Radiation-induced changes in flavin fluorescence of dihydroorotate dehydrogenase

The effect of gamma-radiation on the flavin moiety of dihydroorotate dehydrogenase was studied by fluorescence spectroscopy. Irradiation of aerated solutions (0-7 Gy) led to a small increase in fluorescence intensity, but with higher doses a decrease in intensity was observed. The increase in fluorescence intensity after irradiation at low doses may be attributed to protein unfolding, leading to greater exposure of flavin groups and a concomitant increase in separation between the flavin moiety and the iron-sulphur centre. This was confirmed by fluorescence quenching studies using potassium iodide as quencher. The Stern-Volmer constant calculated for iodide quenching indicates a two-fold increase in the fraction of flavin moiety being accessible to the quencher after 6.6 Gy. No spectral change was observed when unirradiated or irradiated enzyme was denatured with guanidine hydrochloride. However, a decrease in fluorescence intensity in the case of irradiated samples indicated a radiation-induced decrease in the flavin fluorophore. The flavin fluorophore loss in dihydroorotate dehydrogenase was also determined using aerated, argon-saturated or nitrous oxide-saturated solutions. H and OH radicals were found to have nearly equal contributions in damaging the flavin moiety of dihydroorotate dehydrogenase.