Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper...

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu²⁺ to Cu⁺. Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis (LsAA9_A) and one from Thermoascus auranti-acus (TaAA9_A), as the active-site Cu is photoreduced in the X-ray beam. For LsAA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu²⁺ form use a total X-ray dose below 3 × 10⁴ Gy, while the Cu⁺ form can be attained using 1 × 10⁶ Gy. In all cases, we observe the transition from a hexa-coordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ₂ parameter and a decrease of the θ₃ parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θ_T. Thus, the θ₂ and θ₃ parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to LsAA9_A, regardless of the production source, the θ_T parameter increases, making the Cu site less planar, while the active-site Tyr-Cu distance decreases reproducibly for the Cu²⁺ form. Thus, the θ_T increase found on copper reduction may bring LsAA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced LsAA9_A for cellulosic substrates.