Discovery of a Copper-Binding Carbohydrate-Binding Module Regulating the Activity of Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Like other redox enzymes, LPMOs face challenges in handling reactive oxygen species (ROS) generated at their active site, which must be controlled to prevent damaging off-pathway reactions. In the case of copper catalysts, oxidative damage can become self-reinforcing, as released copper from damaged catalytic centers promotes additional ROS formation via abiotic redox cycling. Here we show that a subgroup of carbohydrate-binding modules (CBM2s), exclusively tethered to cellulose-active LPMOs, has evolved the ability to bind copper. The copper site, found on the opposite side of the CBM from the cellulose-binding surface, confers redox protection through two distinct mechanisms: preferential binding of free Cu(I) and direct interaction with the reduced catalytic copper site of the LPMO. Together, these mechanisms prevent off-pathway reactions in the absence of substrate. The protective role of these copper-binding CBM2s is demonstrated by thorough characterization of the kinetics and redox stability of a series of engineered LPMO variants. Predicted models of CBM2-LPMO interactions revealed an interdomain copper-site resembling the Cu(B) site in particulate methane monooxygenase. Notably, this spatial arrangement enables substrate-dependent regulation of copper site reactivity: the CBM2-LPMO interactions that inhibit damaging redox chemistry in the absence of substrate are relieved upon cellulose binding. This work shows a road toward improved redox stability and tunable reactivity of copper catalysts.