ELECTRON SPIN-ECHO SPECTROSCOPY OF THE IRON SULFUR CLUSTERS OF XANTHINE-OXIDASE FROM MILK

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 N-14 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 (H2O)-H-2 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 N-14.