Combined QM/MM calculations and MD simulations are utilized to investigate the detailed mechanisms of reactions catalyzed by wild-type nickel-dependent quercetin 2,4-dioxygenase (Ni-QueD) and its Glu76Asp and Glu76Gln mutants. The conserved nickel-ligating Glu76 residue in the deprotonated form is found to be essential for initiating the catalytic reaction by proton coupled electron transfer process. The generated protonated Glu76 promotes the subsequent reaction by regulating hydrogen-bonding (H-bonding) interaction with the carbonyl groups of quercetin. Investigations of Glu76Gln and Glu76Asp mutants show that mutation of Glu76 suppresses such H-bonding interaction and results in the lower catalytic activity observed experimentally. Thus, our results reveal the critical role of Glu76 residue in steering the reactivity of Ni-QueD. This work is not only useful for better understanding the mechanisms of reactions catalyzed by other metal ion-dependent QueDs, but also provides insights into how enzymes achieve specific reactions by utilizing H-bonding interaction from the metal center-ligating residues. (C) 2020 Elsevier Inc. All rights reserved.