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.