Electron addition to xanthine oxidase. An electron spin resonance study of the effects of ionizing radiation

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Abstract
Exposure of aqueous xanthine oxidase to 60Co γ-rays at 77 K resulted in electron addition to an iron–sulphur centre at low doses. At higher doses, addition to the MoVI unit was also observed, using ESR detection. The results are interpreted in terms of rapid electron transfer from MoV to the nearest Fe/S cluster, with permanent trapping at molybdenum only when a second electron is generated in a given molecule. The ESR spectrum for the primary MoV centre closely resembles that for a centre previously only detected in the presence of a substrate molecule, such as xanthine, and known as the ‘very rapid’ centre. Hence we conclude that there is no major covalent bonding between MoV and the substrate in this complex. On annealing, the first change involved conversion of the primary MoV centre to a secondary MoV centre, exhibiting a 13 G proton hyperfine coupling. The ESR parameters closely resemble those for a centre previously described as the ‘rapid’ centre. This is the first detectable species in rapid-freeze experiments in the absence of specific substrates. When D2O was used, the proton coupling was lost. The ESR parameters are compatible with the postulate that the initial species has a sulphide ligand (Mo—S)– and the second species is protonated on sulphur (Mo—SH). Further annealing results in irreversible loss of the MoV signal and concomitant growth of a second Fe/S cluster signal. These results are discussed in terms of the remarkable selectivity of electrons ejected within the protein by γ-rays, and the very different rates of electron transfers between the different centres. There was also evidence for electron capture at an RS—SR unit giving, initially, an RS—SR– radical anion, followed by an irreversible conversion into a characteristic centre, probably formed from the anion by protonation. This centre is of interest since it is also formed from solvated electrons in pulse-radiolysis studies.