Dose-dependent structural and electron-density features in the lytic polysaccharide monooxygenase NcAA9D

Structural studies of copper-containing lytic polysaccharide monooxygenases (LPMOs) by X-ray crystallography are often complicated by radiation damage. In this study, we analyze a series of 36 X-ray crystal structures of NcAA9D, a Neurospora crassa AA9-family LPMO, determined from data collected at cryogenic temperature from a single crystal to investigate the progressive effects of radiation damage at the active site of this enzyme. We report new insights into the dose-dependence of active-site geometry in LPMOs and utilize the unique pre-bound dioxygen site of NcAA9D to analyze the impact of X-ray dose on the electron density of this species. It is well established that photoreduction of the LPMO active-site copper(II) leads to expulsion of its water ligands. We further characterize this displacement and the corresponding electron-density smearing, a phenomenon that can lead to the erroneous modeling of copper-bound dioxygen species. These findings suggest that radiation-dose series collected from a single crystal provide invaluable data to support unambiguous assignment of radiation-sensitive intermediates at the active site of LPMOs and other radiation-sensitive redox enzymes.