similar to those seen in other [2Fe-2S] proteins, which were attributed to nitrogens of the polypeptide chain; there was no evidence for histidine imidazole coordination. Spectra of samples exchanged into ²H₂O gave evidence for exchangeable protons in close proximity to Fe–S clusters. By these criteria the environment of the [2Fe-2S] clusters in this complex protein is similar to those in the plant-type ferredoxins. In addition, ESEEM spectra of molybdenum (V) in the desulpho-inhibited form of the enzyme did not show modulations due to ¹⁴N.