Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme c omposed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. irradiation of the holoenzyme yi elded simple exponential decay curves and an estimated functional target si ze of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yie lded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple i nactivation curves but considerably different target sizes of 223 kDa and 1 9 kDa, respectively; competition for radioligand binding in irradiated R2 s ubunits yielded two species, one with a target size of similar to 210 kDa a nd the other of similar to 20 kDa. These results are consistent with a mode l in which there is radiation energy transfer between the two monomers of b oth Fl and R2 only in the holoenzyme, a radiation-induced loss of free radi cal only in the isolated R2, and an alteration of the tertiary structure of R2.