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AA9 lytic polysaccharide monooxygenases (LPMOs) are copper-dependent metalloenzymes that play a major role in cellulose degradation and plant infection. Understanding the AA9 LPMO mechanism would facilitate the improvement of plant pathogen control and the industrial application of LPMOs. Herein, via point mutation, we investigated the role of glycine 2 residue in cellulose degradation by <i>Thermoascus aurantiacus</i> AA9 LPMOs (<i>Ta</i>AA9). A computational simulation showed that increasing the steric properties of this residue by replacing glycine with threonine or tyrosine altered the H-bonding network of the copper center and copper coordination geometry, decreased the surface charge of the catalytic center, weakened the <i>Ta</i>AA9-substrate interaction, and enhanced <i>Ta</i>AA9-product binding. Compared with wild-type <i>Ta</i>AA9, G2T-<i>Ta</i>AA9 and G2Y-<i>Ta</i>AA9 variants showed attenuated copper affinity, reduced oxidative product diversity and decreased substrate Avicel binding, as determined using ITC, MALDI-TOF/TOF MS and cellulose binding analyses, respectively. Consistently, the enzymatic activity and synergy with cellulase of the G2T-<i>Ta</i>AA9 and G2Y-<i>Ta</i>AA9 variants were lower than those of <i>Ta</i>AA9. Hence, the investigated residue crucially affects the catalytic activity of AA9 LPMOs, and we propose that the electropositivity of copper may correlate with AA9 LPMO activity. Thus, the relationship among the amino acid at position 2, surface charge and catalytic activity may facilitate an understanding of the proteins in AA9 LPMOs.