Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions.

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper-oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.