Specific radiation damage frequently compromises structural analysis in macromolecular crystallography. However, it can also offer key mechanistic information. Here, we investigate the X-ray-induced radiolysis of the catalytic C5-peroxide adduct in crystals of the cofactor-independent enzyme urate oxidase. Using a top-hat X-ray beam to ensure homogeneous dose distribution, we monitored the occupancy of the peroxide species across extensive dose series at 100 K and room temperature (RT). We observe a fundamental kinetic phase transition between these thermal regimes. At RT, the peroxide decays rapidly following zero-order kinetics, consistent with a flux-limited regime where the radiolytically cleaved O2 product diffuses out of the active site to be replaced by water. Conversely, at 100 K the decay is markedly retarded and follows first-order kinetics. Bayesian kinetic modelling demonstrates that this cryoprotection arises from a recombination mechanism: the cleaved O2 molecule remains trapped in the active site with the organic species, enabling efficient recombination that competes with irreversible degradation, effectively resulting in radiation-induced in crystallo catalysis.
Koulas et al. (Wed,) studied this question.