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

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 Mo^VI unit was also observed, using ESR detection. The results are interpreted in terms of rapid electron transfer from Mo^V 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 Mo^V 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 Mo^V and the substrate in this complex.

On annealing, the first change involved conversion of the primary Mo^V centre to a secondary Mo^V 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 Mo^V 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.