Investigation Into the Role of Reductants and Cosubstrates in Lytic Polysaccharide Monooxygenase Thermothielavioides terrestris AA9E Binding to Cellulose by Single-Molecule Imaging

Cellulose-active Lytic Polysaccharide Monooxygenases (LPMO) facilitate plant cell wall deconstruction by attacking ordered regions of cellulose. In vitro, reductants (e.g., ascorbic acid) reduce LPMOs to Cu(I)-LPMO, and hydrogen peroxide (H₂O₂) serves as co-substrate for oxidative cleavage of cellulose glycosidic bonds. Super-resolution single-molecule imaging by total internal reflection fluorescence microscopy was used to visualize and enumerate binding events of fluorescently-labeled Thermothielavioides terrestris AA9E (TtAA9E) on highly ordered cellulose fibrils in oxygen-scavenging buffer systems. In the glucose oxidase/catalase (GODCAT) system, oxygen is converted to H₂O₂, then removed by catalase. Adding ascorbic acid to the GODCAT system promoted rapid binding to cellulose by TtAA9E. In contrast, absent both oxygen and H₂O₂ in the protocatechuic acid/protocatechuate 3,4-dioxygenase (PCA/PCD) oxygen-scavenging system, adding ascorbic acid nearly eliminated cellulose binding by TtAA9E. Our results suggest that in the GODCAT system, TtAA9Es are reduced by ascorbic acid and activated by H₂O₂, facilitating binding to cellulose. In the PCA/PCD system, reduced TtAA9Es are not activated due to the lack of H₂O₂, suggesting that reduced Cu(I)-TtAA9E cannot bind to cellulose without H₂O₂. Notably, in the PCA/PCD system with ascorbic acid, oxidized sugar release initially lagged but was observed at longer reaction times, suggesting that H₂O₂ could be a limiting reactant generated in situ as oxygen becomes absorbed into solution. Binding durations of LPMO to cellulose were independent of experimental conditions: (82% ± 6%) of cellulose-bound LPMOs resided briefly for 14 ± 2.5 s, while 16% ± 5% of the bound enzymes remained for 60 ± 9 s.